TW202417614A - Photoreceptor rescue cell (prc) compositions and methods for treatment of ocular disorders - Google Patents

Abstract

The present invention provides populations of photoreceptor rescue cells (PRC) possessing unique marker profiles and generated by in vitrodifferentiation from earlier progenitors including pluripotent stem cells and embryonic stem cells (ESCs). Methods of generating the populations of photoreceptor rescue cells and using them for treatment of ocular disorders are also provided.

Description

Photoreceptor rescue cell (PRC) compositions and methods for treating ocular diseases

Related applications

This application claims the benefit of U.S. Provisional Application No. 63/373,298 filed on August 23, 2022 and U.S. Provisional Application No. 63/432,948 filed on December 15, 2022, the entire contents of each of which are incorporated herein by reference.

There are a variety of retinal diseases or conditions that can lead to vision loss or even blindness. Retinal diseases or conditions include rod or cone dystrophies, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration (such as age-related macular degeneration (wet or dry), AMD secondary to pattern atrophy, Leber congenital amaurosis, and Stargardt disease. Several retinal diseases or conditions are the result of cell loss in the nuclear layer, primarily in addition to the photoreceptor cells. Replacing degenerated photoreceptor cells with new cells offers a potential way to slow or stop cell degeneration and vision loss.

Potential sources of photoreceptor cell replacement include stem cells. Early studies included the use of mouse cells, mouse stem cells, or heterogeneous retinal progenitor cell populations as possible cell sources for replacing lost photoreceptor cells. These early studies described transplantation of photoreceptor progenitor cells from the retina of postnatal day 1 mice (Maclaren et al., Nature 444(9):203-207, 2006); generation of retinal progenitor cells in vitro from mouse embryonic stem cells (Ikeda et al., Proc. Natl. Acad. Sci. 102(32):11331-11336, 2005); generation of retinal progenitor cells from the retina of postnatal day 1 mice (Klassen et al., Invest. Ophthal. Vis. Sci. 45(11):4167-4175, 2004); implantation of bone marrow mesenchymal stem cells into the RCS retinal degeneration rat model (Inoue et al., Exp. Eye Res. 8(2):234-241, 2006). 2007); generation of retinal progenitor cells from the H1 human embryonic stem cell line, including ganglion cells, axonal neurons, and photoreceptor cells (of which 0.01% of all cells expressed S-opsin or rhodopsin), bipolar cells, and horizontal cells (Lamba et al., Proc. Natl. Acad. Sci. 10(34):12769-12774, 2006); and induction of induced enriched-potential stem cells (iPS) from human fibroblasts to generate retinal progenitor cells (Lamba et al., PLoS ONE 5(1):e8763. Digital object identifier: 10.1371/journal.pone.0008763). None of these approaches produce a homogenous population of photoreceptor progenitor cells or photoreceptor cells that can be implanted. None of these approaches produce a population of photoreceptor progenitor cells or photoreceptor cells that exhibit rod or cone function in vivo, such as can be detected by conferring improved visual acuity. Stem cell differentiation into photoreceptor rescue cell populations may provide an alternative to currently available therapeutic approaches.

The present invention provides a photoreceptor rescue cell (PRC) composition, which comprises a plurality of heterogeneous photoreceptor rescue cells with a unique marker combination; and a method for using the same to treat eye diseases.

Accordingly, in one aspect, the present invention provides a photosensitive rescue cell composition comprising a plurality of heterogeneous photosensitive rescue cells, wherein the plurality of heterogeneous photosensitive rescue cells cumulatively express at least two of the markers selected from the group consisting of: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA.

In one embodiment, the photorescive cell composition further comprises a culture medium suitable for maintaining cell viability.

In one embodiment, the cells are generated by differentiation of high-potential cells in vitro.

In a specific example, the high-potential cells are embryonic stem cells (ESCs) or induced high-potential stem cells (iPSCs).

In a specific example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the composition are light-sensitive rescue cells.

In one specific example, multiple heterogeneous cells cumulatively expressed FOXG1 and MAP2.

In one embodiment, (i) about 50% to about 100%, about 50% to about 90%, about 55% to about 85%, or about 55% to about 73% of the cells in the composition express FOXG1; and/or (ii) at least about 50%, 55%, 57%, 60%, 65%, 70%, 71%, 75%, 78%, 79%, 80%, 81%, 85%, 87%, 90%, 92%, 95%, 97%, or 100% of the cells in the composition express FOXG1, and/or (iii) the cells in the composition express about 55 transcripts per million (TPM) to about 200 TPM, about 60 TPM to about 170 TPM, about 140 TPM to about 165 TPM, or about 149 TPM to about 170 TPM. and/or (iv) the cells in the composition express at least 55 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM or 200 TPM of the FOXG1 transcript.

In one embodiment, (i) about 75% to about 100%, about 75% to about 98%, about 75% to about 95%, about 77% to about 93% of the cells in the composition express MAP2; and/or (ii) at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the composition express MAP2; and/or (iii) the cells in the composition express about 250 transcripts per million (TPM) to about 700 TPM, about 290 TPM to about 650 TPM, about 450 TPM to about 625 TPM, or about 490 TPM to about 615 TPM of MAP2 transcripts; and/or (iv) the cells in the composition express at least 250 MAP2 transcript of 100 TPM, 200 TPM, 300 TPM, 350 TPM, 400 TPM, 450 TPM, 475 TPM, 490 TPM, 510 TPM, 525 TPM, 550 TPM, 575 TPM, 600 TPM, 610 TPM, 625 TPM, 650 TPM, 675 TPM, or 700 TPM.

In one embodiment, the plurality of heteroplasmic cells cumulatively express at least one other marker selected from the group consisting of STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In one embodiment, the plurality of heteroplasmic cells cumulatively express at least 3, 4, 5, 6, or 7 of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

In a specific example, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 75% to about 95%, about 75% to about 95%, about 75% to about 90%, about 70% to about 85%, about 70% to about 80%, about 75% to about 95%, about 75% to about 95%, about 75% to about 90%, about 70% to about 85%, about 70% to about 80%, about 75% to about 95%, about 75% to about 90 ... to about 90%, about 75% to about 85%, or about 75% to about 80% of the cells in the composition express STMN2; and/or (ii) at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 100% of the cells in the composition express STMN2; and/or (iii) the cells in the composition express from about 150 transcripts per million (TPM) to about 600 TPM and/or (iv) the cells in the composition express at least 150 TPM, 185 TPM, 200 TPM, 250 TPM, 300 TPM, 350 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 525 TPM, 550 TPM, 575 TPM, or 600 TPM of the STMN2 transcript.

In one embodiment, (i) about 65% to about 95%, about 65% to about 85%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 75% to about 95%, about 75% to about 90%, or about 75% to about 89% of the cells in the composition express DCX; and/or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of the cells in the composition express DCX; and/or (iii) the cells in the composition express about 200 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 900 TPM, about 600 TPM to about 900 TPM, or about 750 TPM to about 850 TPM. and/or (iv) the cells in the composition express at least 200 TPM, 250 TPM, 350 TPM, 400 TPM, 450 TPM, 500 TPM, 550 TPM, 600 TPM, 650 TPM, 700 TPM, 750 TPM, 800 TPM, 850 TPM or 900 TPM of the DCX transcript.

In one embodiment, (i) about 65% to about 98%, about 65% to about 95%, about 70% to about 98%, about 70% to about 95%, about 70% to about 90%, about 75% to about 98%, about 75% to about 90%, or about 80% to about 95% of the cells in the composition express LINC00461; and/or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 98% of the cells in the composition express LINC00461; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 100 TPM, about 50 TPM to about 95 TPM, about 85 TPM to about 95 TPM, or about 87 TPM to about 93 TPM. and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 87 TPM, 89 TPM, 90 TPM, 92 TPM, 95 TPM, or 100 TPM of the LINC00461 transcript.

In one embodiment, (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 1% to about 8%, about 1% to about 7%, about 1% to about 5%, or about 2% to about 4% of the cells in the composition express NEUROD2; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, or 25% of the cells in the composition express NEUROD2; and/or (iii) the cells in the composition express about 0 transcripts per million (TPM) to about 10 TPM, about 0.01 TPM to about 9 TPM, about 0.2 TPM to about 2 TPM, or about 0.4 and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.4 TPM, 0.6 TPM, 0.8 TPM, 1.0 TPM, 1.2 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of the NEUROD2 transcript.

In a specific example, (i) from about 35% to about 70%, from about 35% to about 68%, from about 35% to about 67%, from about 35% to about 66%, from about 35% to about 65%, from about 40% to about 70%, from about 40% to about 68%, from about 40% to about 67%, from about 40% to about 66%, from about 40% to about 65%, from about 42% to about 70%, from about 42% to about 68%, from about 42% to about about 67%, about 42% to about 66%, or about 42% to about 65% of the cells in the composition express GAD1; and/or (ii) at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, or 70% of the cells in the composition express GAD1; and/or (iii) the cells in the composition express from about 10 transcripts per million (TPM) to about 50 and/or (iv) the cells in the composition express at least 10 TPM, 12 TPM, 16 TPM, 18 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 47 TPM, or 50 TPM of the GAD1 transcript.

In a specific example, (i) from about 60% to about 95%, from about 60% to about 90%, from about 60% to about 89%, from about 60% to about 88%, from about 60% to about 87%, from about 60% to about 86%, from about 65% to about 95%, from about 65% to about 90%, from about 65% to about 89%, from about 65% to about 88%, from about 65% to about 87%, from about 65% to about 86%, from about 69% to about 90%, from about 69% to about 89%, from about 69% to about and/or (ii) at least about 50%, 55%, 60%, 65%, 67%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, or 95% of the cells in the composition express NFIA; and/or (iii) the cells in the composition express from about 30 transcripts per million (TPM) to about 120 TPM. and/or (iv) the cells in the composition express at least 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM or 120 TPM of the NFIA transcript.

In a specific example, a plurality of heteroplasmic cells cumulatively express each of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In a specific example, each of the heteroplasmic cells individually expresses at least one of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, or NFIA.

In one embodiment, the plurality of allogeneic photoreceptor rescue cells include one or more cell types selected from the group consisting of inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and alternative neurons. In one embodiment, the plurality of allogeneic photoreceptor rescue cells include each of inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and alternative neurons.

In one embodiment, the plurality of heterogeneous photosensitive rescue cells comprises inhibitory neurons expressing one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4 and CALB2. In one embodiment, the plurality of heterogeneous photosensitive rescue cells comprises inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4 and CALB2.

In one embodiment, (i) about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 52% to about 69%, about 52% to about 75%, about 54% to about 69%, about 54% to about 68%, or about 54% to about 66% of the cells in the composition express DLX5; and/or (ii) at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 72%, 74%, 76%, 78%, or 80% of the cells in the composition express DLX5; and/or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 150 TPM, about 50 TPM to about 140 TPM, about 80 and/or (iv) the cells in the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 135 TPM, 140 TPM or 150 TPM of the DLX5 transcript.

In a specific example, (i) from about 60% to about 95%, from about 70% to about 95%, from about 72% to about 95%, from about 75% to about 95%, from about 76% to about 94%, from about 77% to about 93%, from about 78% to about 93%, from about 70% to about 90%, from about 72% to about 89%, from about 73% to about 88%, from about 74% to about 87%, from about 75% to about 87%, from about 76% to about 86%, from about 77% to about 86%, or from about 79% and/or (ii) at least about 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93% or 95% of the cells in the composition express TUBB3; and/or (iii) the cells in the composition express about 150 transcripts per million (TPM) to about 500 TPM. or (iv) the cells in the composition express at least 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 430 TPM, 450 TPM, 475 TPM, or 500 TPM of the TUBB3 transcript.

In one embodiment, (i) about 45% to about 70%, about 45% to about 65%, about 50% to about 70%, about 50% to about 65%, about 50% to about 64%, about 50% to about 63%, about 50% to about 62%, about 50% to about 61%, about 46% to about 52%, about 47% to about 51%, about 48% to about 51%, or about 48% to about 50% of the cells in the composition express SCGN; and/or (ii) at least about 45%, 47%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 65%, 67%, or 70% of the cells in the composition express SCGN; and/or (iii) the cells in the composition express from about 50 transcripts per million (TPM) to about 200 transcripts per million (TPM). and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 171 TPM, 173 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, or 200 TPM of the SCGN transcript.

In a specific example, (i) about 60% to about 85%, about 60% to about 80%, about 60% to about 79%, about 60% to about 78%, about 60% to about 77%, about 60% to about 76%, about 63% to about 75%, about 63% to about 70%, about 63% to about 79%, about 63% to about 78%, about 63% to about 77%, about 63% to about 75%, about 63% to about 73%, about 63% to about 72%, about 63% to about 71% of the composition , about 65% to about 72%, or about 66% to about 71% of the cells in the composition express ERBB4; and/or (ii) at least about 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 75%, 80%, or 85% of the cells in the composition express ERBB4; and/or (iii) the cells in the composition express from about 15 transcripts per million (TPM) to about 120 or (iv) the cells in the composition express at least 15 TPM, 30 TPM, 50 TPM, 70 TPM, 73 TPM, 75 TPM, 80 TPM, 82 TPM, 85 TPM, 88 TPM, 90 TPM, 91 TPM, 93 TPM, 95 TPM, 100 TPM, 110 TPM, or 120 TPM of the ERBB4 transcript.

In one embodiment, about 35% to about 75%, about 40% to about 70%, about 40% to about 65%, about 41% to about 75%, about 41% to about 70%, about 41% to about 65%, about 41% to about 64%, about 41% to about 63%, about 41% to about 62%, about 35% to about 55%, about 40% to about 52%, or about 41% to about 52% of the cells in the composition express CALB2; and/or (ii) at least about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 45%, 47%, 50%, 51%, 52%, 55%, 60%, 65%, 70%, or 75% of the cells in the composition express CALB2; and/or (iii) the cells in the composition express from about 30 transcripts per million (TPM) to about 220 TPM. and/or (iv) the cells in the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 195 TPM, 196 TPM, 220 TPM, 210 TPM, or 220 TPM of the CALB2 transcript.

In one embodiment, the plurality of heterogeneous photoreceptor rescue cells include excitatory neurons that express one or more markers selected from the group consisting of NEUROD2, NEUROD6, SLA, NELL2, and SATB2. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells include a plurality of excitatory neurons that cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

In one embodiment, (i) about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 24%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 24%, about 0.8% to about 30%, about 0.8% to about 25%, about 0.8% to about 24%, about 1% to about 5%, about 1% to about 4.5%, about 2% to about 5%, about 2% to about 4.5%, or about 2% to about 4% of the cells in the composition express NE and/or (ii) at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 2%, 4%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, 23%, 24%, 26%, 28%, or 30% of the cells in the composition express NEUROD6; and/or (iii) the cells in the composition express from about 1 transcript per million (TPM) to about 210 TPM, about 1 TPM to about 205 TPM, about 1 TPM to about 25 TPM, or about 10 TPM to about 20 TPM of the NEUROD6 transcript; and/or (iv) the cells in the composition express at least 1 TPM, 5 TPM, 10 TPM, 12 TPM, 15 TPM, 19 TPM, 50 TPM, 100 TPM, 150 TPM, 175 TPM, 200 TPM, 203 TPM, or 210 TPM of the NEUROD6 transcript.

In one embodiment, (i) about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express SLA; and/or (ii) at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 15%, 16%, 18%, or 20% of the cells in the composition express SLA; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 60 TPM, about 0.1 TPM to about 50 TPM, about 1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 3 TPM to about 6 TPM of the SLA transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 10 TPM, 20 TPM, 30 TPM, 40 TPM, 50 TPM or 60 TPM of the SLA transcript.

In one embodiment, (i) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 30%, or about 20% to about 28% of the cells in the composition express NELL2; and/or (ii) at least about 10%, 12%, 15%, 17%, 20%, 21%, 24%, 25%, 27%, 30%, 32%, 35%, 40%, or 45% of the cells in the composition express NELL2; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 150 TPM, about 4 TPM to about 130 TPM, about 4 TPM to about 35 TPM, about 20 and/or (iv) the cells in the composition express at least 1 TPM, 5 TPM, 15 TPM, 20 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 120 TPM or 150 TPM of the NELL2 transcript.

In one embodiment, (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 12%, about 1% to about 11%, about 2% to about 20%, about 2% to about 15%, about 2% to about 12%, about 2% to about 11%, about 3% to about 20%, about 3% to about 15%, about 3% to about 12%, about 3% to about 11%, about 2% to about 6%, about 2% to about 5%, or about 3% to about 4% of the cells in the composition express SATB2; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 17%, or 20% of the cells in the composition express SATB2; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 30 TPM, about 0.5 TPM to about 20 TPM, about 1 TPM to about 5 TPM, or about 2 TPM to about 3 TPM of the SATB2 transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 25 TPM or 30 TPM of the SATB2 transcript.

In one embodiment, the plurality of heterogeneous light-sensitive rescue cells include progenitor cells that express one or more markers selected from the group consisting of VIM, MKI67, CLU and GLI3. In one embodiment, the plurality of heterogeneous light-sensitive rescue cells include a plurality of progenitor cells that cumulatively express each of the markers VIM, MKI67, CLU and GLI3.

In one embodiment, (i) about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 40% to about 75%, about 40% to about 70%, about 40% to about 69%, about 40% to about 60%, or about 42% to about 47% of the cells in the composition express VIM; and/or (ii) at least about 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 69%, 72%, 75%, or 80% of the cells in the composition express VIM; and/or (iii) the cells in the composition express about 250 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 865 TPM, about 200 TPM to about 1000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 TPM to about 2000 TPM, about 150 and/or (iv) the cells in the composition express at least 250 TPM, 260 TPM, 270 TPM, 300 TPM, 320 TPM, 350 TPM, 370 TPM, 400 TPM, 500 TPM, 600 TPM, 700 TPM, 800 TPM or 900 TPM of the VIM transcript.

In one embodiment, (i) about 5% to about 20%, about 5% to about 15%, about 5% to about 12%, about 6% to about 15%, about 6% to about 12%, about 7% to about 15%, about 7% to about 12%, or about 6% to about 8% of the cells in the composition express MKI67; and/or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the cells in the composition express MKI67; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 10 TPM to about 35 TPM, about 15 TPM to about 25 TPM, or about 18 TPM to about 22 TPM. and/or (iv) the cells in the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 33 TPM, 35 TPM, 37 TPM or 40 TPM of the MKI67 transcript.

In one embodiment, (i) about 10% to about 60%, about 15% to about 55%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 40%, about 20% to about 35%, or about 25% to about 32% of the cells in the composition express CLU; and/or (ii) at least about 10%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 55%, or 60% of the cells in the composition express CLU; and/or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 400 TPM, about 40 TPM to about 150 TPM, about 60 TPM to about 150 TPM, or about 60 TPM to about 105 TPM. and/or (iv) the cells in the composition express a CLU transcript of at least 30 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 365 TPM, 375 TPM, or 400 TPM.

In one embodiment, (i) about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 12% to about 50%, about 12% to about 35%, about 12% to about 29%, about 15% to about 29%, about 15% to about 29%, or about 15% to about 17% of the cells in the composition express GLI3; and/or (ii) at least about 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GLI3; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 60 TPM, about 10 TPM to about 45 TPM, or about 15% to about 17% of the cells in the composition. and/or (iv) the cells in the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM or 60 TPM of the GLI3 transcript.

In one embodiment, the plurality of heterogeneous photoreceptor rescue cells include astrocytes that express one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG, and FBXL7. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells include astrocytes that cumulatively express each of the markers GFAP, LUCAT1, MIR99AHG, and FBXL7.

In one embodiment, (i) about 1% to about 50%, about 1% to about 20%, about 1% to about 15%, about 1% to about 13%, about 1% to about 10%, about 1% to about 7%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express GFAP; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GFAP; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 150 TPM, about 0.1 TPM to about 125 TPM, about 1 TPM to about 20 TPM, or about 3 TPM to about 15 and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.5 TPM, 1 TPM, 5 TPM, 7 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 30 TPM, 40 TPM, 50 TPM, 80 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 140 TPM or 150 TPM of the GFAP transcript.

In a specific example, (i) about 5% to about 20%, about 5% to about 17%, about 5% to about 15%, about 5% to about 13%, about 7% to about 20%, about 7% to about 17%, about 7% to about 15%, about 7% to about 13%, about 5% to about 12%, or about 7% to about 10% of the cells in the composition express LUCAT1; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17%, or 20% of the cells in the composition express LUCAT1.

In one embodiment, (i) about 50% to about 100%, about 50% to about 90%, about 50% to about 88%, about 60% to about 100%, about 60% to about 90%, about 60% to about 88%, about 70% to about 90%, about 70% to about 88%, or about 75% to about 82% of the cells in the composition express MIR99AHG; and/or (ii) at least about 50%, 60%, 65%, 70%, 72%, 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 96%, 98%, or 100% of the cells in the composition express MIR99AHG; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM, or 40 TPM of the MIR99AHG transcript.

In one embodiment, (i) about 20% to about 70%, about 20% to about 60%, about 25% to about 70%, about 25% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 40%, about 32% to about 39%, or about 34% to about 39% of the cells in the composition express FBXL7; and/or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 60%, 65%, or 70% of the cells in the composition express FBXL7; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 30 TPM, about 7 TPM to about 10 TPM, about 15 TPM to about 20 TPM, about 10 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM, about 15 TPM to about 20 TPM and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM, or 40 TPM of the FBXL7 transcript.

In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises selective neurons that express one or more markers selected from the group consisting of MEIS2, PBX3, GRIA2 and CACNA1C. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises a plurality of selective neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2 and CACNA1C.

In one embodiment, (i) about 30% to about 80%, about 30% to about 90%, about 40% to about 90%, about 45% to about 85%, about 45% to about 80%, about 49% to about 79%, or about 50% to about 78% of the cells in the composition express MEIS2; and/or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 81%, or 82% of the cells in the composition express MEIS2; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 200 TPM, about 10 TPM to about 180 TPM, about 50 TPM to about 180 TPM, or about 60 and/or (iv) the cells in the composition express at least 5 TPM, 10 TPM, 15 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 135 TPM, 136 TPM, 138 TPM, 140 TPM, 145 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM or 200 TPM of the MEIS2 transcript.

In one embodiment, (i) about 30% to about 90%, about 35% to about 85%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, or about 49% to about 75% of the cells in the composition express PBX3; and/or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 80%, 85%, or 90% of the cells in the composition express PBX3; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 100 TPM, about 5 TPM to about 90 TPM, about 25 TPM to about 90 TPM, or about 29 TPM to about 88 TPM. and/or (iv) the cells in the composition express at least 5 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM or 100 TPM of the PBX3 transcript.

In one embodiment, (i) about 20% to about 60%, about 20% to about 50%, about 20% to about 50%, about 20% to about 48%, about 22% to about 55%, about 22% to about 50%, about 22% to about 47%, or about 23% to about 47% of the cells in the composition express GRIA2; and/or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 46%, 47%, 50%, 52%, 54%, 56%, 58%, or 60% of the cells in the composition express GRIA2; and/or (iii) the cells in the composition express about 2 transcripts per million (TPM) to about 40 TPM, about 2 TPM to about 30 TPM, about 4 TPM to about 35 TPM, about 5 TPM to about 50 TPM, about 6 TPM to about 60 TPM, about 8 TPM to about 80 TPM, about 10 TPM to about 15 TPM, about 15 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM, about 10 TPM to about 15 TPM and/or (iv) the cells in the composition express at least 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 17 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 26 TPM, 27 TPM, 28 TPM, 29 TPM, 30 TPM, 35 TPM or 40 TPM of the GRIA2 transcript.

In one embodiment, (i) about 20% to about 70%, about 30% to about 60%, about 35% to about 70%, about 35% to about 60%, about 33% to about 60%, about 35% to about 60%, or about 39% to about 60% of the cells in the composition express CACNA1C; and/or (ii) at least about 20%, 25%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, or 70% of the cells in the composition express CACNA1C; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 15 TPM, about 1 TPM to about 10 TPM, about 1 TPM to about 7 TPM, or about 3 and/or (iv) cells in the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 10 TPM, 12 TPM or 15 TPM of the CACNA1C transcript.

In a specific example, the plurality of heterogeneous photosensitive rescue cells include: (i) inhibitory neurons expressing one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4 and CALB2; (ii) excitatory neurons expressing one or more markers selected from the group consisting of NEUROD2, NEUROD6, SLA, NELL2 and SATB2; (iii) progenitor cells expressing one or more markers selected from the group consisting of VIM, MKI67, CLU and GLI3; (iv) astrocytes expressing one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG and FBXL7; and (v) selective neurons expressing one or more markers selected from the group consisting of MEIS2, PBX3, GRIA2 and CACNA1C.

In a specific example, the plurality of heterogeneous photorescive rescue cells include: (i) a plurality of inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4 and CALB2; (ii) a plurality of excitatory neurons that cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2 and SATB2; (iii) a plurality of progenitor cells that cumulatively express each of the markers VIM, MKI67, CLU and GLI3; (iv) a plurality of astrocytes that cumulatively express each of the markers GFAP, LUCAT1, MIR99AHG and FBXL7; and (v) a plurality of selective neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2 and CACNA1C.

In one embodiment, the composition comprises: (i) about 25% to about 55%, about 25% to about 50%, about 30% to about 55%, about 30% to about 50%, about 35% to about 55%, about 35% to about 50%, or about 38% to about 49% inhibitory neurons; and/or (ii) about 0% to about 15%, about 0% to about 12%, about 0% to about 10%, about 0% to about 8%, or about 0.5% to about 9% excitatory neurons; and/or (iii) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 17% to about 45%, about 17% to about 40%, about 17% to about 35% , or about 20% to about 35% of progenitor cells; and/or (iv) about 0% to about 6%, about 0% to about 5%, about 0% to about 4%, about 0% to about 3%, about 0% to about 2%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, or about 0.5% to about 1.5% of astrocytes; and/or (v) about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, or about 17% to about 37% of mixed neurons.

In one embodiment, the cells in the composition further express one or more eye movement area progenitor cell markers, rod/cone photoreceptor cell markers and/or neuron markers. In one embodiment, the eye movement area progenitor cell markers are selected from the group consisting of PAX6, LHX2, SIX3, NES and SOX2. In one embodiment, the plurality of heterogeneous cells cumulatively express at least 1, 2, 3 or 4 of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES or SOX2. In one embodiment, the plurality of heterogeneous cells cumulatively express at least each of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2. In one embodiment, the plurality of heterogeneous cells cumulatively express SOX2. In one embodiment, the composition is substantially free of cells expressing eye movement field progenitor cell markers RAX, SIX6 and/or TBX3.

In one embodiment, (i) about 25% to about 60%, about 25% to about 55%, about 25% to about 52%, about 25% to about 45%, about 30% to about 60%, about 30% to about 55%, about 30% to about 45%, or about 30% to about 42% of the cells in the composition express PAX6; and/or (ii) at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the cells in the composition express PAX6; and/or (iii) the cells in the composition express about 20 transcripts per million (TPM) to about 125 TPM, about 30 TPM to about 110 TPM, about 35 TPM to about 100 TPM, about 70 TPM to about 80 TPM, or about 73 TPM to about 78 TPM. and/or (iv) the cells in the composition express at least 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 78 TPM, 80 TPM, 82 TPM, 85 TPM, 87 TPM, 90 TPM, 92 TPM, 95 TPM, 97 TPM, 100 TPM, 105 TPM, 110 TPM, 115 TPM, 120 TPM or 125 TPM of the PAX6 transcript.

In one embodiment, (i) about 3% to about 35%, about 5% to about 35%, about 5% to about 35%, about 6% to about 35%, about 7% to about 35%, about 3% to about 30%, about 5% to about 30%, about 6% to about 30%, about 7% to about 30%, about 3% to about 25%, about 5% to about 25%, about 6% to about 25%, or about 7% to about 9% of the cells in the composition express LHX2; and/or (ii) at least about 3%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 32%, or 35% of the cells in the composition express LHX2; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 36 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 TPM to about 5 TPM, about 5 and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 36 TPM, 38 TPM or 40 TPM of the LHX2 transcript.

In one embodiment, (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or 2% to about 10% of the cells in the composition express SIX3; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, or 25% of the cells in the composition express SIX3; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 50 TPM, about 2 TPM to about 30 TPM, about 1 TPM to about 40 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about 1 TPM to about 50 TPM, about and/or (iv) the cells in the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, or 50 TPM of a SIX3 transcript.

In one embodiment, (i) about 15% to about 40%, about 15% to about 35%, about 15% to about 34%, about 15% to about 33%, about 15% to about 32%, about 15% to about 31%, about 18% to about 40%, about 18% to about 35%, about 18% to about 34%, about 18% to about 33%, about 18% to about 32%, or about 18% to about 31% of the cells in the composition express the NES; and/or (ii) at least about 15%, 17%, 20%, 25%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, or 40% of the cells in the composition express the NES; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 35 TPM, about 5 TPM to about 28 TPM, about 7 TPM to about 11 TPM, about 7 TPM to about 13 TPM, about 7 TPM to about 15 TPM, about 7 TPM to about 16 TPM, about 7 TPM to about 17 TPM, about 7 TPM to about 18 TPM, about 7 TPM to about 19 TPM, about 8 TPM to about 20 TPM, about 8 TPM to about 21 TPM, about 8 TPM to about 22 TPM, about 8 TPM to about 23 TPM, about 8 TPM to about 24 TPM, about 8 TPM to about 25 TPM, about 8 TPM to about 26 TPM, about 8 TPM to about 27 TPM, about 8 TPM to about 28 TPM, about 8 TPM to about 29 TPM, about 8 TPM to about 29 TPM, about 8 TPM and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 22 TPM, 24 TPM, 25 TPM, 26 TPM, 27 TPM, 30 TPM, or 35 TPM of the NES transcript.

In one embodiment, (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 75% of the cells in the composition express SOX2; and/or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or 90% of the cells in the composition express SOX2; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 250 TPM. and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, 200 TPM, 210 TPM, 220 TPM, 230 TPM, 240 TPM, or 250 TPM of the CACNA1C transcript.

In one embodiment, the rod/cone photoreceptor cell marker is selected from the group consisting of ASCL1, RORB, NR2E3 and NRL. In one embodiment, a plurality of heterogeneous cells cumulatively express each of the rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL. In one embodiment, the composition is substantially free of cells expressing the rod/cone photoreceptor cell markers CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1 and GNAT2.

In one embodiment, (i) about 10% to about 60%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 22% to about 45%, about 22% to about 43%, about 25% to about 420%, or about 28% to about 30% of the cells in the composition express ASCL1; and/or (ii) at least about 10%, 15%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 41%, 42%, 45%, 50%, 55%, or 60% of the cells in the composition express ASCL1; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 150 TPM, about 60 TPM to about 140 TPM, about 65 TPM to about 130 TPM, or about 95 TPM to about 130 TPM. and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 125 TPM, 130 TPM, 140 TPM or 150 TPM of the ASCL1 transcript.

In one embodiment, (i) about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 10% to about 25%, or about 11% to about 22% of the cells in the composition express RORB; and/or (ii) at least about 5%, 7%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express RORB; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 20 TPM, about 0.1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 1 and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 18 TPM or 20 TPM of the RORB transcript.

In one embodiment, (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or about 2% to about 5% of the cells in the composition express NR2E3; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of the cells in the composition express NR2E3; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 2 TPM, 5 TPM, 7 TPM, 9 TPM or 10 TPM of the NR2E3 transcript.

In one embodiment, (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, or about 2% to about 8% of the cells in the composition express NRL; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of the cells in the composition express NRL; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 7 TPM, or about 0.1 TPM to about 2 TPM. and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of the NRL transcript.

In one embodiment, the neuronal marker is selected from the group consisting of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. In one embodiment, a plurality of heteroplasmic cells cumulatively express each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

In one embodiment, (i) between about 50% to about 100%, between about 60% to about 100%, between about 60% to about 95%, between about 70% to about 100%, between about 70% to about 95%, between about 80% to about 100%, between about 80% to about 95%, or between about 80% to about 90% of the cells in the composition express NFIB; and/or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 100% of the cells in the composition express NFIB; and/or (iii) the cells in the composition express between about 150 transcripts per million (TPM) and about 650 TPM. or (iv) the cells in the composition express at least 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 550 TPM, 600 TPM, 625 TPM, or 650 TPM of the NFIB transcript.

In one embodiment, (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, or about 2% to about 6% of the cells in the composition express OTX2; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of the cells in the composition express OTX2; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 50 TPM, about 8 TPM to about 40 TPM, about 8 TPM to about 25 TPM, or about 12 TPM to about 15 TPM. and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 26 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM or 50 TPM of the OTX2 transcript.

In one embodiment, (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 78%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 78%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 78% of the cells in the composition express ELAVL3; and/or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or 90% of the cells in the composition express ELAVL3; and/or (iii) the cells in the composition express from about 10 transcripts per million (TPM) to about 120 TPM. or (iv) the cells in the composition express at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, or 120 TPM of the ELAVL3 transcript.

In one embodiment, (i) about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 78%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 78%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, or about 50% to about 65% of the cells in the composition express ELAVL4; and/or (ii) about 30% to about 90% of the cells in the composition express ELAVL4. about 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 75%, 80%, 85% or 90% of the cells express ELAVL4; and/or (iii) the cells in the composition express from about 10 transcripts per million (TPM) to about 120 or (iv) the cells in the composition express at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 82 TPM, 84 TPM, 86 TPM, 90 TPM, 100 TPM, 110 TPM, or 120 TPM of the ELAVL4 transcript.

In one embodiment, (i) about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 73%, about 51% to about 73%, or about 52% to about 66% of the cells in the composition express SLC1A2; and/or (ii) at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 75%, 80%, 85% or 90% of the cells express SLC1A2; and/or (iii) the cells in the composition express from about 10 transcripts per million (TPM) to about 120 or (iv) the cells in the composition express at least 10 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, or 120 TPM of the SLC1A2 transcript.

In one embodiment, (i) about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 11% to about 19%, or about 10% to about 12% of the cells in the composition express SLC1A3; and/or (ii) at least about 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 25%, 30%, 35%, or 40% of the cells in the composition express SLC1A3; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 200 TPM, about 70 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about 190 TPM, about 120 TPM to about or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 79 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 175 TPM, 180 TPM, or 200 TPM of the SLC1A3 transcript.

In one embodiment, (i) about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 3% to about 4% of the cells in the composition express HCN1; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the cells in the composition express HCN1; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 5 TPM, about 0.1 TPM to about 1 TPM, or about 0.2 TPM to about 0.6 TPM of HCN1 transcripts; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, or 10% of the cells in the composition. TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1.0 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM, or 10 TPM of HCN1 transcript.

In one embodiment, (i) about 5% to about 30%, about 5% to about 25%, about 1% to about 30%, about 1% to about 25%, about 5% to about 25%, about 10% to about 25%, or about 10% to about 15% of the cells in the composition express HES5; and/or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or 30% of the cells in the composition express HES5; and/or (iii) the cells in the composition express about 10 transcripts per million (TPM) to about 60 TPM, about 5 TPM to about 50 TPM, about 12 TPM to about 46 TPM, or about 15 TPM to about 39 TPM. and/or (iv) the cells in the composition express at least 10 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM of the HES5 transcript.

In a specific example, the plurality of heterogeneous cells cumulatively express: (i) one or more of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2; (ii) one or more of the rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL; and (iii) one or more of the neuron markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5.

In a specific example, the plurality of heterogeneous cells cumulatively express: (i) each of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2; (ii) each of the rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL; and (iii) each of the neuron markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5.

In one embodiment, the composition is substantially free of at least one cell type selected from the group consisting of: high potential stem cells, retinal ganglion cells, photoreceptor cells, and axonal neurons. In one embodiment, the composition is substantially free of high potential stem cells, retinal ganglion cells, photoreceptor cells, and axonal neurons.

In one embodiment, the composition is substantially free of retinal progenitor cells expressing VSX2 and/or POU5F1. In one embodiment, the composition has less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of cells expressing SSEA4, as determined by flow cytometry, or wherein the composition is free of cells expressing SSEA4, as determined by flow cytometry.

In one embodiment, the cells in the composition have phagocytic activity, optionally the ability to engulf isolated photoreceptor outer segments, dye conjugates, or both.

In one embodiment, the cells in the composition secrete one or more neuroprotective factors. In one embodiment, the neuroprotective factor is selected from the group consisting of CNTF, MIF, S100B, GFAP, TAU, NCAM1 and TNC.

In a specific example, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells in the composition are viable. In a specific example, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable. In a specific example, at least 50% of the cells in the composition are viable. In one embodiment, at least 55% of the cells in the composition are alive. In one embodiment, at least 60% of the cells in the composition are alive. In one embodiment, at least 65% of the cells in the composition are alive. In one embodiment, at least 68% of the cells in the composition are alive.

In one embodiment, a composition is produced according to a method comprising: 1) culturing enriched potential stem cells in rescue induction medium (RIM) and Noggin; 2) culturing the cells obtained from step 1 in neural differentiation medium (NDM) and Noggin; 3) expanding the cells obtained from step 2 in NDM lacking Noggin, comprising a) culturing the cells in NDM without Noggin under low-adherence or non-adherence conditions, and b) culturing the cells in NDM without Noggin under adhesion conditions.

In a specific example, the high-potential stem cells are embryonic stem cells (ESCs) or induced high-potential stem cells (iPSCs).

In a specific example, step 3 is performed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In a specific example, the cells in the composition are collected after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In a specific example, the cells in the composition are collected after the fifth repetition of step 3.

In a specific example, the collected cells in the composition are cryopreserved. In a specific example, the composition is cryopreserved between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3. In a specific example, the composition is cryopreserved after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In a specific example, step 3 is repeated five times and the composition is cryopreserved after the fifth repetition of step 3.

In a specific example, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells are viable after cryopreservation and thawing. In a specific example, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells are viable after cryopreservation and thawing. In one embodiment, at least about 50% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, at least about 55% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, at least about 60% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, at least about 65% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, at least about 68% of the cells in the composition are alive after cryopreservation and thawing.

In one embodiment, the composition comprises spheroids. In one embodiment, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are spheroids.

In one embodiment, during steps 1, 2 and/or 3, the cells in the composition are cultured in a cell culture container selected from the group consisting of a culture dish, a culture bottle and a culture chamber. In one embodiment, the cell culture container has a size of about 50 cm ² to about 800 cm ² , about 60 cm ² to about 800 cm ² , about 100 cm ² to about 800 cm ² , about 150 cm ² to about 800 cm ² , about 175 cm ² to about 800 cm ² , about 200 cm ² to about 800 cm ² , about 250 cm ² to about 800 cm ² , about 300 cm ² to about 800 cm 2, about 400 cm 2 to about 800 cm ² , about 500 cm ² to about 800 cm ² , about 600 cm ² to about 800 cm ² , about 700 cm ² to about 800 cm ² , about 30 cm ² to about 100 cm ² , about 50 cm ² to about 100 cm ² , or about 150 cm ² to about 800 cm ^2. , about 100 cm ² to about 300 cm ² , about 150 cm ² to about 250 cm ² , or about 150 cm ² to about 200 cm ² of cell growth area. In a specific example, the cell culture container has a cell growth area of at least about 60 cm ² , about 175 cm ² , or about 636 cm ² .

In a specific example, the culture chamber is a stackable rectangular chamber. In a specific example, step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5, or 1 to 2 culture chambers. In a specific example, step 3 has at least 1, 2, 5, 10 or 40 culture chambers.

In one embodiment, the cell culture container is coated with a low-attachment or non-attachment cell culture medium in step 3a and/or coated with an adherent cell culture medium in step 3b. In one embodiment, the cells are dissociated from the culture dish using an enzyme into a cell suspension. In one embodiment, the enzymatic dissociation uses an enzyme selected from the group consisting of thermolysin, liberase, accutase, and combinations thereof. In one embodiment, the enzyme used to dissociate the cells is accutase. In one embodiment, dissociating the cells from the culture dish does not involve manual scraping.

Accordingly, in another aspect, the present invention provides a pharmaceutical preparation comprising the photosensitive rescue cell composition of various embodiments of the above aspects or any other aspects of the present invention described herein and a pharmaceutically acceptable formulation. In a specific example, the pharmaceutically acceptable formulation is suitable for ocular delivery.

Accordingly, in another aspect, the present invention provides a formulation comprising: a) a composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein; and b) about 4-10% (v/v) cryoprotectant, about 2-3% (w/v) albumin, about 0-1.5% (w/v) glucose and a buffer.

In one embodiment, the low temperature protective agent is selected from DMSO, glycerol and ethylene glycol. In one embodiment, the low temperature protective agent is DMSO. In one embodiment, the formulation comprises about 4-6% (v/v) low temperature protective agent. In one embodiment, the formulation comprises about 0.08-0.10% (w/v) glucose. In one embodiment, the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.09% (w/v) glucose and buffer. In one embodiment, the formulation comprises about 0.6% (w/v) glucose. In one embodiment, the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose and buffer.

In a specific example, the albumin is human albumin. In a specific example, the albumin is recombinant human albumin.

In one embodiment, the buffer is saline buffer. In one embodiment, the buffer is phosphate buffered saline (PBS). In one embodiment, the saline buffer contains Ca2+ and Mg2+. In one embodiment, the saline buffer does not contain Ca2+ and Mg2+. In one embodiment, the formulation is stored at low temperature.

Accordingly, in another aspect, the present invention provides a formulation comprising: a) a composition of the various embodiments of the above or any other aspects of the present invention described herein, or a pharmaceutical preparation of the various embodiments of the above or any other aspects of the present invention described herein; and b) a solution comprising (1) a buffer that maintains the solution at physiological pH; and (2) at least 2 mM or at least 0.05% (w/v) glucose; and (3) an osmotic active agent that maintains the solution at a physiological osmotic pressure.

In one embodiment, the solution comprises at least 5 mM or at least 0.1% (w/v) glucose; or at least 7.5 mM or at least 0.14% (w/v) glucose; or at least 10 mM or at least 0.2% (w/v) glucose; or at least 15 mM or at least 0.25% (w/v) glucose; or at least 20 mM or at least 0.4% (w/v) glucose; or at least 25 mM or at least 0.5% (w/v) glucose.

In one embodiment, the solution further comprises (4) a divalent cation source, optionally wherein the divalent cation source comprises a calcium and/or magnesium source, and/or (5) an acetate buffer and/or a citrate buffer.

Accordingly, in another aspect, the present invention provides a formulation comprising: a) a composition of the various embodiments of the above or any other aspects of the present invention described herein, or a pharmaceutical preparation of the various embodiments of the above or any other aspects of the present invention described herein; and b) a solution comprising (1) a buffer that maintains the solution at physiological pH, wherein the buffer is not a bicarbonate buffer; and (2) glucose; and (3) an osmotic active agent that maintains the solution at a physiological osmotic pressure; and (4) a divalent cation source, optionally wherein the divalent cation source comprises a calcium source and/or a magnesium source, and/or wherein the buffer comprises an acetate buffer and/or a citrate buffer.

In one embodiment, the calcium source comprises a pharmaceutically acceptable calcium salt, and/or the magnesium source comprises a pharmaceutically acceptable magnesium salt. In one embodiment, the pharmaceutically acceptable calcium and/or the pharmaceutically acceptable magnesium salt are selected from the group of calcium and/or magnesium salts formed with an acid, and the acid is selected from the group comprising the following: acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid. In one embodiment, the calcium source comprises calcium chloride, optionally wherein the calcium source comprises calcium chloride dihydrate. In one embodiment, the magnesium source comprises magnesium chloride, optionally wherein the magnesium source comprises magnesium chloride hexahydrate. In one embodiment, the citrate buffer is provided in the form of sodium citrate. In one embodiment, the glucose is D-glucose (dextrose).

In one embodiment, the osmotic active agent is a salt, optionally wherein the osmotic active agent is a sodium salt, and optionally wherein the osmotic active agent is sodium chloride. In one embodiment, the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride and glucose.

In one embodiment, the pH of the solution is 6.8-7.8, or 7.4-7.5, or about 7.5.

In one embodiment, the solution is isotonic or hypertonic.

In a specific example, the solution exhibits an osmotic pressure of about 270-345 mOsm/1 or about 315 mOsm/1.

In a specific example, the concentration of the calcium source is (a) 0.25-0.75 mM, or 0.4-0.65 mM, or 0.5-0.6 mM, or about 0.6 mM; or (b) 0.5-0.9 mM, or 0.6-0.8 mM, or about 0.7 mM.

In one embodiment, the magnesium source concentration is 0.05-5 mM, or 0.1-0.3 mM, or about 0.3 mM. In one embodiment, the glucose concentration is 5-50 mM, or 10-25 mM, or 10-20 mM, or about 16 mM. In one embodiment, the concentration of the osmotic active agent is about 100-200 mM, or about 125-175 mM, or about 150 mM. In one embodiment, the citrate or acetate concentration is 0.1-5 mM, or 0.5-2 mM, or about 1 mM.

In one embodiment, the solution further comprises a potassium salt, optionally wherein the potassium salt is potassium chloride, and optionally wherein the KCl concentration is 0.2-5 mM, or 1-2.5 mM, or about 2 mM.

In one embodiment, the solution comprises (a) about 0.7 mM CaCl ₂ (calcium chloride), about 0.3 mM MgCl ₂ (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose and about 145 mM NaCl, or (b) about 0.5-0.9 mM CaCl ₂ (calcium chloride), about 0.2-0.4 mM MgCl ₂ (magnesium chloride), about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose and about 116-174 mM NaCl, or (c) about 0.85% NaCl, about 0.01% CaCl ₂ dihydrate (calcium chloride dihydrate), about 0.006% MgCl ₂ hexahydrate. (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate and about 0.29% dextrose, or (d) about 0.68-1.02% NaCl, about 0.008-0.012% CaCl ₂ dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl ₂ hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate and about 0.23-0.35% dextrose.

In one embodiment, the solution further comprises (a) about 2 mM KCl, and/or (b) a viscoelastic polymer, optionally wherein the polymer is hyaluronic acid or a salt or solvent thereof, and optionally wherein the polymer is sodium hyaluronate.

In one embodiment, the polymer is present at a concentration effective to reduce exposure of cells in solution to shear stress, optionally wherein the concentration of the polymer is 0.005-5% w/v or about 0.05% w/v.

In one embodiment, the solution comprises (a) about 0.7 mM CaCl ₂ (calcium chloride), about 0.3 mM MgCl ₂ (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl and about 0.05% hyaluronic acid; or (b) about 0.5-0.8 mM CaCl ₂ (calcium chloride), about 0.2-0.4 mM MgCl ₂ (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl and about 0.04-0.06% hyaluronic acid.

In one embodiment, the solution does not contain (a) a carbonate buffer, and/or (b) glutathione or glutathione disulfide (GSSG), and/or (c) a zwitterionic organic buffer.

In a specific example, the solution can be stored (a) at 25°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least one week, at least two weeks, at least three weeks, or at least one month without measurable precipitation of solutes and/or without measurable loss of the ability of the solution to support the viability and viability of cells stored in the solution; and/or (b) at 2-8°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least one week, at least two weeks, at least three weeks, or at least one month without measurable precipitation of solutes and/or without measurable loss of the ability of the solution to support the viability and viability of cells stored in the solution.

In one embodiment, the solution is (a) suitable for administration to a subject, suitable for administration to the eye of a subject, and/or suitable for transplanting cells into the eye of a subject, and/or (b) substantially free of pyrogens, and/or (c) sterile, and/or (d) suitable for irrigation, cell recovery, cell storage, cell transport, and/or administration to a subject.

Accordingly, in another aspect, the present invention provides a method for making a composition of various embodiments of the above aspects or any other aspects of the present invention described herein, comprising 1) culturing high-potential stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells obtained from step 1 in neural differentiation medium (NDM) and noggin; 3) expanding the cells obtained from step 2 in NDM without noggin, comprising a) culturing the cells in NDM without noggin under low attachment or non-attachment conditions, and b) culturing the cells in NDM without noggin under non-attachment conditions, thereby differentiating the high-potential stem cells into photosensitive rescue cells.

Accordingly, in another aspect, the present invention provides a method for producing a photosensitive rescue cell composition comprising a plurality of heterogeneous cells, the method comprising: 1) culturing high-potential stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells in neural differentiation medium (NDM) and noggin; 3) expanding the cells in NDM without noggin, comprising a) culturing the cells in NDM without noggin under low attachment or non-attachment conditions, and b) culturing the cells in NDM without noggin under attachment conditions, thereby differentiating the high-potential stem cells into a plurality of cells in the photosensitive rescue cell composition.

In a specific example, the high-potential stem cells are embryonic stem cells (ESCs) or induced high-potential stem cells (iPSCs).

In a specific example, step 3 is performed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In a specific example, the cells in the composition are collected after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In a specific example, the composition is collected after the fifth repetition of step 3.

In one embodiment, the collected cells in the composition are cryopreserved.

In a specific example, the composition is cryopreserved between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3. In a specific example, the composition is cryopreserved after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In a specific example, step 3 is repeated five times and the composition is cryopreserved after the fifth repetition of step 3.

In a specific example, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells in the composition are viable after cryopreservation and thawing. In a specific example, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, less than about 50% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, less than about 55% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, less than about 60% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, less than about 65% of the cells in the composition are alive after cryopreservation and thawing. In one embodiment, less than about 68% of the cells in the composition are alive after cryopreservation and thawing.

In one embodiment, the composition comprises spheroids. In one embodiment, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are spheroids.

In one embodiment, during steps 1, 2 and/or 3, the cells in the composition are cultured in a cell culture container selected from the group consisting of a culture dish, a culture bottle and a culture chamber.

In one embodiment, the cell culture container has a size of about 50 cm ² to about 800 cm ² , about 60 cm ² to about 800 cm ² , about 100 cm ² to about 800 cm ² , about 150 cm ² to about 800 cm ² , about 175 cm ² to about 800 cm ² , about 200 cm ² to about 800 cm ² , about 250 cm ² to about 800 cm ² , about 300 cm ² to about 800 cm 2, about 400 cm 2 to about 800 cm ² , about 500 cm ² to about 800 cm ² , about 600 cm ² to about 800 cm ² , about 700 cm ² to about 800 cm ² , about 30 cm ² to about 100 cm ² , about 50 cm ² to about 100 cm ² , or about 150 cm ² to about 800 cm ^2. , about 100 cm ² to about 300 cm ² , about 150 cm ² to about 250 cm ² , or about 150 cm ² to about 200 cm ² of cell growth area. In a specific example, the cell culture container has a cell growth area of at least about 60 cm ² , about 175 cm ² , or about 636 cm ² .

In one embodiment, the culture chamber is a stackable rectangular chamber.

In one embodiment, step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5 or 1 to 2 culture chambers. In a specific example, step 3 has at least 1, 2, 5, 10 or 40 culture chambers.

In one embodiment, the cell culture container is coated with a low-attachment or non-attachment cell culture medium in step 3a and/or coated with an adherent cell culture medium in step 3b.

In one embodiment, the cells are dissociated from the culture dish using an enzyme into a cell suspension. In one embodiment, the enzyme used to dissociate the cells is thermolysin, liberase and/or acurase. In one embodiment, the enzyme used to dissociate the cells is acurase. In one embodiment, dissociating the cells from the culture dish does not involve manual scraping.

Accordingly, in another aspect, the present invention provides a photosensitive rescue cell composition produced by the methods of the various embodiments of the above aspects or any other aspects of the present invention described herein.

Accordingly, in another aspect, the present invention provides a method for treating an eye disease or disorder in an individual, comprising administering to the individual a photorescrutin cell composition of the various embodiments of the above or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above or any other aspects of the present invention described herein.

Accordingly, in another aspect, the present invention provides a method for increasing the secretion of neuroprotective factors in the eye of an individual suffering from an ocular disease or disorder, comprising administering to the individual a photosensitive rescue cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the method increases the secretion of the neuroprotective factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the secretion of the neuroprotective factor before administration.

Accordingly, in another aspect, the present invention provides a method for improving visual acuity in an individual suffering from a retinal disease or disorder, comprising administering to the individual a photoreceptor rescue cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the method increases visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to visual acuity prior to administration.

In one specific example, visual acuity is measured based on optomotor response (OMR) and/or electroretinogram (ERG).

In a specific example, the method increases the spatial frequency threshold measured by OMR by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the spatial frequency threshold before administration.

In a specific example, the method increases the amplitude of the glimmer b-wave measured according to the ERG by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the glimmer b-wave amplitude before administration.

Accordingly, in another aspect, the present invention provides a method for preventing or slowing photoreceptor cell loss in an individual suffering from a retinal disease or disorder, comprising administering to the individual a photoreceptor rescue cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In one embodiment, prevention of photoreceptor cell loss is measured by expression of CNFT. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to CNFT expression in the eye prior to administration.

Accordingly, in another aspect, the present invention provides a method for enhancing phagocytic activity in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescrutin cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the method increases the phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the phagocytic activity before administration.

Accordingly, in another aspect, the present invention provides a method of inhibiting microglial cell activation in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photosensitive rescue cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In one embodiment, inhibition of microglial cell activation is measured by the expression of CNFT and/or MIF. In one embodiment, the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of CNFT before administration. In one embodiment, the method increases the expression of MIF by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of MIF before administration.

Accordingly, in another aspect, the present invention provides a method of reducing oxidative stress in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescive cell composition of the various embodiments of the above or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above or any other aspects of the present invention described herein.

In one embodiment, the reduction of oxidative stress is measured by the expression of CNFT. In one embodiment, the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of CNFT before administration.

Accordingly, in another aspect, the present invention provides a method for enhancing the expression of anti-apoptotic factors in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescrutin cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of the anti-apoptotic factor before administration.

In one embodiment, the anti-apoptotic factor is S100B.

Accordingly, in another aspect, the present invention provides a method for preventing degeneration of the outer nuclear layer (ONL) in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescive cell composition of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical preparation of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, secondary map atrophy of macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber's congenital amaurosis, or Stargardt's disease. In a specific example, the eye disease is macular degeneration or retinitis pigmentosa. In a specific example, the disease is a retinal degenerative disease.

In one specific instance, the disease is associated with the loss of photoreceptor cells.

In one embodiment, the disease is associated with loss of photoreceptor cells in the outer nuclear layer of the retina. In one embodiment, the photoreceptor rescue cell composition, formulation or pharmaceutical preparation is administered to the subretinal space, suprachoroidal space, to the eye via a reservoir, or to other parts of the individual's body via systemic delivery.

In one embodiment, the cell preparation or formulation is administered by injection or implantation.

In one embodiment, the injection is administered intraocularly.

In one embodiment, intraocular administration comprises injecting an aqueous solution, an isotonic solution and/or a saline solution into the subretinal space to form an anterior blister; and removing the aqueous solution, and then administering the photoreceptor rescue cell composition in the form of the aqueous solution into the same subretinal space.

In one embodiment, the cell preparation or formulation is administered within at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years of symptom onset.

In a specific example, (i) the individual has intermediate or nearly advanced disease; (ii) the individual has a best corrected visual acuity (BCVA) in the range of 20/50 to 20/200; (iii) the individual has a BCVA worse than 20/200 but maintains light perception; or (iv) the individual is diagnosed with retinitis pigmentosa by genotyping.

In one embodiment, the method further comprises administering to the individual one or more anti-inflammatory agents.

In one embodiment, one or more anti-inflammatory agents are administered concurrently. In one embodiment, one or more anti-inflammatory agents are administered separately.

In one embodiment, one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before the one or more anti-inflammatory agents. In one embodiment, one or more anti-inflammatory agents are administered before and after the cell preparation or formulation is administered. In one embodiment, the cell preparation or formulation is administered without administering one or more anti-inflammatory agents. In one embodiment, the one or more anti-inflammatory agents include dexamethasone and/or cyclosporine.

Accordingly, in another aspect, the present invention provides a population of extracellular vesicles (EVs) secreted by the photosensitive rescue cell composition of various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the EVs secreted by the photoresc1 cells express at least one of the proteins selected from the group consisting of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA, DLX5, TUBB3, SCGN, ERBB4, CALB2, NEUROD2, NEUROD6, SLA, NELL2, SATB2, VIM, MKI67, CLU, GLI3, GFAP, LUCAT1, MIR99 AHG, FBXL7, MEIS2, PBX3, GRIA2, CACNA1C, PAX6, LHX2, SIX3, NES, SOX2, ASCL1, RORB, NR2E3, NRL, TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, HES5, AGT, ACBLN2, CDH7, DNAH11, EGR1, FAM216B, FOS, KCNC 2. LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, Nestin, TRbeta2, MASH1, RORbeta, MAP2, ELAVL3, NFIA, DCX, LHX2, SLC1A2, ELAVL4, PAX6, E MX2, ASCL1, DLL1, NFIB, ENOX1, TUBB3, MAP2, DCLK1/2, DCX, KALRN, LINC00461, C1orf61, NCAM1, SETBP1, PAK3, AKAP6, RTN1, CRMP1, FOXG1, TRIM2, BACH2, Recoverin, Opsin, Rhodopsin, Rod and Cone cGMP phosphodiesterase.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising the EV population of various embodiments of the above aspects or any other aspects of the present invention described herein and a pharmaceutically acceptable carrier.

Accordingly, in another aspect, the present invention provides a formulation comprising: a) an EV population of various embodiments of the above aspects or any other aspects of the present invention described herein, or a pharmaceutical composition of various embodiments of the above aspects or any other aspects of the present invention described herein; and b) about 4-10% (v/v) cryoprotectant, about 2-3% (w/v) albumin, about 0-1.5% (w/v) glucose and a buffer.

In one embodiment, the low temperature protective agent is selected from DMSO, glycerol and ethylene glycol. In one embodiment, the low temperature protective agent is DMSO. In one embodiment, the formulation comprises about 4-6% (v/v) low temperature protective agent. In one embodiment, the formulation comprises about 0.08-0.10% (w/v) glucose. In one embodiment, the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.09% (w/v) glucose and buffer. In one embodiment, the formulation comprises about 0.6% (w/v) glucose. In one embodiment, the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose and buffer.

In a specific example, the albumin is human albumin. In a specific example, the albumin is recombinant human albumin.

In a specific example, the buffer is saline buffer. In a specific example, the buffer is phosphate buffered saline (PBS). In a specific example, the saline buffer contains Ca2+ and Mg2+. In a specific example, the saline buffer does not contain Ca2+ and Mg2+.

In one embodiment, the formulation is stored at low temperatures.

Accordingly, in another aspect, the present invention provides a method for treating an eye disease in an individual, comprising: administering to the individual an EV group of the various embodiments of the present invention described herein or any other aspect, a pharmaceutical composition comprising the EV group of the various embodiments of the present invention described herein or any other aspect, or a formulation comprising the EV group of the various embodiments of the present invention described herein or any other aspect.

Accordingly, in another aspect, the present invention provides a method for increasing the secretion of neuroprotective factors in the eye of an individual, comprising: administering to the individual an EV population of the various embodiments of the present invention described above or any other aspect, a pharmaceutical composition comprising the EV population of the various embodiments of the present invention described above or any other aspect, or a formulation comprising the EV population of the various embodiments of the present invention described above or any other aspect.

In a specific example, the method increases the secretion of the neuroprotective factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the secretion of the neuroprotective factor before administration.

Accordingly, in another aspect, the present invention provides a method for improving visual acuity in an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical composition comprising the EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation comprising the EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In a specific example, the method increases visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to visual acuity before administration. In a specific example, visual acuity is measured according to optomotor response (OMR) and/or electroretinogram (ERG). In a specific example, the method increases the spatial frequency threshold measured according to OMR by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the spatial frequency threshold before administration. In a specific example, the method increases the amplitude of the glimmer b-wave measured according to the ERG by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the glimmer b-wave amplitude before administration.

Accordingly, in another aspect, the present invention provides a method for preventing or slowing down photoreceptor cell loss in an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical composition comprising the EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation comprising the EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In one embodiment, the prevention or reduction of photoreceptor cell loss is measured by the expression of CNFT. In one embodiment, the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the expression of CNFT in the eye before administration.

Accordingly, in another aspect, the present invention provides a method for enhancing phagocytic activity in the eye of an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of the various embodiments of the present invention described herein or any other aspect, a pharmaceutical composition comprising the EV population of the various embodiments of the present invention described herein or any other aspect, or a formulation comprising the EV population of the various embodiments of the present invention described herein or any other aspect.

In a specific example, the method increases the phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the phagocytic activity before administration.

Accordingly, in another aspect, the present invention provides a method for inhibiting microglial cell activation in the eye of an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein, a pharmaceutical composition comprising the EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein, or a formulation comprising the EV population of the various embodiments of the above aspects or any other aspects of the present invention described herein.

In one embodiment, inhibition of microglial cell activation is measured by the expression of CNFT and/or MIF. In one embodiment, the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of CNFT before administration. In one embodiment, the method increases the expression of MIF by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of MIF before administration.

Accordingly, in another aspect, the present invention provides a method for reducing oxidative stress in the eye of an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of the various embodiments of the present invention described above or any other aspect, a pharmaceutical composition comprising the EV population of the various embodiments of the present invention described above or any other aspect, or a formulation comprising the EV population of the various embodiments of the present invention described above or any other aspect.

In one embodiment, the reduction of oxidative stress is measured by the expression of CNFT. In one embodiment, the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of CNFT before administration.

Accordingly, in another aspect, the present invention provides a method for enhancing the expression of anti-apoptotic factors in the eye of an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of the various embodiments of the present invention as described herein or any other aspect, a pharmaceutical composition comprising the EV population of the various embodiments of the present invention as described herein or any other aspect, or a formulation comprising the EV population of the various embodiments of the present invention as described herein or any other aspect.

In a specific example, the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of the anti-apoptotic factor before administration. In a specific example, the anti-apoptotic factor is S100B.

Accordingly, in another aspect, the present invention provides a method for preventing degeneration of the outer nuclear layer (ONL) in the eye of an individual suffering from a retinal disease or disorder, comprising: administering to the individual an EV population of various embodiments of the present invention as described herein or any other aspect, a pharmaceutical composition comprising the EV population of various embodiments of the present invention as described herein or any other aspect, or a formulation comprising the EV population of various embodiments of the present invention as described herein or any other aspect.

In a specific example, the disease or condition is rod or cone dystrophy, retinal degeneration, pigmentary retinitis, diabetic retinopathy, macular degeneration, secondary map-like atrophy of macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber's congenital amaurosis, or Stargardt's disease. In a specific example, the eye disease is macular degeneration or pigmentary retinitis. In a specific example, the disease is a retinal degenerative disease. In a specific example, the disease is associated with loss of photoreceptor cells. In a specific example, the disease is associated with loss of photoreceptor cells in the outer nuclear layer of the retina.

In one specific example, an EV population, a pharmaceutical composition comprising an EV population, or a formulation comprising an EV population is administered into the subretinal space or supracortal space of an individual.

In one embodiment, the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population is administered by injection or implantation.

In one embodiment, the injection is administered intraocularly.

In one embodiment, intraocular administration comprises injecting an aqueous solution, an isotonic solution and/or a saline solution into the subretinal space to form an anterior blister; and removing the aqueous solution, and then administering the photoreceptor rescue cell composition in the form of the aqueous solution into the same subretinal space.

In one specific example, the EV population, a pharmaceutical composition comprising the EV population, or a formulation comprising the EV population is administered within at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years of symptom onset.

In one embodiment, the method further comprises administering to the individual one or more anti-inflammatory agents.

In one embodiment, one or more anti-inflammatory agents are administered concurrently with an EV population, a pharmaceutical composition comprising an EV population, or a formulation comprising an EV population.

In one embodiment, one or more anti-inflammatory agents are administered separately from the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population.

In one specific example, 1) one or more anti-inflammatory agents are administered before an EV population, a pharmaceutical composition comprising an EV population, or a formulation comprising an EV population; or 2) an EV population, a pharmaceutical composition comprising an EV population, or a formulation comprising an EV population is administered before one or more anti-inflammatory agents.

In one specific example, one or more anti-inflammatory agents are administered before and after administration of an EV population, a pharmaceutical composition comprising an EV population, or a formulation comprising an EV population.

In one embodiment, an EV population, a pharmaceutical composition comprising an EV population, or a formulation comprising an EV population is administered without administering one or more anti-inflammatory agents. In one embodiment, the one or more anti-inflammatory agents include dexamethasone and/or cyclosporine.

The present invention relates to a photoreceptor rescue cell composition, which includes a plurality of heterogeneous photoreceptor rescue cells with a unique marker profile. The heterogeneous photoreceptor rescue cell (PRC) composition of the present invention cumulatively expresses the markers: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA. The photoreceptor rescue cell composition of the present invention includes inhibitory neurons, excitatory neurons, progenitor cells, astrocytes and mixed neurons. PRC can be defined according to phenotype, for example, according to the expression of intracellular or extracellular markers. Photoreceptor rescue cells can be further characterized by the expression or non-expression of eye movement area progenitor cell markers, neural markers and/or rod/cone photoreceptor cell markers.

These photosensitive rescue cell compositions can be generated by in vitro differentiation of early progenitor cells, including high-potential stem cells, such as embryonic stem cells (ESCs), cells that have undergone transdifferentiation or partial reprogramming to reach a progenitor cell state, and induced high-potential stem cells (iPSCs). The present invention provides compositions of photosensitive rescue cells that have not been obtained or not obtained from an initial source and thus have a unique non-natural marker profile.

Photoreceptor rescue cell compositions can be used in a variety of in vivo and in vitro methods. For example, photoreceptor rescue cells can be used to treat retinal conditions, including but not limited to macular degeneration (including age-related macular degeneration (AMD), such as wet and dry AMD; retinitis pigmentosa and AMD secondary to geographic atrophy). Photoreceptor rescue cells can be used in vitro in screening assays to identify putative therapeutic or preventive treatment candidates.

Functionally, PRCs exhibit the ability to treat eye diseases and improve visual acuity in individuals with retinal diseases or disorders, for example, by increasing the secretion of neuroprotective factors, by preventing or slowing the loss of photoreceptor cells, by increasing phagocytic activity (e.g., the ability to phagocytose isolated photoreceptor outer segments), by inhibiting microglial activation (e.g., by increasing the expression of CNFT and/or MIF), by reducing oxidative stress (e.g., by increasing the expression of CNFT), by increasing the expression of anti-apoptotic factors, and/or by preventing outer nuclear layer degeneration. Definition

As defined herein, the singular form is provided for illustrative purposes, but may also apply to the plural form of the phrase. The following definitions are intended to supplement the learned definitions of the terms as they would be understood by a person of ordinary skill.

As used herein, the term "photosensitive rescue cell composition" refers to a composition comprising a heterogeneous photosensitive rescue cell (PRC) composition. As used herein, the photosensitive rescue cell composition includes heterogeneous cells, including but not limited to inhibitory neurons, excitatory neurons, mixed neurons, progenitor cells and astrocytes. The cells in the photosensitive rescue cell composition cumulatively express at least 2, 3, 4, 5, 6 or 7 of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and/or NFIA. In a specific example, the cells in the composition cumulatively express at least FOXG1 and MAP2. In another embodiment, the cells in the composition cumulatively express at least one of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

As used herein, the excitatory neurons in the light-sensitive rescue cell composition of the present invention refer to cells expressing one or more of the markers NEUROD2, NEUROD6, SLA, NELL2 and/or SATB2.

As used herein, the inhibitory neurons in the light-sensitive rescue cell composition of the present invention refer to cells expressing one or more of the markers DLX5, TUBB3, SCGN, ERBB4 and CALB2.

As used herein, selective neurons refer to cells expressing one or more markers MEIS2, PBX3, GRIA2, and CACNA1C in the photosensitive rescue cell composition.

As used herein, when referring to specific cell types contained in the photosensitive rescue cell composition of the present invention, progenitor cells refer to cells expressing one or more of the markers VIM, MKI67, CLU and GLI3.

As used herein, the photosensitive rescue cell composition of the present invention expresses one or more of the astrocyte markers GFAP, LUCAT1, MIR99AHG and FBXL7.

The photoreceptor rescue cell composition may further contain photoreceptor rescue cells expressing eye movement zone progenitor cell markers, rod/cone photoreceptor cell markers and/or neuron markers.

Exemplary eye region progenitor cell markers expressed by cells in the photoreceptor rescue cell composition of the present invention include PAX6, LHX2, SIX3, NES or SOX2. Several eye region progenitor cell markers, such as PAX6, LHX2, SOX2, NES, are widely expressed in a variety of neural progenitor cells and are also highly expressed in the PRCs of the composition of the present invention. Thus, in a specific example, the composition of the present invention includes heterogeneous photoreceptor rescue cells that cumulatively express at least PAX6, LHX2, SOX2, NES and, if necessary, SIX3. However, certain genes that are markers of specific eye movement area progenitor cells in neural progenitor cells, such as RAX, SIX6 and TBX3, are hardly expressed in the PRC of the composition of the present invention. Accordingly, in a specific embodiment, the composition of the present invention is substantially free of photoreceptor rescue cells or generally free of cells expressing RAX, SIX6 or TBX3. In a specific embodiment, the composition of the present invention is substantially free of photoreceptor rescue cells or generally free of cells expressing RAX and/or TBX3.

Exemplary rod/cone photoreceptor cell markers expressed by cells in the photoreceptor rescue cell composition of the present invention include Mash1/ASCL1 and RORB. Several rod/cone photoreceptor cell markers, such as Mash1/ASCL1 and RORB, are widely expressed in the neuroectoderm and neural progenitor cells derived from the neuroectoderm and are also highly expressed in the PRCs of the composition of the present invention. Thus, in a specific example, the composition of the present invention includes heterogeneous photoreceptor rescue cells that cumulatively express at least Mash1/ASCL1 and RORB. However, certain genes, such as NRL and NR2E3, which are markers of specific rod/cone photoreceptor cells in the neuroectoderm and neural progenitor cells derived from the neuroectoderm, are hardly expressed in the PRCs of the compositions of the present invention. Accordingly, in a specific embodiment, the compositions of the present invention are substantially free of photorescrutin cells, or generally free of cells expressing CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1 and GNAT2.

Exemplary neuronal markers expressed by cells in the photosensitive rescue cell composition of the present invention include TUBB3, NFIA, DCX, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. Several neuronal markers, such as TUBB3, NFIA, DCX, and NFIB, are stably expressed in the PRCs of the composition of the present invention. Thus, in a specific example, the composition of the present invention includes heterogeneous photosensitive rescue cells that cumulatively express TUBB3, NFIA, DCX, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. However, certain genes, such as OTX2, which are specific neuron markers in neural progenitor cells, are underexpressed in the PRCs of the composition of the present invention. Accordingly, in a specific embodiment, the composition of the present invention is substantially free of light-sensitive rescue cells or generally free of cells expressing OTX2.

In other embodiments, the compositions of the present invention are substantially free of photorescive cells or generally free of cells expressing the high potential markers SSEA4 and/or OCT4.

"Retina cells" refers to the nerve cells of the retina, which are stratified into three nuclear layers, which include photoreceptor cells, horizontal cells, bipolar cells, axonal neurons, Muller neuroglia cells and ganglion cells. Retina cells include neural retinal cells (also referred to as photoreceptor cells herein), retinal pigment epithelium (RPE) cells, iris epithelial cells and their precursors. As used herein, "retinal pigment epithelium (RPE) cells" refers to the outermost cells of the retina. The role of RPE cells is to provide support to the retinal photoreceptor cells and is responsible for the metabolic digestion of the discarded neural retinal outer segments. As used herein, "neuro-retinal cells" refers to the layer of photoreceptor cells (i.e., rods and cones) underlying the RPE cell layer in the retina. Neuro-retinal (NR) cells are engineered photosensitive neurons. As used herein, the term "cumulatively" and grammatical variations thereof refer to the expression of a marker throughout a heterogeneous population of cells in a composition. Specifically, cumulative expression refers to a composition in which at least one cell in the composition expresses a marker such that the cells in the composition collectively express all of the genes enumerated. For example, the statement "a plurality of heterogeneous cells cumulatively express FOXG1 and MAP2" may refer to a composition in which at least one cell expresses FOXG1 and at least one cell expresses MAP2, or a composition in which at least a single cell expresses FOXG1 and MAP2, such that a plurality of cells in the composition cumulatively express FOXG1 and MAP2. For another example, the statement "a plurality of heterogeneous cells cumulatively express FOXG1, MAP2, STMN2, and DCX" may refer to, but is not limited to, a composition in which at least one cell expresses FOXG1, at least one cell expresses MAP2, at least one cell expresses STMN2, and at least one cell expresses DCX. Alternatively, such phrases are intended to encompass compositions wherein at least one cell expresses FOXG1 and MAP2 and at least one other cell expresses STMN2 and DCX. As another example, such phrases are intended to encompass compositions wherein at least one cell expresses FOXG1 and STMN2, at least one cell expresses DCX, and at least one cell expresses MAP2.

The term "human neural stem cells" or "hNSCs" is used herein to refer to cells that are self-renewing and arise during adult life through neurogenesis. These multipotent adult stem cells give rise to the major phenotypes of the nervous system, differentiating into neurons, astrocytes, and oligodendrocytes.

The term "neural progenitor cell" or "NPC" is used herein to refer to the progenitor cells of the central nervous system (CNS) that give rise to many, if not all, of the neuroglia and neuronal cell types that populate the CNS.

The term "plurality" is used herein to refer to a state of being plural, ie, at least two, such as a cell type, such as a plurality of allelic photorescrutin cells.

The term "substantially free" or "essentially free" is used herein to refer to more than about 95%, 96%, 97%, 98%, 99% or 100% free. For example, the phrase "wherein the composition is substantially free of cells expressing the progenitor cell markers RAX, SIX6 and/or TBX3" refers to a composition wherein at least 95%, 96%, 97%, 98%, 99% or 100% of the cells do not express any of the aforementioned markers.

The compositions of the invention are characterized in that at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the cells in the composition are PRCs. In certain embodiments, the methods described herein can produce a composition of the invention that can be characterized by about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, 55% to about 100%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 50% to about 55%, about 55% to about 100%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, 60% to about 100%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, 65% to about 100%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, 70% to about 100%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, 75% to about 100%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, From about 80% to about 100%, from about 80% to about 95%, from about 80% to about 90%, from about 80% to about 85%, from about 85% to about 100%, from about 85% to about 95%, from about 85% to about 90%, from 90% to about 100%, from about 90% to about 95%, or from about 95% to about 100% of the cells are PRCs.

The compositions of the invention may be characterized by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% of the cells in the composition being viable. In certain embodiments, the methods described herein can produce a composition of the invention characterized by about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 50%, about 50% to about 60%, about 50% to about 55%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about In some embodiments, the cells of the present invention may be viable in an amount of at least about 50%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 60%, at least about 70%, at least about 70%, at least about 8 ...

As used herein, the phrase "substantially pure photosensitive rescue cell composition" refers to a heterogeneous photosensitive rescue cell composition (e.g., a composition comprising cells), wherein the composition includes a substantial percentage of cells, such as at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, which share the same expression when it comes to a specific marker profile. All or most PRC cells are able to contact their surrounding culture medium and thus contact factors in such culture medium to approximately the same extent, so that those progenitor cells differentiate at similar times and to similar degrees. This similar differentiation timeline of the PRC cell population indicates that such cells are synchronized. The cell cycle of PRCs can also be synchronized in some cases. Such synchronization can make subpopulations of cells homogeneous or nearly homogeneous with respect to their expression profiles of specific markers.

For example, purity can refer to the percentage of photoreceptor cells in a composition that exhibit a particular expression profile. For example, with respect to the expression of FOXG1 and MAP2, a substantially pure photoreceptor rescue cell composition can refer to a heterogeneous photoreceptor rescue cell composition, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the photoreceptor rescue cells in the composition express FOXG1 and MAP2. In some embodiments, the heterogeneous photosensitive rescue cell composition is at least 50% pure, at least 55% pure, at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, at least about 80% pure, at least 85% pure, at least 90% pure, or at least 95% pure. In some embodiments, the cells have a purity of about 50% to about 95% purity, about 55% to about 95% purity, about 60% to about 95% purity, about 70% to about 95% purity, about 75% to about 95% purity, about 80% to about 95% purity, about 85% to about 95% purity, about 90% to about 95% purity, about 50% to about 90% purity, about 55% to about 90% purity, about 60% to about 95% purity, about 80% to about 95% purity, about 85% to about 95% purity, about 90% to about 95% purity, about 50% to about 90% purity, about 55% to about 90% purity, about 60% to about 90% pure, about 65% pure to about 90% pure, about 70% pure to about 90% pure, about 75% pure to about 90% pure, about 80% pure to about 90% pure, about 85% pure to about 90% pure, about 50% pure to about 85% pure, about 55% pure to about 85% pure, about 60% pure to about 85% pure, about 65% pure to about 85% pure, about 70% pure to about 85% pure, about 75% pure to about 85% pure, about 80% to about 85% pure, about 50% to about 80% pure, about 55% to about 80% pure, about 60% to about 80% pure, about 65% to about 80% pure, about 70% to about 80% pure, about 75% to about 80% pure, about 50% to about 75% pure, about 55% to about 75% pure, about 60% to about 75% pure, about 65% to about 75% pure, about 70% to about In some embodiments, the invention relates to a novel molecule having a purity of about 75% pure, about 50% pure to about 70% pure, about 55% pure to about 70% pure, about 60% pure to about 70% pure, about 65% pure to about 70% pure, about 70% pure to about 75% pure, about 50% pure to about 65% pure, about 55% pure to about 65% pure, about 60% pure to about 65% pure, about 50% pure to about 60% pure, about 55% pure to about 60% pure, or about 50% pure to about 55% pure.

As used herein, a majority of cells means at least 50%, and depending on the specific example, can include at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the cells.

The purity that can be achieved using the methods of the present invention is particularly important where such cell populations are to be used for in vivo therapeutic or preventive purposes. Being able to obtain a highly pure cell population avoids having to perform another manipulation, such as an enrichment or selection step, which can result in unnecessary cell loss. This is particularly important where the cell population may be small or the number of cells may be limited.

For example, the level of purity can be quantified by determining the ratio of cells in a preparation that express one or more markers, such as those of PRC (including those identified in the present application), relative to the total number of cells in the preparation, for example, by detecting cells that express or do not express the one or more markers. If desired, the expression of markers indicative of non-PRC cells can also be detected to facilitate the detection and/or quantification of such cells. Exemplary methods that can be used to detect and/or quantify marker expression include, but are not limited to, flow cytometry, fluorescence activated cell sorting (FACS), immunohistochemistry, in situ hybridization, scRNAseq, immunofluorescence, single cell proteomics (e.g., via LC-MS), possible single cell metabolomics, liposomics, scPCR, and other suitable methods known in the art. If desired, the purity of a composition can be determined based on the percentage of viable cells present in the composition.

"Embryoid bodies" refer to clumps or clusters of high-potential cells (e.g., iPSCs or ESCs) that can be formed by culturing high-potential cells under non-attachment conditions (e.g., on a low-attachment medium or in "hanging drops"). In such cultures, high-potential cells can form clumps or clusters of cells, which are named embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb;6(2):88-95, which is incorporated herein by reference in its entirety. Typically, EBs initially form solid aggregates or clusters of potential-rich cells, and over time some EBs include fluid-filled compartments, the former referred to in the literature as "simple" EBs and the latter as "cystic" EBs.

Based on the ability of the disclosed methods to direct differentiation of progenitor cells, such as, but not limited to, high-potential stem cells (e.g., ESCs and iPSCs), the present invention provides compositions of heterogeneous PRCs. As used herein, direct differentiation means that a population of progenitor cells differentiates into or toward a desired lineage, in part due to factors or other stimuli provided to such progenitor cells, thereby avoiding differentiation into other undesirable lineages and thereby avoiding potential contamination of lineages. In some embodiments, the methods provided herein drive differentiation of, for example, high-potential stem cells into PRCs without generating embryoid bodies (EBs). EBs, as described below, are three-dimensional cell clusters that can be formed during the differentiation of high-potential stem cells (including but not limited to embryonic stem (ES) cells and iPSCs) and typically contain cells, including progenitor cells of the mesoderm, ectoderm, and endoderm lineages. The three-dimensional nature of EBs can establish a different environment from the non-EB-based methods described herein, including different cell-cell interactions and different cell-cell signaling. In addition, cells within EBs may not all receive similar doses of exogenously added agents, such as differentiation factors present in the surrounding culture medium, and this can cause various differentiation events and decisions during EB development.

In contrast, the PRC cell culture methods of the present invention do not require the formation of EBs and preferably avoid the formation of EBs. In practice, these methods culture cells under conditions that provide the cells with equal contact with the surrounding culture medium (including factors in such culture medium). In certain specific examples, PRCs can be grown as a monolayer or near-monolayer (e.g., adherent conditions) attached to the culture surface. In some specific examples, the culture methods disclosed herein provide that PRCs are cultured under non-adhesive or low-adhesive conditions (e.g., suspension).

The term "embryonic stem cell" (ES cell or ESC) is used herein as it is used in this technology. This term includes cells derived from the inner cell mass of a human blastocyst or morula, including cells that have been continuously reproduced as a cell line. ES cells can be derived from the fertilization of an egg cell by a sperm, and using DNA, nuclear transfer, parthenogenesis, or by producing ES cells with homozygosity in the HLA region. ES cells are also cells derived from fertilized eggs, blastomeres, or blastocyst-stage mammalian embryos produced by: fusion of sperm and egg cells, nuclear transfer, parthenogenesis, androgenesis, or chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane to produce cells. Embryonic stem cells, regardless of their origin or the specific method used to generate them, can be identified based on: (i) the ability to differentiate into cells of all three germ layers; (ii) the expression of at least OCT 4 and alkaline phosphatases; and (iii) the ability to produce teratomas when transplanted into immunodeficient animals. Embryonic stem cells that can be used in specific embodiments of the present invention include, but are not limited to, human ES cells ("ESCs" or "hES cells"), such as MA01, MA09, ACT-4, No.3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Additional exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also the NIH Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that can be used is MA09 cells. The isolation and preparation of MA09 cells were previously described in Klimanskaya et al. (2006) "Human Embryonic Stem Cell lines Derived from Single Blastomeres." Nature 444: 481-485. The human ES cells used in the exemplary embodiments of the present invention can be obtained and maintained according to GMP standards.

The term "ES cell" does not and should not be inferred to mean that the cell is produced by destroying an embryo. Instead, various methods are available and can be used to produce ES cells without destroying an embryo (such as a human embryo). For example, ES cells can be produced from a single blastomere derived from an embryo in a manner similar to the extraction of blastomeres for preimplantation genetic diagnosis (PGD). Examples of such cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. An exemplary human embryonic stem cell line that can be used is MA09 cells. Isolation and preparation of MA09 cells were previously described in Klimanskaya et al. (2006) "Human Embryonic Stem Cell lines Derived from Single Blastomeres." Nature 444: 481-485. See also Chung et al., 2008, Cell Stem Cell, 2: 113. All of these lines were generated without destroying the embryo. As used herein, the term "rich-potential stem cells" includes but is not limited to tissue-derived stem cells, embryonic stem cells, embryonic stem cells, induced rich-potential stem cells, and stimulation-triggered rich-potential (STAP) cells, regardless of the method by which the rich-potential stem cells are obtained. The term also includes high-potential stem cells that have the functional and phenotypic characteristics of the aforementioned cells, regardless of the method used to generate such cells. High-potential stem cells are functionally defined as stem cells that: (a) are capable of inducing teratomas when transplanted into SCID mice; (b) are capable of differentiating into cell types of all three germ layers (e.g., can differentiate into ectoderm, mesoderm, and endoderm cell types); (c) express one or more markers of embryonic stem cells (e.g., express OCT4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.); and d) are capable of self-renewal. The term "rich potential" refers to all lineages of cells that are capable of forming a body or a soma (i.e., an embryo). In certain specific examples, the rich potential stem cells express one or more markers selected from the group consisting of: OCT-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary rich potential stem cells include embryonic stem cells derived from the ICM of blastocyst stage embryos, and embryonic stem cells derived from one or more blastomeres of cleavage stage or morula stage embryos (optionally without destroying the rest of the embryo). Other exemplary enriched potential stem cells include induced enriched potential stem cells (iPSCs) produced by reprogramming somatic cells by expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be produced using fetal, postnatal, newborn, juvenile or adult somatic cells. As used herein, the term "enriched potential stem cells", "PS cells" or "PSCs" includes embryonic stem cells, induced enriched potential stem cells and embryonic enriched potential stem cells, regardless of the method by which the enriched potential stem cells are obtained. For example, embryonic stem cells and induced high-potential stem cells are a type of high-potential stem cells that can form cells from each of the three germ layers (ectoderm, mesoderm, and endoderm). High potential is a continuous developmental efficiency, ranging from incomplete or partial high-potential cells that cannot produce a complete organism to more primitive, more high-potential cells (such as embryonic stem cells) that can produce a complete organism. Exemplary high-potential stem cells can be produced using methods known in the art, for example. Exemplary high-potential stem cells include, but are not limited to, embryonic stem cells derived from the inner cell mass of a blastocyst stage embryo, embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the rest of the embryo), induced high-potential stem cells generated by reprogramming somatic cells to a high-potential state, and high-potential cells generated from embryonic germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF). Such embryonic stem cells can be generated from embryonic material generated by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis.

In some embodiments, factors that can be used to reprogram somatic cells into high-potential stem cells include, for example, a combination of OCT 4 (sometimes referred to as OCT 3/4), SOX2, c-Myc, and KLF4. In other embodiments, factors that can be used to reprogram somatic cells into high-potential stem cells include, for example, a combination of OCT 4, SOX2, Nanog, and Lin28. In some embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, other reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into high-potential stem cells. Induced high-potential stem cells are functionally defined and include cells reprogrammed using any of a variety of methods (integrating vectors, non-integrating vectors, chemical means, etc.). The high-potential stem cells may be genetically modified or otherwise modified to increase lifespan, efficacy, homing, prevent or reduce alloimmune response, or deliver desired factors via cells differentiated from such high-potential cells (e.g., photoreceptor rescue cells, photoreceptor progenitor cells, rods, cones, etc., and other cell types described herein (e.g., in specific examples). In a specific example, the high-potential stem cells may be genetically engineered or otherwise modified, for example, to increase lifespan, efficacy, homing, prevent or reduce alloimmune response, or deliver desired factors via cells derived from such high-potential cells (e.g., photoreceptor rescue cells or cells present in a photoreceptor rescue cell composition). For example, the enriched potential stem cells and the differentiated cells derived therefrom can be engineered or otherwise modified to lack or reduce the expression of beta 2 microglobulin, class I genes (including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G), TAP1, TAP2, alpha protein, CTIIA, RFX5, TRAC or TRAB genes. As described in WO2012145384 and WO2013158292, which are incorporated herein by reference in their entirety, in some embodiments, cells, such as enriched potential stem cells and differentiated cells derived therefrom, such as photosensitive rescue cells or cells present in a photosensitive rescue cell composition, contain a genetically engineered disruption in the beta-2 microglobulin (B2M) gene. In some embodiments, the cell further comprises a polynucleotide capable of encoding a single-chain fusion human leukocyte antigen (HLA) class I protein comprising at least a portion of a B2M protein covalently linked directly or via a linker sequence to at least a portion of an HLA-1 alpha chain. In some embodiments, the HLA-1 alpha chain is selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the cell comprises a genetically engineered break in a human leukocyte antigen (HLA) class II-associated gene. In some embodiments, the HLA class II-associated gene is selected from regulatory factor X-associated anchor protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), class II transactivator (CIITA), HLA-DPA (alpha chain), HLA-DPB (beta chain), HLA-DQA, HLA-DQB, HLA-DRA, HLA-DRB, HLA-DMA, HLA-DMB, HLA-DOA and HLA-DOB. In some embodiments, the cell comprises one or more polynucleotides encoding a single-chain fusion HLA class II protein or an HLA class II protein. In some embodiments, the cell further comprises one or more factors selected from the group consisting of CD200, CD24, PD-L 1, HLAG or H2-M3, Cd47, FASLG or Fasl, Ccl21 or Ccl21b, Mfge8, serpin B9 or Spi6, or DUX4.

"Induced enriched potential stem cells" (iPS cells or iPSCs) can be generated by protein transduction of reprogramming factors in somatic cells. In certain embodiments, at least two reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. In other embodiments, at least three reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. In other embodiments, at least four reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells.

High-potential stem cells can be derived from any species. Embryonic stem cells have been successfully obtained from a variety of species such as mice, non-human primates, and humans, and embryonic stem cell-like cells have been generated from many other species. Therefore, those skilled in the art can generate embryonic stem cells and embryonic stem cells from any species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild), cats (domestic and wild, such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.) and the like. In some specific examples, the species is an endangered species. In some specific examples, the species is a currently extinct species.

Similarly, iPS cells can be from any species. These iPS cells have been successfully generated using mouse and human cells. In addition, iPS cells have been successfully generated using embryonic, fetal, newborn, and adult tissues. Therefore, iPS cells can be easily generated using donor cells from any species. Thus, iPS cells from any species can be generated, including (but not limited to) humans, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild), cats (domestic and wild, such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), and the like. In some embodiments, the species is an endangered species. In some embodiments, the species is a currently extinct species.

Induced high potential stem cells can be generated using any somatic cell at almost any stage of development as a starting point. For example, the cell can come from an embryo, placenta, fetus, newborn, infant or adult donor. Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained from skin samples or sections, synovial cells from synovial tissue, foreskin cells, buccal cells or lung fibroblasts. In some specific examples, the somatic cell is a placental cell. Although skin and buccal provide an easily accessible and easily accessible source of suitable cells, almost any cell can be used. In some embodiments, the somatic cell is not a fibroblast. In some embodiments, the somatic cell is from the placenta.

Induced high-potential stem cells can be generated by expressing one or more reprogramming factors or inducing the expression of one or more reprogramming factors in somatic cells. The somatic cells are fibroblasts, such as dermal fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblast somatic cells. Somatic cells can be reprogrammed by causing at least 1, 2, 3, 4, 5 reprogramming factors to express (e.g., via viral transduction, integrating or non-integrating vectors, etc.) and/or contacting the reprogramming factors (e.g., using protein transduction domains, electroporation, microinjection, cationic amphiphilic molecules, fusion with lipid-containing bilayers, detergent permeation, etc.). The reprogramming factors can be selected from OCT 3/4, SOX2, NANOG, LIN28, C-MYC, and KLF4. The expression of reprogramming factors can be induced by contacting the somatic cells with at least one agent that induces the expression of reprogramming factors (e.g., small organic molecule agents).

Other exemplary high-potential stem cells include induced high-potential stem cells produced as follows: somatic cells are reprogrammed by combining or inducing the expression of expression factors ("reprogramming factors"). iPS cells can be obtained from a cell bank. The preparation of iPS cells can be an initial step in the production of differentiated cells. iPS cells can be specifically produced using materials from specific patients or matched donors, with the purpose of producing tissue-matched photosensitive rescue cells. iPSCs can be produced from cells that are substantially non-immunogenic in the intended recipient, such as autologous cells of the intended recipient or cells that are tissue compatible with the intended recipient.

Somatic cells can also be reprogrammed using combinatorial methods, where reprogramming factors are expressed (e.g., using viral vectors, plasmids, and the like) and reprogramming factor expression is induced (e.g., using small organic molecules). For example, reprogramming factors can be expressed in somatic cells by infection with a viral vector, such as a retroviral vector or a lentiviral vector. In addition, reprogramming factors can be expressed in somatic cells using non-integrating vectors, such as episomal plasmids. See Yu et al., Science. 2009 May 8; 324(5928):797-801, which is incorporated herein by reference in its entirety. When non-integrating vectors are used to express reprogramming factors, the factors can be expressed in the cells by introducing the vector into somatic cells using electroporation, transfection, or transformation. For example, in mouse cells, expressing four factors (OCT3/4, SOX2, C-MYC, and KLF4) using integrating viral vectors is sufficient to reprogram somatic cells. In human cells, expressing four factors (OCT3/4, SOX2, NANOG, and LIN28) using integrating viral vectors is sufficient to reprogram somatic cells.

After the reprogramming factors are expressed in the cells, the cells can be cultured. Over time, cells with ES characteristics appear in the culture dish. Cells can be selected based on, for example, ES morphology or based on the expression of selectable or detectable markers and continued in culture. The cells can be cultured to produce a culture of cells that resemble ES cells.

To confirm the rich potential of iPS cells, the cells can be tested in one or more rich potential assays. For example, the cells can be tested for expression of ES cell markers; the cells can be assessed for their ability to generate teratomas when transplanted into SCID mice; the cells can be assessed for their ability to differentiate to generate cell types of all three germ layers. Once rich potential iPSCs are obtained, they can be used to generate cell types disclosed herein, such as light-sensitive rescue cells.

Stimulus-triggered acquired high-potential (STAP) cells are high-potential stem cells generated by reprogramming somatic cells in response to sublethal stimulation (e.g., low pH exposure). Reprogramming does not require nuclear transfer into somatic cells or genetic manipulation of somatic cells. See Obokata et al., Nature, 505:676-680, 2014.

As used herein, the term "stem cell" refers to a master cell that can regenerate indefinitely to form specialized cells of tissues and organs. Stem cells are developmentally potent or multipotent cells. Stem cells can divide to produce two daughter stem cells, or one daughter stem cell and a progenitor ("transition") cell, which then proliferate to produce mature, fully formed cells of tissues.

As used herein, the term "adult stem cells" refers to stem cells (e.g., bone marrow stem cells, cord blood stem cells, and adipose stem cells) isolated from tissues or organs of animals (e.g., humans) at a growth stage later than the embryonic stage. In one aspect, the stem cells of the present invention can be isolated at the postnatal stage. Preferably, cells can be isolated from mammals (e.g., humans). Adult stem cells are different from embryonic stem cells, which are defined according to their origin (the inner cell mass of the blastocyst). Adult stem cells according to the present invention can be isolated from any non-embryonic tissue, and include newborns, young children, adolescents, and adult patients. Generally, the stem cells of the present invention will be isolated from non-neonatal mammals, and more preferably from non-neonatal humans. These adult stem cells are characterized in that, in an undifferentiated state, they express telomerase and they do not display gap junctional intercellular communication (GJIC) and do not have a transformed phenotype.

As used herein, a "sign" of disease refers broadly to any abnormality that is detectable upon examination of a patient and that is indicative of disease; an objective indicator of disease, in contrast to symptoms, which are subjective indicators of disease.

As used herein, disease "symptoms" refer broadly to any pathological phenomena or deviations from normal in structure, function, or sensation that a patient experiences and that are indicative of disease.

As used herein, "therapy", "therapeutic", "treating", "treat", and "treatment" broadly refer to treating a disease, arresting or reducing the development of a disease or its clinical symptoms, and/or relieving a disease, or causing the regression of a disease or its clinical symptoms. Therapy covers preventing, preventing, curing, curing, rescuing, reducing, alleviating, and/or alleviating a disease, sign, and/or symptom of a disease. Therapy covers the relief of signs and/or symptoms in patients who have persistent signs and/or symptoms of a disease. Therapy also covers "prevention" and "prevention". Prevention includes the prevention of a disease in a patient after treatment for a disease, or reducing the incidence or severity of a disease in a patient. The term "reduce" for the purpose of treatment broadly refers to a clinically significant decrease in signs and/or symptoms. Treatment includes treating recurring or relapsing signs and/or symptoms. Treatment includes, but is not limited to, preventing the appearance of signs and/or symptoms at any time, as well as reducing existing signs and/or symptoms and eliminating existing signs and/or symptoms. Treatment includes treating chronic conditions ("maintenance") and acute conditions. For example, treatment includes treating or preventing the recurrence or recurrence of signs and/or symptoms.

Conditions to be treated according to the present invention and thus treated using one or more of the formulations provided herein include, but are not limited to, macular degeneration, including age-related macular degeneration, and such macular degeneration may be early or late stage macular degeneration. Other conditions to be treated include, but are not limited to, retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, age-related macular degeneration (e.g., wet or dry), AMD secondary to map atrophy, congenital retinal dystrophy, rod dystrophy, cone dystrophy, cone-rod dystrophy, Leber's congenital amaurosis, Stargardt's disease, retinal detachment, glaucoma, optic neuropathy, and trauma to the infected eye. Cell Markers:

The photosensitive rescue cell composition comprising a plurality of heterogeneous photosensitive rescue cells exhibits a unique marker pattern, thereby distinguishing it from naturally occurring cells. Specifically, the composition of the present invention is characterized by the expression of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA or a combination thereof. In some specific examples, the plurality of photosensitive rescue cells in the composition of the present invention are characterized by the cumulative expression of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA. In some specific examples, the cells in the photosensitive rescue cell composition of the present invention are characterized by the expression of FOXG1 and/or MAP2. In some embodiments, the plurality of light-sensitive rescue cells in the compositions of the invention are characterized by the cumulative expression of FOXG1 and MAP2.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention include inhibitory neurons and are therefore characterized by the expression of DLX5, TUBB3, SCGN, ERBB4, CALB2 or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of DLX5, TUBB3, SCGN, ERBB4 and CALB2.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention include excitatory neurons and are therefore characterized by the expression of NEUROD2, NEUROD6, SLA, NELL2, SATB2 or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of NEUROD2, NEUROD6, SLA, NELL2 and SATB2.

In some embodiments, the cells in the light-sensitive rescue cell composition of the present invention include progenitor cells and are therefore characterized by the expression of VIM, MKI67, CLU, GLI3 or a combination thereof. In some embodiments, a plurality of light-sensitive rescue cells in the composition of the present invention are characterized by the cumulative expression of VIM, MKI67, CLU and GLI3.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention include astrocytes and are thus characterized by the expression of GFAP, LUCAT1, MIR99AHG, FBXL7 or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of GFAP, LUCAT1, MIR99AHG and FBXL7.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention include selective neurons and are therefore characterized by the expression of MEIS2, PBX3, GRIA2, CACNA1C or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of MEIS2, PBX3, GRIA2 and CACNA1C.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are characterized by the expression of an eye movement area progenitor cell marker selected from the group consisting of: PAX6, LHX2, SIX3, NES, SOX2 or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of PAX6, LHX2, SIX3, NES and SOX2.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing the eye movement area progenitor cell markers RAX, SIX6 and/or TBX3. In some embodiments, the plurality of photoreceptor rescue cells in the composition of the present invention are characterized by almost no cumulative expression of RAX, SIX6 and TBX3.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are characterized by the expression of a rod/cone photoreceptor cell marker selected from the group consisting of: ASCL1, RORB, NR2E3, NRL or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of ASCL1, RORB, NR2E3 and NRL.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are characterized by the expression of a neuronal marker selected from the group consisting of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, HES5 or a combination thereof. In some embodiments, a plurality of photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5.

In some embodiments, the cells in the photosensitive rescue cell composition of the present invention are substantially free of cells expressing CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1, GNAT2 or a combination thereof. In some embodiments, the plurality of photosensitive rescue cells in the composition of the present invention are characterized by almost no cumulative expression of CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1 and GNAT2.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing VSX2, POU5F1 or a combination thereof. In some embodiments, the plurality of photoreceptor rescue cells in the composition of the present invention are characterized by almost no cumulative expression of VSX2 and POU5F1.

In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing OCT4, SSEA4 or a combination thereof. In some embodiments, the plurality of photoreceptor rescue cells in the composition of the present invention are characterized by almost no cumulative expression of OCT4 and SSEA4.

In some specific examples, the cells in the light-sensitive rescue cell composition of the present invention are characterized by AGT, ACBLN2, CDH7, DNAH11, EGR1, FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, nestin, TRbeta2, MASH1, RORbeta, MAP2, ELAVL3, NFI Expression of A, DCX, LHX2, SLC1A2, ELAVL4, PAX6, EMX2, ASCL1, DLL1, NFIB, ENOX1, TUBB3, MAP2, DCLK1/2, DCX, KALRN, LINC00461, C1orf61, NCAM1, SETBP1, PAK3, AKAP6, RTN1, CRMP1, FOXG1, TRIM2, BACH2, recoverin, opsin, rhodopsin, rod, and cone cGMP phosphodiesterase, which can be assessed at the protein and/or mRNA level (see Fischer et al., 2009). A J, Reh T A, Dev Neurosci. 2001; 23(4-5):268-76; Baumer et al., Development. 2003 Jul; 130(13):2903-15, Swaroop et al., Nat Rev Neurosci. 2010 Aug; 11(8):563-76, Agathocleous and Harris, Annu. Rev. Cell Dev. Biol. 2009. 25:45-69, each of which is incorporated herein by reference in its entirety).

In some embodiments, cells in the light-sensitive rescue cell composition of the present invention are characterized by evaluating the expression of cell markers compared to the expression of enriched potential stem cells (e.g., iPSCs or embryonic PSCs). In some embodiments, cells in the light-sensitive rescue cell composition of the present invention are characterized by reduced expression of FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4c, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, and SSEA4 compared to enriched potential stem cells (e.g., iPSCs or embryonic PSCs). In some specific examples, the cells in the light-sensitive rescue cell composition of the present invention are characterized by increased expression of AGT, ACBLN2, DCH7, DNA11 and EGR1 compared to ESC or iPSC. The marker is generally, for example, a human marker, except where otherwise indicated by the context. Cell markers can be identified using known immunocytochemistry methods, known PCR methods, total transcript analysis (including RNAseq), quantitative real-time PCR, flow cytometry, FACS, scRNAseq, bulk RNAseq, single cell or bulk qRT-PCR, immunocytochemistry, immunofluorescence, single cell or bulk proteomics (e.g., via LC-MS), metabolomics when possible, liposomics, and other suitable methods known in the art.

As used herein, the term "scRNAseq" refers to single cell RNA sequencing. In some embodiments, scRNAseq provides data for clustering single cells in a cell population based on the expression of a genetic marker. In some embodiments, scRNAseq provides data for determining the percentage of single cells in a population (e.g., a PRC cell population) that express a genetic marker. The mapped sequence data of scRNAseq is filtered according to quality control metrics, undergoes dimensionality reduction for visualization, and clustered using the common nearest neighbor method combined with the Louvain algorithm. Differential representation is thereby performed on the cluster, and the most differentially represented genes are compared between the cluster and several public data sources. We used a number of reviews of human developmental biology and datasets of human retina (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE142526), human hippocampus (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119212), human midbrain (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76381), and human cortex (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE132672) to assign the best guessed cellular attributes to clusters based on differentially expressed genes, as well as the expression profiles during development and in the reviewed datasets.

As used herein, the term bulk "RNAseq" refers to RNA sequencing analysis of a population of cells. Bulk RNAseq provides transcripts per million (TPM), where the amount of RNA molecules per 1,000,000 in an RNA-seq sample is derived from a gene of interest. Cell Culture Medium:

In a specific embodiment of the present invention, cells are stored, proliferated or differentiated in various cell culture media. Rescue induction medium is used to differentiate stem cells into early neural progenitor cells. Rescue induction medium (RIM) may include D-glucose, N2 supplement (e.g., 0.1-5%), B27 supplement (e.g., 0.005 to 0.2%), MEM non-essential amino acid solution and optionally include insulin and/or noggin, and may be present in DMEM/F12 (Invitrogen) or similar basic medium. For example, the rescue induction medium may include at least insulin. In addition, the insulin concentration may be changed or increased, which may promote cell survival and/or the yield of differentiated cells. For example, insulin concentrations can be varied over a range and survival and/or differentiation monitored to identify insulin concentrations that improve either or both of these properties. The addition of mitogen-activated protein kinases is not required, but has been observed to increase the expression of transcription factors associated with neural progenitor cells.

The components of DMEM/F12, neurobasal medium, N2 serum supplement, and B27 serum supplement are provided in Tables 1-4. It should be understood that the present invention contemplates the use of these specific media and supplements or media or supplements comprising, consisting essentially of, or consisting of these components. Table 1 DMEM/F12 components Molecular weight Concentration ( mg/L ) mM Amino Acids Glycine 75 18.75 0.25 L-Alanine 89 4.45 0.05 L-Arginine Hydrochloride 211 147.5 0.699 L-Asparagine-H2O 150 7.5 0.05 L-Aspartic Acid 133 6.65 0.05 L-Cysteine Hydrochloride-H2O 176 17.56 0.0998 L-Cysteine 2HCl 313 31.29 0.1 L-Glutamine 147 7.35 0.05 L-Glutamine 146 365 2.5 L-Histidine Hydrochloride-H2O 210 31.48 0.15 L-Isoleucine 131 54.47 0.416 L-Leucine 131 59.05 0.451 L-Lysine Hydrochloride 183 91.25 0.499 L-Methionine 149 17.24 0.116 L-Phenylalanine 165 35.48 0.215 L-Proline 115 17.25 0.15 L-Serine 105 26.25 0.25 L-Threonine 119 53.45 0.449 L-Tryptophan 204 9.02 0.0442 L-Tyrosine disodium salt dihydrate 261 55.79 0.214 L-Valine 117 52.85 0.452 Vitamins Biotin 244 0.0035 1.43E-05 Choline chloride 140 8.98 0.0641 D-Calcium Pantothenate 477 2.24 0.0047 Folic acid 441 2.65 0.00601 Niacinamide 122 2.02 0.0166 Pyridoxine Hydrochloride 206 2 0.00971 Riboflavin 376 0.219 0.000582 Thiamine hydrochloride 337 2.17 0.00644 Vitamin B12 1355 0.68 0.000502 i-Inositol 180 12.6 0.07 Inorganic salt Calcium chloride (CaCl2) (anhydrous) 111 116.6 1.05 Copper sulfate (CuSO4-5H2O) 250 0.0013 5.2E-06 Iron nitrate (Fe(NO3)3-9H2O) 404 0.05 0.000124 Iron sulfate (FeSO4-7H2O) 278 0.417 0.0015 Magnesium chloride (anhydrous) 95 28.64 0.301 Magnesium sulfate (MgSO4) (anhydrous) 120 48.84 0.407 Potassium chloride (KCl) 75 311.8 4.16 Sodium bicarbonate (NaHCO3) 84 1200 14.29 Sodium chloride (NaCl) 58 6995.5 120.61 Anhydrous sodium hydrogen phosphate (Na2HPO4) 142 71.02 0.5 Sodium dihydrogen phosphate (NaH2PO4-H2O) 138 62.5 0.453 Zinc sulfate (ZnSO4-7H2O) 288 0.432 0.0015 Sodium bicarbonate (NaHCO3) 84 1200 14.29 Sodium chloride (NaCl) 58 6995.5 120.61 Anhydrous sodium hydrogen phosphate (Na2HPO4) 142 71.02 0.5 Sodium dihydrogen phosphate (NaH2PO4-H2O) 138 62.5 0.453 Zinc sulfate (ZnSO4-7H2O) 288 0.432 0.0015 Other components D-Glucose (Dextrose) 180 3151 17.51 HEPES 238 3574.5 15.02 Sodium Hypoxanthine 159 2.39 0.015 Linoleic acid 280 0.042 0.00015 Lipole Acid 206 0.105 0.00051 Phenol red 376.4 8.1 0.0215 Putrescine 2HCl 161 0.081 0.000503 Sodium pyruvate 110 55 0.5 Thymidine 242 0.365 0.00151 Table 2 Neural basis components Molecular weight Concentration ( mg/L ) mM Amino Acids Glycine 75 30 0.4 L-Alanine 89 2 0.0225 L-Arginine Hydrochloride 211 84 0.398 L-Asparagine-H2O 150 0.83 0.00553 L-Cysteine 121 31.5 0.26 L-Histidine Hydrochloride-H2O 210 42 0.2 L-Isoleucine 131 105 0.802 L-Leucine 131 105 0.802 L-Lysine Hydrochloride 183 146 0.798 L-Methionine 149 30 0.201 L-Phenylalanine 165 66 0.4 L-Proline 115 7.76 0.0675 L-Serine 105 42 0.4 L-Threonine 119 95 0.798 L-Tryptophan 204 16 0.0784 L-Tyrosine 181 72 0.398 L-Valine 117 94 0.803 Vitamins Choline chloride 140 4 0.0286 D-Calcium Pantothenate 477 4 0.00839 Folic acid 441 4 0.00907 Niacinamide 122 4 0.0328 Pyridoxine Hydrochloride 204 4 0.0196 Riboflavin 376 0.4 0.00106 Thiamine hydrochloride 337 4 0.0119 Vitamin B12 1355 0.0068 0.000005 i-Inositol 180 7.2 0.04 Inorganic salt Calcium chloride (CaCl2) (anhydrous) 111 200 1.8 Iron nitrate (Fe(NO3)3-9H2O) 404 0.1 0.000248 Magnesium chloride (anhydrous) 95 77.3 0.814 Potassium chloride (KCl) 75 400 5.33 Sodium bicarbonate (NaHCO3) 84 2200 26.19 Sodium chloride (NaCl) 58 3000 51.72 Sodium dihydrogen phosphate (NaH2PO4-H2O) 138 125 0.906 Zinc sulfate (ZnSO4-7H2O) 288 0.194 0.000674 Other components D-Glucose (Dextrose) 180 4500 25 HEPES 238 2600 10.92 Phenol red 376.4 8.1 0.0215 Sodium pyruvate 110 25 0.227 table 3 N2 Supplement Components Molecular weight Concentration ( mg/L ) mM Protein Human iron transporter protein (Holo) 10000 10000 1 Insulin recombinant chain 5807.7 500 0.0861 Other components Progesterone 314.47 0.63 0.002 Putrescine 161 1611 10.01 Selenite 173 0.52 0.00301 Table 4 B27 component Vitamins Protein Other components Biotin BSA, fatty acid free fraction V Corticosterone Linoleic acid DL-alpha-tocopheryl acetate Human recombinant insulin D-Galactose Linolenic acid DL α-Tocopherol Vitamin A (Acetate) Catalase Ethanolamine HCl Progesterone Human ferritin Glutathione (reduced) Sodium Putrescine 2HCl Superoxide dismutase L-Carnitine HCl

The methods described herein may use human factors, such as human Noggin, human insulin, and their analogs.

Noggin is a secreted bone morphogenetic protein (BMP) inhibitor that reportedly binds BMP2, BMP4, and BMP7 with high affinity to block TGFβ family activity. SB431542 is a small molecule that reportedly inhibits TGFβ/activin/Nodal by blocking ACTRIB, TGFβR1, and ACTRIC receptor phosphorylation. SB431542 is believed to destabilize activin- and Nanog-mediated potential-rich networks and inhibit BMP-induced trophoblast, mesoderm, and endoderm cell fate by blocking endogenous activin and BMP signaling. It is expected that agents having one or more of the aforementioned activities can replace or enhance the function of one or both of Noggin and SB431542, for example when used in the context of the disclosed methods. For example, the applicants envision that one or more inhibitors that affect any or all of the following three target areas may displace or enhance the protein Noggin and/or small molecule SB4312542: 1) prevent ligands (e.g., bone morphogenetic proteins (BMPs), such as BMP2, BMP4, BMP5, BMP6, BMP7, BMP13, and BMP14) from binding to receptors (e.g., bone morphogenetic protein receptors); 2) block receptor (e.g., dorsomorphin) activation; and 3) inhibit SMAD intracellular proteins/transcription factors. Potentially suitable exemplary factors include the natural secreted BMP inhibitors tenascin (which blocks BMP4) and follistatin (which blocks activin), as well as analogs or mimetics thereof. Other exemplary factors that can mimic the effects of noggin include the use of dominant negative receptors or blocking antibodies that isolate BMP2, BMP4 and/or BMP7. In addition, doxorubicin (or Compound C) has been reported to have similar effects on stem cells in terms of blocking receptor phosphorylation. Inhibition of SMAD proteins can also be achieved using soluble inhibitors such as SIS3 (6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enyl))-1,2,3,4-tetrahydroisoquinoline, a specific inhibitor of Smad3, SIS3), overexpression of one or more inhibitors of one of the receptor SMADs (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8/9) (e.g., SMAD6, SMAD7, SMAD10), or RNAi. Another combination of factors expected to be suitable for generating neural progenitor cells includes leukemia inhibitory factor (LIF), GSK3 inhibitor (CHIR 99021), Compound E (γ-secretase inhibitor XXI) and TGFβ inhibitor SB431542, which have previously been shown to be effective for generating neural spinal stem cells (Li et al., Proc Natl Acad Sci USA. 2011 May 17; 108(20):8299-304). Other exemplary factors may include SB431542 derivatives, such as molecules that include one or more added or different substituents, similar functional groups, etc., and have similar inhibitory effects on one or more SMAD proteins. Suitable factors or combinations of factors can be identified, for example, by contacting high-potential cells with the factor or factors and monitoring early neural progenitor cells. The use of a phenotype, such as characteristic gene expression (including expression of a marker described herein, expression of a reporter gene coupled to an early neural progenitor cell promoter, or the like) or the ability to form a cell type disclosed herein (such as early neural progenitor cells, late neural progenitor cells, retinal neural progenitor cells, photoreceptor progenitor cells, rod progenitor cells, cones, rods and/or photoreceptor rescue cells).

Preferably, the cells are treated or cultured in a rescue induction medium before being cultured with a neural differentiation medium (NDM). NDM can be used to promote further maturation of early neural progenitor cells. In a specific embodiment, the neural differentiation medium is used to promote the differentiation and development of early neural progenitor cells. The neural differentiation medium may contain D-glucose, penicillin, streptomycin, GlutaMAX™, N2 supplement, B27 supplement, MEM non-essential amino acid solution and, if necessary, noggin. The neural differentiation medium can also be used for the differentiation and maturation of early neural progenitor cells with transcriptional markers of excitatory or inhibitory neurons and neurons or "photosensitive rescue cells", but does not contain noggin. In certain embodiments, noggin is not required when PSCs and PSC subpopulations are no longer present in the mature cell product.

The components of neural differentiation medium are as follows: N2: 1% (1 ml N2/100 ml); B27: 2% (2 ml B27/100 ml); and Noggin: 50 ng/ml.

After the cells have all become early neural progenitor cells, noggin is not needed. Stem cells, embryonic stem cells ( ESC ) or adult stem cells or induced enriched potential stem cells ( iPS ):

As used herein, ESCs or adult stem cells or iPS cells can be propagated in a feeder-free system, such as in Matrigel™ (a soluble preparation of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) or another matrix. Additionally or alternatively, the high-potential cells may be cultured on a matrix selected from the group consisting of laminins (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332/laminin-3A32, laminin-3B32, laminin-311/laminin-3A11, laminin-321/laminin-3A21, laminin-411, laminin-421, laminin-511 (e.g., iMatr The matrix may comprise, consist of, or consist essentially of Matrigel™ (a soluble preparation of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells). In some embodiments, the stem cells do not form embryoid bodies in culture, which is an improvement over prior art techniques. In one embodiment, stem cells (e.g., ESCs or iPSCs) are differentiated into light-sensitive rescue cells in the presence of noggin.

In the developing embryo, stem cells give rise to all the specialized embryonic tissues. In the adult organism, stem cells and progenitor cells serve as the body's repair system, replenishing specialized cells and also maintaining the normal turnover of regenerating organs such as blood, skin or intestinal tissue.

Rich-potential stem cells, such as human embryonic stem cells (ESCs) and induced rich-potential stem cells (iPSCs), are able to proliferate for long periods of time in vitro while retaining the potential to differentiate into all cell types of the body, including photorescienct cells. Therefore, these cells can potentially provide an unlimited supply of patient-specific functional photorescienct cells for drug development and transplantation therapy. Differentiation of rich-potential stem cells into photorescienct cells in vitro may involve the addition of different growth factors at different stages of differentiation and may require differentiation for about 10-30 days (see, e.g., FIG. 12 ). Potentially enriched stem cells with unlimited proliferation capacity offer advantages over somatic cells as the starting cell population for generating photorescienct cells.

Potential-rich stem cells, such as embryonic stem (ES) cells or iPS cells, can be the starting material for the disclosed method. In any specific example herein, the potential-rich stem cells can be human potential-rich stem cells (hPSC). Potential-rich stem cells (PSC) can be cultured in any manner known in the art, such as in the presence or absence of feeder cells. In addition, PSCs produced using any method can be used as starting materials for generating photosensitive rescue cells. For example, hES cells can be derived from blastocyst stage embryos, which are the product of in vitro fertilization of eggs and sperm. Alternatively, hES cells can be derived from one or more blastomeres removed from early cleavage stage embryos, without destroying the rest of the embryo as necessary. In yet other embodiments, nuclear transfer can be used to generate hES cells. In another embodiment, iPSCs can be used. As starting material, previously cryopreserved PSCs can be used. In another embodiment, PSCs that have not been cryopreserved can be used.

In one aspect of the present invention, PSCs are seeded on an extracellular matrix with or without feeder cells. In one embodiment, PSCs can be cultured on an extracellular matrix including but not limited to laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the extracellular matrix is laminin with or without epithelial calcineurin. In some embodiments, laminin can be selected from the group consisting of laminin 521, laminin 511, or iMatrix 511. In some embodiments, the feeder cells are human feeder cells, such as human dermal fibroblasts (HDFs). In other embodiments, the feeder cells are mouse embryonic fibroblasts (MEFs).

In some embodiments, the culture medium used when culturing PSCs can be selected from any culture medium suitable for culturing PSCs. In some embodiments, any culture medium capable of supporting PSC culture can be used. For example, a person skilled in the art can select from commercially available or proprietary culture media.

The medium supporting the enriched potential can be any such medium known in the art. In some embodiments, the medium supporting the enriched potential is Nutristem™. In some embodiments, the medium supporting the enriched potential is TeSR™. In some embodiments, the medium supporting the enriched potential is StemFit™. In other embodiments, the medium supporting the enriched potential is Knockout™ DMEM (Gibco), which can be supplemented with Knockout™ serum replacement (Gibco), LIF, bFGF, or any other factor. Each of these exemplary mediums is known and commercially available in the art. In other embodiments, the medium supporting the enriched potential can be supplemented with ROCK inhibitor, bFGF, or any other factor. In one embodiment, bFGF can be supplemented at a low concentration (e.g., 4 ng/mL). In another embodiment, bFGF can be supplemented at a higher concentration (e.g., 100 ng/mL), which can induce PSC differentiation.

The concentration of PSCs used in the production method of the present invention is not particularly limited. For example, when a 10 cm culture dish is used, 1×10 ⁴ to 1×10 ⁸ cells are used per culture dish, preferably 5×10 ⁴ to 5×10 ⁶ cells are used per culture dish, and more preferably 1×10 ⁵ to 1×10 ⁷ cells are used per culture dish. For example, when a CellSTACK® container is used, 1×10 ⁴ to 1×10 ⁸ cells are used per container, preferably 5×10 ⁴ to 5×10 ⁶ cells are used per container, or 1.5×10 ⁶ to 2.2×10 ⁶ cells are used per container, and more preferably 1×10 ⁵ to 1×10 ⁷ cells are used per container. For example, when using flasks (e.g., PDL pre-coated T175 flasks), 1×10 ⁴ to 1×10 ⁸ cells are used per flask, preferably 5×10 ⁴ to 5×10 ⁶ cells are used per flask, or 4.3×10 ⁵ to 6.2×10 ⁶ cells are used per flask, and more preferably 1×10 ⁵ to 1×10 ⁷ cells are used per flask.

In some embodiments, hESCs are expanded using a target seed density of 3×10 ³ and 6×10 ³ cells/cm ^2. In some embodiments, hESCs are preferably expanded using 6-well plates (3×10 ⁴ to 6×10 ⁴ cells per container, such as 2×10 ⁴ to 7×10 ⁴ cells per container) or in T75 flasks (2×10 ⁵ to 5×10 ⁵ cells per container, such as 1×10 ⁵ and 6×10 ⁵ cells per container). In some embodiments, hESCs are expanded using CellSTACK® containers or T175 flasks.

In some embodiments, hESCs are seeded to initiate differentiation into PRCs. In some embodiments, the target seeding density is 3,000 cells/cm ² . This target density is approximately 1.5×10 ⁶ to 2.2×10 ⁶ cells per CellSTACK® container and 4.3×10 ⁵ to 6.2×10 ⁶ cells per T175 flask.

In some embodiments, the PSCs are seeded at a cell density of about 1,000 to 100,000 cells/cm ^2. In some embodiments, the PSCs are seeded at a cell density of about 5000 to 100,000 cells/cm ² , about 5000 to 50,000 cells/cm ² , or about 5000 to 15,000 cells/cm ^2. In other embodiments, the PSCs are seeded at a density of about 10,000 cells/cm ² .

In some embodiments, a culture medium (e.g., StemFit™ or other similar culture medium) supporting rich potential is replaced with a differentiation medium to differentiate cells into photosensitive rescue cells. In some embodiments, the culture medium can be replaced from a culture medium supporting rich potential to a differentiation medium at different time points during the culture of PSC cells, and this can also depend on the initial inoculation density of PSC. In some embodiments, the replacement of the culture medium can be performed after the PSC is cultured in a rich potential medium for 2 to 14 days. In some embodiments, the replacement of the culture medium can be performed on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some specific examples, stem cells suitable for use in the methods described herein include, but are not limited to, embryonic stem cells, induced high-potential stem cells, mesenchymal stem cells, bone marrow-derived stem cells, hematopoietic stem cells, chondrocyte progenitor cells, epidermal stem cells, gastrointestinal stem cells, neural stem cells, hepatic stem cells, adipose-derived mesenchymal stem cells, pancreatic progenitor cells, hair follicle stem cells, endothelial progenitor cells, and smooth muscle progenitor cells.

In some embodiments, stem cells used in the methods described herein are isolated from umbilical cord, placenta, amniotic fluid, villous villi, blastocyst, bone marrow, adipose tissue, brain, peripheral blood, gastrointestinal tract, umbilical cord blood, blood vessels, skeletal muscle, skin, liver, and menstrual blood.

Detailed procedures for isolating human stem cells from various sources are described in Current Protocols in Stem Cell Biology (2007), which is incorporated herein by reference in its entirety. Methods for isolating and culturing stem cells from various sources are also described in U.S. Patents Nos. 5,486,359, 6,991,897, 7,015,037, 7,422,736, 7,410,798, 7,410,773, 7,399,632; each of which is incorporated herein by reference in its entirety. Somatic Cells

In certain aspects of the present invention, transdifferentiation methods may also be provided, that is, directly converting one somatic cell type into another somatic cell, such as obtaining light-sensitive rescue cells from other somatic cells.

However, the supply of human somatic cells may be limited, especially from living donors. In order to provide an unlimited supply of starting cells for light-rescued cell differentiation, somatic cells can be immortalized by introducing immortalizing genes or proteins (such as hTERT and/or other oncogenes). Cell immortalization can be reversible (e.g., using removable expression cassettes) or inducible (e.g., using inducible promoters).

In certain aspects of the present invention, somatic cells may be primary cells (non-immortalized cells), such as cells freshly isolated from an animal, or may be derived from a cell line (immortalized cells). Cells may be maintained in cell culture after being isolated from an individual. In certain embodiments, cells are passaged once or more than once (e.g., 2 to 5, 5 to 10, 10 to 20, 20 to 50, 50 to 100 times or more) before being used in the methods of the present invention. In some embodiments, cells have been passaged no more than 1, 2, 5, 10, 20, or 50 times before being used in the methods of the present invention.

The somatic cells used or described herein may be native somatic cells or engineered somatic cells, i.e., somatic cells that have been genetically altered. The somatic cells of the present invention are typically mammalian cells, such as human cells, primate cells, or mouse cells. They can be obtained by well-known methods and can be obtained from any organ or tissue containing living cells, such as blood, bone marrow, skin, lung, pancreas, liver, stomach, intestines, heart, reproductive organs, bladder, kidney, urethra and other urinary organs.

Mammalian cells suitable for use in the present invention include, but are not limited to, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiac myocytes and other muscle cells.

The methods described herein can be used to program one or more somatic cells (e.g., a population or group of somatic cells) into light-sensitive rescue cells. In some embodiments, the cell population of the present invention is substantially homogeneous in that at least 90% of the cells display the phenotype or characteristic of interest. In some embodiments, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9, 99.95% or more of the cells display the phenotype or characteristic of interest. In certain embodiments of the present invention, the somatic cells have the ability to divide, that is, the somatic cells are not post-mitotic cells.

Somatic cells can be partially or fully differentiated. As described herein, both partially differentiated somatic cells and fully differentiated somatic cells can be differentiated to produce photorescienct rescue cells. Photorescienct rescue cells ( PRCs ):

PRCs can be differentiated from high-potential stem cells (e.g., ESCs or iPSCs lacking Noggin and present in a neural differentiation medium). PRCs are FOXG1(+) and MAP2(+), as determined by flow cytometry. In a specific example, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of PRCs are FOXG1(+) and MAP2(+). PRCs may also be STMN2(+), DCX(+), LINC00461(+), NEUROD2(+), GAD1(+) and/or NFIA(+). PRCs may also be SSEA4(-) and/or OCT4(-), as determined by flow cytometry. The cells can be grown as spheroids or neurospheres (e.g. on low attachment plates or, if desired, in hanging drop cultures, in low gravity environments, aggrewells or other suitable culture conditions).

Schematic diagrams of alternative PRC production methods are shown in Figures 12A and 12B . In some embodiments, hESCs or high-potential cells are expanded prior to PRC differentiation. Expansion of hESCs or high-potential cells can be performed using a culture chamber (e.g., a culture dish, a culture flask, such as an iMatrix-coated T75 culture flask, or a culture container, such as a TC-coated CellSTACK®). In some specific examples, the expansion of hESCs or high-potential cells comprises: thawing the hESCs or high-potential cells on day -10; counting and plating on a culture vessel coated with iMatrix (laminin-511, Matrixome) and culturing in a feeder cell-free condition with StemFit™ medium (Ajinomoto) supplemented with 100 ng/mL bFGF (Peprotech) and 10 µM ROCK inhibitor (Y-27632; Fujifilm/Wako). After four days of daily medium replacement (StemFit+bFGF without ROCK inhibitor), hESCs or high-potential cells were collected using cell dissociation buffer (Gibco) and replated using the above culture conditions.

After another 4 days of culture (day -2), hESCs were collected as described above, the survival rate percentage and live cell count were obtained, and hESCs were seeded at 3,000 cells/ ^cm2 in StemFit+bFGF medium supplemented with ROCK inhibitor (Y-27632) in a culture chamber (e.g., a culture dish, a culture flask, such as an iMatrix-coated T75 culture flask, or a culture container, such as a TC-coated CellSTACK®) for PRC differentiation. After inoculation on day -2, the cultures were fed: (1) on day -1 - StemFit + bFGF (without ROCK inhibitor); (2) on days 0 to 3 (daily) - rescue induction medium (RIM: DMEM/F12 + B27 + N2 + non-essential amino acids (all from Gibco) + glucose (Sigma) + insulin (Akron Biotech) + Noggin (Gibco)); and (3) on days 4 to 19 (every 2 to 3 days) - neural differentiation medium + Noggin (NDM+: neural basal medium + B27 + N2 + non-essential amino acids + glucose + glutamax (Gibco) + Noggin).

On day 19, cultures were "raised" to suspension cultures (2D to 3D) by incubation with a combination of liberase and thermolysin (Roche Custom Labs) and plated onto culture chambers (e.g., culture dishes, culture flasks, such as ultra-low attachment T75 flasks, or culture vessels, such as ultra-low attachment CellSTACK®). 3D cultures were maintained for 3 to 4 days using Noggin-free NDM (NDM-) to allow neural spheroids ("spheres") to form. Then, spheres in suspension were inoculated (3D to 2D) onto poly-D-lysine (Advanced Biomatrix), virus-inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina)-coated culture vessels to initiate passage 0 (P0d0) and cultured in 2D conditions using NDM- (fed every 2 to 3 days) for 14 days (until P0d14).

The 2D to 3D to 2D transition was repeated three times (through P1, P2 and P3, 3 to 4 days of 3D culture and 14 days of 2D culture) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Akuzyme (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. P3 spheres were then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to vapor phase liquid nitrogen for storage. Cryopreserved P3 spheres are referred to as cell stock (CS). In some embodiments, cells in the composition are expanded by growing under culture, attachment conditions and then under low attachment conditions. In some embodiments, the repeated culturing under attachment conditions and then under low attachment conditions is performed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In some embodiments, the cells in the composition are collected after the third repeated culturing under attachment conditions and then under low attachment conditions, after the fourth repeated culturing under attachment conditions and then under low attachment conditions, or after the fifth repeated culturing under attachment conditions and then under low attachment conditions.

In some embodiments, the cells in the composition are collected after the fifth repetition of culturing under attachment conditions and then under low attachment conditions. In some embodiments, the collected cells in the composition are cryopreserved.

In some embodiments, suspension cultures to attached cultures (2D to 3D) are grown in culture dishes, culture bottles, or culture containers. In some embodiments, the culture container is a CellSTACK® container from Corning. In some embodiments, the CellSTACK® is treated with a low attachment or an attachment coating. In one embodiment, the cells are cultured under low attachment conditions and the CellSTACK® is coated with an ultra-low attachment coating. In one embodiment, the cells are cultured under attachment conditions and the CellSTACK® is coated with a TC-treated coating.

Examples of methods for "lifting" cells into suspension include, but are not limited to, those shown in Table 13. Table 13 Time point method 1 Method 2 Method 3 Days Inheritance days 19 Cell scraper Release enzyme + Thermolysin Akubin + ROCK inhibitor 37 P0d14 Cell scraper Release enzyme + Thermolysin Akuzyme 54 P1d14 Cell scraper Release enzyme + Thermolysin Akuzyme 72 P2d14 Cell scraper Release enzyme + Thermolysin Akuzyme 90 P3d14 Akuzyme Akuzyme Akuzyme 107 P4d14 Akuzyme Akuzyme Akuzyme

To produce the composition of the present invention, the CS vials were thawed in a 37°C water bath, resuspended in NDM- and transferred to ultra-low attachment T75 flasks and cultured in 3D suspension for 2 to 3 days, then seeded on poly-D-lysine/fibronectin/laminin-521 coated T75 flasks and cultured in 2D conditions with NDM- for 14 days to complete the 4th passage. At P4d14, cells were harvested using acurase, resuspended in NDM-, wet-triturated and filtered through a 40 µm cell filter to obtain a single cell suspension constituting the PRC composition of the present invention, which can then be formulated with a cryopreservative.

The PRC manufacturing protocol can be modified to allow for scale-up. For example, the flasks used can be changed from T75 flasks to T225 flasks or cell stacks (e.g., Corning® CellSTACK®).

In addition, the PRC manufacturing scheme may include an intermediate low temperature storage step between P0 and P1, between P1 and P2, between P2 and P3, and/or between P3 and P4. In some specific examples, the PRC manufacturing scheme may exclude the intermediate low temperature storage step.

The intermediate cryopreservation step may include 5% DMSO instead of 10% DMSO. In some embodiments, the intermediate cryopreservation step may include about 1% DMSO, about 2% DMSO, about 3% DMSO, about 4% DMSO, about 5% DMSO, about 6% DMSO, about 7% DMSO, about 8% DMSO, about 9% DMSO, about 10% DMSO, about 11% DMSO, about 12% DMSO, about 14% DMSO, or about 15% DMSO.

The intermediate cryopreservation step may include cryopreservation formulations as described herein. The PRC production protocol may include thermolysin and liberase and acurase at D19, followed by overnight incubation with Rock inhibitor. The raised cells or cell clusters may be plated in Aggrewells™ to allow for the development of uniformly sized spheres. On day 19, the cultures were "raised" to suspension cultures (2D to 3D) by incubation with acurase (Innovative Cell Technologies) and plated on ultra-low attachment T75 flasks using NDM (NDM-) medium without Noggin supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako). After 24 hours, the growth medium was replaced with NDM-free Y-27632 and 3D cultures were maintained for another 2 to 3 days using NDM- to allow for the formation of neural spheroids ("spheres"). Spheres in suspension were then seeded (3D to 2D) onto poly-D-lysine (Advanced Biomatrix), virus-inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) coated culture vessels to initiate passage 0 (P0d0) and cultured under 2D conditions using NDM- (fed every 2 to 3 days) for 14 days (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2 and P3, 3-4 days of 3D culture and 14 days of 2D culture) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Akuzyme (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. P3 spheres were then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to vapor phase liquid nitrogen for storage. Cryopreserved P3 spheres are called cell stock (CS).

In some embodiments, the PRC production protocol uses Aggrewells. According to this method, on day 19, the culture is "upgraded" to suspension culture (2D to 3D) by incubation with acurase (Innovative Cell Technologies) and plated on Aggrewell plates (Stemcell Technologies) using NDM (NDM-) medium without Noggin, which is supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako) to allow neural spheres ("spheres") to form. After 24 hours, cells are collected from the Aggrewell plates and transferred to ultra-low attachment T75 flasks, where 3D cultures are maintained for an additional 2 to 3 days using NDM- without Y-27632. Then, spheres in suspension were seeded (3D to 2D) onto poly-D-lysine (Advanced Biomatrix), virus-killed human fibronectin (Akron Biotech), and laminin-521 (Biolamina) coated culture vessels to start passage 0 (P0d0) and cultured in 2D conditions with NDM- (fed every 2 to 3 days) for 14 days (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2, and P3, 3D culture for 3 to 4 days and 2D culture for 14 days) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Akuzyme (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. The P3 spheres are then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to vapor phase liquid nitrogen for storage. The cryopreserved P3 spheres are referred to as cell stock solution (CS). The PRC composition can be pretreated with sucrose before being formulated with a cryoprotectant, for example, at D14 and P4.

Finally, the cryopreservation step of the final PRC preparation may include poloxamer 188 (a nonionic block linear copolymer) and/or sucrose. Extracellular vesicles ( EVs )

The present invention also provides extracellular vesicles secreted from photosensitive rescue cells and their use in methods for treating eye diseases in individuals. Extracellular vesicles secreted from photosensitive rescue cells

The present invention also provides extracellular vesicles isolated, derived, secreted or released from cells (e.g., the photosensitive rescue cells of the present invention).

As used herein, the term "extracellular vesicle" or "EV" refers to a lipid-bound vesicle secreted by a cell into the extracellular space. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, based on their biogenesis, release pathways, size, content, and function (Zaborowski MP et al., Bioscience . 2015; 65: 783-797). In general, the diameter of an extracellular vesicle ranges from 20 nm to 5000 nm, and may contain a variety of macromolecular payloads within the inner space (i.e., lumen), which are presented on the outer surface of the extracellular vesicle and/or span the membrane. The payload may include nucleic acids, such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs), mRNAs, DNA fragments; proteins, carbohydrates, lipids, small molecules, and/or combinations thereof. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived/secreted from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesicled organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived/secreted from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and/or cultured cells.

As used herein, the term "exosome" refers to a cell-derived vesicle that contains a membrane that encloses an internal space (i.e., lumen) and is formed by the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane (Yáñez-Mó M. et al., J. Extracell. Vesicles. 2015;4:27066). Specifically, exosomes are involved in protein sorting, recycling, storage, transport, and release. Exosomes generally have a diameter between 20 and 300 nm. Exosomes are secreted by all cell types and have been found in plasma, urine, semen, saliva, bronchial fluid, cerebrospinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, and gastric acid.

Exosomes have been found to be involved in cell-cell communication, cell maintenance, and tumor progression. In addition, exosomes have been found to stimulate immune responses by acting as antigen-presenting vesicles (Bobrie A. et al., Traffic. 2011 ; 12: 1659-1668). In the nervous system, exosomes have been found to help promote myelination, neurite outgrowth, and neuron survival, thereby playing a role in tissue repair and regeneration (Faure J. et al., Mol. Cell. Neurosci. 2006; 31: 642-648). Meanwhile, exosomes in the central nervous system (CNS) have been found to contain pathogenic proteins such as beta amyloid peptide, superoxide dismutase, and alpha synuclein that may contribute to disease progression (Fevrier B. et al., Proc. Natl. Acad. Sci. USA. 2004;101:9683-9688). Exosomes have also been shown to be carriers of disease markers. Since these vesicles are found in body fluids such as blood and urine, the use of exosomes as carriers of biomarkers is ideal, allowing minimally invasive to non-invasive "liquid biopsy" type approaches to diagnose and even monitor a patient's response to treatment.

In addition to its natural role in cell-cell interactions, exosomes can also carry different cargoes, such as drugs and exogenous nucleic acids or proteins, and deliver such cargoes to different cells. The cargoes can be bound to extracellular vesicles, embedded in extracellular vesicles, encapsulated in extracellular vesicles, or carried out in other ways by extracellular vesicles, or any combination thereof. Therefore, as used herein, reference to a cargo "present" in an extracellular vesicle or its lumen should be understood to include any of the aforementioned ways of carrying the cargo.

The cargo may be an endogenous cargo, an exogenous cargo, or a combination thereof. Examples of cargoes that may be bound, embedded, encapsulated, or otherwise delivered into the extracellular vesicles described herein include, but are not limited to, nucleic acid molecules (e.g., DNA, cDNA, antisense oligonucleotides, mRNA, inhibitory RNA (e.g., antisense RNA, miRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), and agomiRs), antagomiRs, primordial miRNA (pri-miRNA), long noncoding RNA (lncRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and microbial RNA), polypeptides (e.g., enzymes, antibodies), lipids, hormones, vitamins, minerals, small molecules, and drugs, or any combination thereof. Importantly, exosomes are natural carriers of miRNA and other noncoding RNAs, and direct membrane fusion with target cells allows the contents to be delivered directly into the cytosol. This makes the exosome an excellent small molecule delivery system (Lai RC et al., Biotechnol. Adv. 2013;31:543-551).

Minicells are EVs formed by direct outward budding or pinching of the plasma membrane. Minicell diameters typically range from 100 nm to 1000 nm. The pathway of minicell formation is not fully understood, however, it is thought to require cytoskeletal components such as actin and microtubules, as well as molecular motors (kinesin and myosin), and fusion machinery (SNAREs and tethering factors) (Cai H. et al., Dev. Cell. 2007;12:671-682). The number of minicells produced depends on the physiological state and microenvironment of the donor cell (Zaborowski MP et al., Bioscience . 2015;65:783-797). Similarly, it has been previously shown that the number of minicells consumed depends on the physiological state and microenvironment of the recipient cell. Like endosomes, microvesicles participate in cell-cell communication between local cells and distant cells. The ability of these EVs to alter recipient cells has been well documented (Harding CV et al., J. Cell Biol. 2013;200:367-371; White IJ et al., EMBO J. 2006;25:1-12). The uniqueness of EVs is that they can encapsulate active cargo (proteins, nucleic acids and lipids) and deliver them to another cell, either nearby or distant, and through their delivery, alter the function of the recipient cell.

Apoptotic bodies are released by dying cells into the extracellular space. Their diameters are reported to range from 50 nm to 5000 nm, with most tending to be on the larger side (Borges F. et al., Braz. J. Med. Biol. Res. 2013;46:824-830). These apoptotic bodies are formed by the separation of the plasma membrane from the cytoskeleton as a result of increased hydrostatic pressure following cell contraction (Wickman G. et al., Cell Death Differ. 2012;19:735-742). The composition of apoptotic bodies is in direct contrast to exosomes and cysteine formation. Unlike exosomes and microsomes, apoptotic bodies contain intact organelles, chromatin, and a small amount of glycosylated proteins (Borges F. et al., Braz. J. Med. Biol. Res. 2013;46:824-830; Théry C. et al., J. Immunol. 2001;166:7309-7318). Methods for isolating extracellular vesicles

EVs of the present invention can be isolated, secreted, derived or separated from culture medium or other source materials (e.g., photosensitive rescue cells of the present invention) using conventional methods known in the art (see, e.g., Taylor et al., Serum/Plasma Proteomics, Chapter 15, "Extracellular vesicle Isolation for Proteomic Analyses and RNA Profiling", Springer Science, 2011; and Tauro et al., Methods 56 (2012) 293-304 and references cited therein) and as described in the Examples section below. The most commonly used methods include multiple centrifugation and ultracentrifugation steps.

The physical properties of EVs can be used for EV separation, purification or enrichment, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular screening, etc.), density (e.g., conventional or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external forces, etc.). Alternatively or in addition, separation can be based on one or more biological properties, and include methods that can use surface labels (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, immunomagnetic capture of EVs using magnetic beads coated with antibodies (these antibodies are directed against proteins exposed on the EV membrane), etc.).

Methods based on the use of volume-excluding polymers such as PEG have recently been described in a variety of different classes (US Patent Application 20130273544, US Patent Application 20130337440). Two such products are ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Life Technologies, Carlsbad, USA). These polymers act by tethering water molecules and extruding low-soluble components such as extracellular vesicles as well as proteins from the solution, allowing them to be collected by brief low-speed centrifugation.

In some embodiments, separation, purification and enrichment can be performed in a general non-selective manner (typically including continuous centrifugation). Alternatively, separation, purification and enrichment can be performed in a more specific selective manner (e.g., using production cell-specific surface markers). For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, or bead-bound ligands for magnetic separation.

In some embodiments, tangential flow filtration may be used to separate or purify EVs.

In some embodiments, size exclusion chromatography can be used to separate or purify EVs. Size exclusion chromatography techniques are known in the art. In some embodiments, density gradient centrifugation can be used to separate EVs. In some embodiments, the separation of EVs may include ion chromatography, such as anion exchange, cation exchange, or mixed mode chromatography. In some embodiments, the separation of EVs may include desalination, dialysis, tangential flow filtration, ultrafiltration or filtration, or any combination thereof. In some embodiments, the separation of EVs may include a combination of methods, including but not limited to differential centrifugation, size-based membrane filtration, concentration and/or rate-zoned centrifugation. In some embodiments, the isolation of EVs may include one or more centrifugation steps. Centrifugation may be performed at about 50,000 to 150,000 × g. Centrifugation may be performed at about 50,000 × g, 75,000 × g, 100,000 × g, 125,000 × g, or 150,000 × g. In another embodiment, EVs are separated from membrane-free particles by taking advantage of their relatively low buoyant density (Raposo et al., 1996; Escola et al., 1998; van Niel et al., 2003; Wubbolts et al., 2003). Kits for such separations are available from, for example, Qiagen, InVitrogen, and SBI. Methods for EV loading of therapeutic agents are known in the art and include liposome transfection, electroporation, and any standard transfection method. Applications and Uses Screening Assays

The present invention provides methods for screening various agents that can regulate the differentiation of retinal progenitor cells. It can also be used to discover therapeutic agents that support and/or rescue mature photoreceptor cells, which are produced by retinal progenitor cells in culture. For the purposes of the present invention, "agents" are intended to include but are not limited to biological or chemical compounds, such as simple or complex organic or inorganic molecules, peptides, proteins (such as antibodies), polynucleotides (such as antisense) or ribonucleases. A large number of compounds can be synthesized, such as polymers, such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and their analogs. It is understood, although not always explicitly stated, that an agent is used alone or in combination with another agent that has the same or different biological activity as the agent identified by screening according to the present invention.

To practice the in vitro screening method, an isolated cell population can be obtained as described herein. When the agent is a component other than DNA or RNA (such as a small molecule as described above), the agent can be added directly to the cells or added to the culture medium. As will be apparent to those skilled in the art, an "effective" amount must be added, which can be determined empirically. When the agent is a polynucleotide, it can be added directly using a gene gun or electroporation. Alternatively, it can be inserted into the cells using a gene delivery vehicle or other methods as described above. Positive and negative controls can be analyzed to confirm the claimed activity of the drug or other agent. Biocompatible carriers for photosensitive rescue cells

The biocompatible carrier of cells can be a biodegradable polyester film carrier of photosensitive rescue cells. The biodegradable polyester can be any biodegradable polyester suitable for use as a substrate or skeleton. The polyester should be able to form a thin film, preferably a micro-textured film, and if used for tissue or cell transplantation, it should be biodegradable. Biodegradable polyesters suitable for use in the present invention include polylactic acid (PLA), polylactide, polyhydroxyalkanoate, homopolymers and copolymers such as polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyhydroxybutyrate-co-hydroxyhexanoate (PHBHx), polyhydroxybutyrate-co-hydroxyoctanoate (PHBO) and polyhydroxybutyrate-co-hydroxyoctadecanoate (PHBOd), polycaprolactone (PCL), polyesteramide (PEA), aliphatic copolyesters such as polybutylene succinate (PBS) and polybutylene succinate/adipate (PBSA), aromatic copolyesters. High molecular weight and low molecular weight polyesters, substituted and unsubstituted polyesters (block, branched or random), and polyester mixtures and blends can be used. The biodegradable polyester is preferably polycaprolactone (PCL).

In certain embodiments, the biocompatible carrier is a poly(p-xylene) polymer, such as poly(p-xylene) N, poly(p-xylene) D, poly(p-xylene)-C, poly(p-xylene) AF-4, poly(p-xylene) SF, poly(p-xylene) HT, poly(p-xylene) VT-4, and poly(p-xylene) CF, and poly(p-xylene)-C is the most preferred.

The polymer carrier can typically be formed into a thin film using known techniques. The film thickness is advantageously about 1 micron to about 50 microns, preferably about 5 microns thick. The film surface can be smooth, or the film surface can be partially or completely microtextured. Suitable surface textures include, for example, microgrooves or micropillars. The film can be cut and shaped to form a shape suitable for implantation.

Photosensitive rescue cells can be directly seeded on the membrane to form a biocompatible scaffold. Alternatively, the polymer film can be coated with a suitable coating material, such as poly-D-lysine, poly-L-lysine, fibronectin, laminin (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332/laminin-3A32, laminin-3B32, laminin-311/laminin-3A11, laminin-3 21/laminin-3A21, laminin-411, laminin-421, laminin-511 (e.g., iMatrix™-511), laminin-521, laminin-213, laminin-432, laminin-522, laminin-532 and/or laminin fragments), collagen I, collagen IV, vitronectin, and Matrigel™. The cells can be seeded at any desired density, but preferably a PRC cell monolayer (PRC monolayer).

Alternatively, photoreceptor rescue cells (PRCs) can be administered with other cell types, including other retinal cell types, such as but not limited to retinal ganglion cells, retinal ganglion progenitor cells, retinal pigment epithelium (RPE) cells or RPE progenitor cells. Photoreceptor rescue cells (PRCs) can be administered with one or any combination of these different cell types (e.g., corneal endothelial cells). Cells can be administered on a matrix or skeleton or membrane as described above, or they can be administered as cell aggregates, or they can be administered as a suspension of dissociated cells. In some specific examples, cells can be administered on top of a monolayer of RPE cells, which may or may not be located on a matrix or substrate. The cells to be administered may all be derived from in vitro differentiation of hES cells or iPS cells, or in some cases, they may be derived from or obtained from other sources. Some cells may be derived from in vitro differentiation of hES cells or iPS cells, while other cells may be derived from or obtained from other sources. Photosensitive rescue cells are at least derived from in vitro differentiation of enriched potential stem cells (such as hES cells or iPS cells). Any of these various cell combinations may be administered in combination with other therapeutic agents (such as those described herein).

Photoreceptor rescue cells can be administered alone or in combination with another cell type (e.g., retinal pigment epithelial cells) via a device, such as a syringe, Oxulumis-illuminated supracortal microcatheter (Oxular), POD3 Gold (Oxular), Oxuspheres (Oxular), SCS microinjector (Clearside Biomedical), Orbit SDS (Gyroscope); via an implant, via a cell-seeded matrix, or an implantable membrane. Therapeutic Uses

The present invention also provides a method for improving the activity/function of photoreceptor cells in a patient in need of such treatment, comprising administering to a patient a pharmaceutical preparation comprising, derived from, or a combination of a plurality of heterogeneous photoreceptor rescue cells of the present invention. As described herein, the pharmaceutical preparation can be a cell suspension or cells that form a transplantable tissue in a test tube or on a matrix. In many cases, the cells will be administered to the subretinal space or supracordial space of a diseased or degenerated retina. However, because the plurality of heterogeneous photoreceptor rescue cells of the present invention also have a neuroprotective effect, the cells can be administered locally to the retina, but outside the retina (such as into the vitreous), on the choroid, or delivered to other parts of the body via a reservoir or systemically. For example, the photoreceptor rescue cells of the present invention can be administered via a patch or other implantable device, wherein the cell population secretes neuroprotective factors. In one embodiment, such a device can be reloaded with photoreceptor cells for repeated administration to increase the duration of the therapeutic effect.

The pharmaceutical formulations of the present invention are useful for a wide range of diseases and conditions that result in deterioration of the visual system, including retinal degeneration-related diseases. Such diseases and conditions may result from aging, such that damage or disease that may be identified as a substantial source of deterioration appears to be absent. One skilled in the art will appreciate existing methods for diagnosing such disease states and/or detecting known signs of such damage. In addition, the literature is replete with information regarding age-related decline or deterioration of the visual system in animals. The term "retinal degeneration-related disease" is intended to refer to any disease caused by congenital or postnatal retinal degeneration or abnormality. Examples of retinal degeneration-related diseases include retinal dysplasia, retinal degeneration, age-related macular degeneration (wet or dry), AMD secondary to geographic atrophy, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, Leber's congenital amaurosis, Stargardt's disease, retinal detachment, glaucoma, optic neuropathy, and trauma.

Additionally or alternatively, deterioration of visual system components such as the neurosensory retina may result from injury, such as trauma to the visual system itself (e.g., the eye), trauma to the head or brain, or trauma to the body more generally. Certain such injuries are known to be age-related injuries, i.e., their likelihood or frequency increases with age. Examples of such injuries include retinal tears, macular holes, epiretinal membranes, and retinal detachments, each of which may occur in animals of any age, but are more likely to occur or occur with greater frequency in aged animals, including otherwise healthy aged animals.

Deterioration of the visual system or its components can also be caused by disease. Disease includes a variety of age-related disorders that affect the visual system. Such disorders are more likely and/or more frequent in older animals than in younger animals. Examples of diseases that can affect the visual system, including, for example, the retinal nerve sensory layer, and lead to its deterioration are various forms of retinitis, optic neuritis, macular degeneration (wet or dry), map-like atrophy secondary to AMD, proliferative or non-proliferative diabetic retinopathy, diabetic macular edema, progressive retinal atrophy, progressive retinal degeneration, suddenly acquired retinal degeneration, immune-mediated retinopathy, retinal dysplasia, choroidal retinitis, retinal ischemia, retinal hemorrhage (pre-retinal, intra-retinal and/or sub-retinal), hypertensive retinopathy, retinal inflammation, retinal edema, retinoblastoma, or pigmented retinitis.

Some of the aforementioned diseases tend to be specific to certain animals (e.g., companion animals, such as dogs and/or cats). Some of the diseases are listed generally, i.e., there can be many types of retinitis, or retinal hemorrhage; therefore, some diseases are not caused by one specific pathogen, but rather describe more the type of disease or outcome. Many of the diseases that can cause the decline or deterioration of one or more components of the visual system can have both major and minor or greater long-term effects on the animal's visual system.

Advantageously, the pharmaceutical formulations of the present invention can be used to compensate for the lack or reduction of photoreceptor cell function. As described in the embodiments, the cells of the present invention (including photoreceptor rescue cells) can be used as cell replacement therapy in individuals who have completely or partially lost photoreceptor cell function. Such individuals (if human) may have vision characterized by 20/60 or worse, including 20/80 or worse, or 20/100 or worse, or 20/120 or worse, or 20/140 or worse, or 20/160 or worse, or 20/180 or worse, or 20/200 or worse. Therefore, the present disclosure contemplates the treatment of individuals with some levels of visual acuity as well as individuals with indistinguishable visual acuity.

The photoreceptor rescue cells of the present disclosure may be characterized by their ability to reconstruct certain levels of visual acuity in animal models, such as mouse models. In some cases, suitable animal models may be those with visual impairment, where the visual impairment is manifested as an optokinetic response that is 10% or less, 20% or less, 30% or less, 40% or less, or 50% or less of the wild-type response. The optokinetic response can be measured using assays such as those described in the examples. Following transplantation of the photoreceptor rescue cells of the present invention, such optokinetic responses will be increased, preferably by a statistically significant amount, as shown in the examples.

The present disclosure therefore contemplates administering a composition comprising a plurality of heterogeneous photorescrutin cells as described herein for the purpose of completely or partially preventing disease progression or improving the recipient's vision, or some combination thereof. The extent to which either mechanism contributes to the improved outcome will depend on the extent of retinal degeneration in the recipient.

Preferably, the photoreceptor rescue cell composition of the present invention is administered to an individual who has a surplus of functional photoreceptor cells at the time of administration for preservation/protection. Thus, the target patient population is individuals with mid-stage disease. In a particular embodiment, the individual has no substantial loss or nearly complete loss of photoreceptor cells.

Examples of retinal dysfunction that can be treated by the retinal stem cell population and photoreceptor rescue cell composition and method of the present invention include, but are not limited to: partial or complete photoreceptor cell degeneration (such as that occurring in pigmentary retinitis, cone dystrophy, cone-rod and/or rod-cone dystrophy and macular degeneration); retinal detachment and retinal trauma; laser or sunlight-induced photopathies; macular holes; macular edema; night blindness and color blindness. blindness; ischemic retinopathy due to diabetes or vascular occlusion; retinopathy due to precocious/premature birth; infectious conditions such as CMV retinitis and toxoplasmosis; inflammatory conditions such as uveitis; tumors such as retinoblastoma and ocular melanoma; and for replacement of intrinsic retinal neurons affected by ocular neuropathies, including glaucoma, traumatic optic neuropathy, and radiation-induced optic neuropathy and retinopathy.

In one aspect, the photoreceptor rescue cells can treat or alleviate the symptoms of pigmentary retinitis in a patient in need of treatment. In another aspect, the cells can treat or alleviate the symptoms of macular degeneration in a patient in need of such treatment, such as age-related macular degeneration (wet or dry), Stargardt's disease, myopic macular degeneration, or the like. For all of these treatments, the cells can be autologous or allogeneic to the patient. In another aspect, the cells of the invention can be administered in combination with other therapies.

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited eye disorders characterized by progressive vision loss due to gradual degeneration of photoreceptor cells. An estimated 100,000 people in the United States have RP. The classification of these disorders under a red heading is based on the clinical features most commonly observed in these patients. RP is marked by night blindness and decreased peripheral vision, narrowing of the retinal blood vessels, and migration of pigment from the ruptured retinal pigment epithelium into the retina to form clots of various sizes, usually adjacent to the retinal blood vessels.

Typically, patients first report difficulty seeing at night (due to loss of rod photoreceptor cells); the remaining cone photoreceptors then become responsible for the majority of visual function. However, over the years and decades, the cones also degenerate, leading to progressive loss of vision. In most patients with RP, visual field defects begin in the equator of the retina (between 30° and 50° of fixation). The area of defect gradually expands, leaving islands of vision in the periphery and a contracted central visual field (called tunnel vision). When the visual field contracts to 200 or less and/or central vision is 20/200 or worse, patients become legally blind.

The inheritance pattern suggests that RP can be transmitted in an X-linked (XLRP), autosomal dominant (ADRP), or recessive (ARRP) manner. Of the three genetic types of RP, ADRP is the mildest. Such patients usually retain good central vision until age 60 and older. In contrast, patients with the XLRP form of the disease are usually legally blind by age 30 to 40. However, among patients with the same genetic type of RP, the severity of symptoms and the age of onset vary greatly. This variation is evident even within the same family, as all affected members may have the same genetic mutation. Many mutations that induce RP have now been described. Among the genes identified so far, many encode photoreceptor cell-specific proteins, and several are involved in phototransduction in the photoreceptor rods, such as rhodopsin, subunits of cGMP phosphodiesterase, and cGMP-gated Ca2+ channels. Multiple mutations have been found in each selected gene. For example, in the case of the rhodopsin gene, 90 different mutations have been identified in ADRP patients.

Regardless of the specific mutation, the most critical visual loss in RP patients is attributed to the gradual degeneration of cones. In many cases, the protein affected by the RP-causing mutation is not even expressed in cones; the main example is rhodopsin, the rod-specific visual pigment. Therefore, cone loss can be an indirect result of rod-specific mutations. Being able to replace damaged photoreceptor cells offers a way to treat this disease.

In a particular embodiment, the individual is diagnosed with RP, for example by genotyping. Specifically, prior to treatment, the individual is diagnosed with RP based on the identification of mutations affecting RPE genes or photoreceptor cells. In a particular embodiment, the patient's vision is reduced, but not to the extent that they are completely blind or have no light perception (NLP) vision. Preferably, the best corrected visual acuity (BCVA) of individuals suitable for treatment is in the range of 20/50 (reduced vision) to 20/200 (legally blind, but not NLP). In other embodiments, individuals suitable for treatment have vision worse than 20/200, but maintain light perception.

Age-related macular degeneration (AMD) results in progressive loss of central vision and is the most common cause of vision loss in people over 55 years of age. The underlying pathology is degeneration of photoreceptor cells. Several studies have implicated genetic factors, cardiovascular disease, environmental factors (such as smoking and light exposure), and nutritional causes for the risk of developing AMD. RPE degeneration is accompanied by variable loss of perfusion of the overlying photoreceptor cells and underlying choroids. Loss of visual acuity or visual field occurs when the RPE atrophies and results in secondary loss of the overlying photoreceptor cells it supplies. The ability to replace the RPE and/or photoreceptor cells offers a means of treating existing AMD.

AMD is diagnosed by fundus examination and early/intermediate/late AMD is determined based on anatomical landmarks of disease stage observed on fundus images. Intermediate AMD is defined by the presence of at least one large drusen (>125 μm) and/or pigment abnormalities. AMD pigment abnormalities are defined as eyes with hyperpigmentation or hypopigmentation within 2 optic disc diameters of the central macula and drusen ≥63 μm in diameter in the absence of a known retinal disease entity or other cause for such abnormalities. (See García-Layana A, Cabrera-López F, García-Arumí J, Arias-Barquet L, Ruiz-Moreno JM. Early and intermediate age-related macular degeneration: update and clinical review. Clin Interv Aging. 2017 Oct 3;12:1579-1587.) This definition encompasses a wide range of clinical manifestations of intermediate AMD, including only one large drusen (>125 μm) or many large drusen and extensive pigmentary abnormalities, as long as the criteria for geographic atrophy or neovascular AMD (two forms of advanced AMD) are not met.

Macular degeneration is broadly divided into two types. In the exudative-neovascular form or "wet" AMD, which accounts for 10% of all cases, abnormal blood vessel growth occurs under the macula. A network of choroidal neovascularization forms under the retina, often associated with intraretinal hemorrhage, subretinal fluid, pigment epithelial desquamation, and hyperpigmentation. Eventually, this complex shrinks and leaves distinct raised scars at the posterior pole. Fluid and blood in these vessels infiltrate the retina and thereby damage the photoreceptor cells. Wet AMD tends to progress rapidly and can cause severe damage; rapid loss of central vision can occur in only a few months.

The remaining 90% of AMD cases are atrophic macular degeneration (dry form), in which pigment disturbances are present in the macular area rather than in raised macular scars and there are no hemorrhages or discharges in the macular area. In these patients, the retinal pigment epithelium (RPE) gradually disappears, creating a surrounding area of atrophy. Because the loss of photoreceptor cells occurs after the RPE disappears, the affected areas of the retina have little or no visual function. Vision loss from dry AMD occurs more gradually over the course of many years. These patients typically retain some central vision, but the loss is severe enough to impair performance for tasks that require seeing detail.

When appropriate age and clinical findings are accompanied by loss of visual acuity, visual field, or other visual functions, the condition is usually classified as AMD. Sometimes, if the patient has characteristic drusen and a relevant family history, AMD is classified as a step before visual loss occurs.

Occasionally, macular degeneration occurs at a much earlier age. Many of these cases are caused by genetic mutations. There are several forms of inherited macular degeneration, each with its own clinical presentation and genetic causes. The most common form of juvenile macular degeneration is called Stargardt's disease, which is inherited in a somatic recessive manner. Patients are usually diagnosed before the age of 20. Although the progression of vision loss can vary, most of these patients are legally blind by the age of 50. Mutations that cause Stargardt's disease have been identified in the ABCR gene, which encodes a protein that transports retinoids across the membranes of photoreceptor cells.

The photosensitive rescue cell composition of the present invention can be used to treat degenerative diseases. The cells are administered in a manner that allows the cells to transplant or migrate to a predetermined retinal site (such as the outer nuclear layer) and reconstruct or regenerate the functionally deficient area.

The examples demonstrate that the photoreceptor rescue cells disclosed herein can regenerate visual acuity in a mouse blindness model caused by photoreceptor cell degeneration. The visual acuity of such mouse models can be assessed using optokinetic response (or optokinetic nystagmus response).

In another aspect, the present invention provides a method of drug delivery, comprising administering to the patient a photosensitive rescue cell composition described herein or produced by any of the methods described herein, wherein the photosensitive rescue cell composition delivers the drug. A wide range of drugs can be used. The engineered photosensitive rescue cell composition can be prepared so that it includes one or more compounds selected from the group consisting of: drugs that act on synapses and neural receptor junctions; drugs that act on the central nervous system; drugs that modulate inflammatory responses, such as anti-inflammatory agents, including non-steroidal anti-inflammatory agents; drugs that affect renal and/or cardiovascular function; drugs that affect gastrointestinal function; antibiotics; antiviral agents, anti-obesity agents and anti-cancer agents; immunomodulators; anesthetics, steroids; Alcoholic drugs, antigens, vaccines, antibodies, decongestants, antihypertensives, sedatives, birth control agents, gestational agents, anticholine stimulants, analgesics, antidepressants, antipsychotics, beta-adrenaline stimulant blocking agents, diuretics, cardiovascular active agents, vasoactive agents, nutrients, drugs acting on the blood and/or hematopoietic organs; hormones; hormone antagonists; drugs affecting calcification and bone turnover, vitamins, gene therapy agents; or other drugs, such as targeted agents, etc.

For example, the light-sensitive rescue cell composition can be prepared so that it includes one or more compounds selected from the group consisting of drugs that act on synapses and neural receptor junctions (e.g., acetylcholine, methacholine, pilocarpine, atropine, scopolamine, physostigmine, succinylcholine, epinephrine, norepinephrine, dopamine, dobutamine, isoproterenol, , albuterol, propranolol, serotonin; drugs that act on the central nervous system (e.g., clonazepam, diazepam, lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, ropivacaine, amitriptyline, fluoxetine, paroxetine, valproic acid); acid, carbamazepine, bromocriptine, morphine, fentanyl, naltrexone, naloxone; drugs that modulate inflammatory responses (e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, sodium cromolyn, theophylline); drugs that affect renal and/or cardiovascular function (e.g., furosemide, thiazide, amiloride, spironolactone, captopril, enalapril, lisinopril); pril, diltiazem, nifedipine, verapamil, digoxin, isordil, dobutamine, lidocaine, quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin, mevalonate; drugs that affect gastrointestinal function (e.g. omeprazole, sucralfate); antibiotics (e.g. tetracycline, clindamycin, amphotericin B); B), quinine, methicillin, vancomycin, penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline, acyclovir, zidovudine (AZT), ddC, ddI, ribavirin, cefaclor, cephalexin, streptomycin, gentamicin, tobramycin, chloram phenicol), isoniazid, fluconazole, amantadine, interferon; anticancer agents (e.g. cyclophosphamide, methotrexate, fluorouracil, cytarabine, mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C); C), hydroxyurea, prednisone, tamoxifen, cisplatin, decarbazine; immunomodulators (e.g., interleukins, interferons, GM-CSF, TNFα, TNFβ, cyclosporine, FK506, azathioprine, steroids); drugs acting on the blood and/or hematopoietic organs (e.g., interleukins, G-CSF, GM-CSF, erythropoietin, vitamins, iron, copper, vitamin B12, folic acid, heparin, warfarin, coumarin); hormones (e.g., Such as growth hormone (GH), prolactin, ovulation hormone, TSH, ACTH, insulin, FSH, CG, somatostatin, estrogen, androgen, progesterone, gonadotropin-releasing hormone (GnRH), thyroxine, triiodothyronine); hormone antagonists; drugs that affect calcification and bone turnover (such as calcium, phosphate, parathyroid hormone (PTH), vitamins D, bisphosphonates, calcitonin, fluoride), vitamins (e.g., riboflavin, niacin, pyridoxine, pantothenic acid, biotin, choline, inositol, carnitine, vitamin C, vitamin A, vitamin E, vitamin K), gene therapy agents (e.g., viral vectors, liposomes carrying nucleic acids, DNA-protein conjugates, antisense agents); or other agents, such as targeting agents, etc. The light-sensitive rescue cell composition of the present invention can be engineered to include one or more therapeutic agents, which are released or secreted by the cells in a passive manner (diffusion from the cells over time) or an active manner (after the cells are intentionally ruptured or lysed). hESCs and/or hiPSCs can be genetically modified and used to generate photoreceptor rescue cells (PRCs) that express a desired agent for treating a disease. In one aspect, hESCs, hiPSCs, and/or multilymphoid progenitor cells (MLPs) can be genetically modified to express anti-tumor agents. Photoreceptor rescue cells (PRCs) generated from such genetically modified hESCs and hiPSCs, adult stem cells, and multilymphoid progenitor cells (MLPs) can be used to deliver such anti-tumor agents to tumors for the treatment of metastatic diseases, including, for example, retinoblastoma.

In some embodiments, the light-sensitive rescue cells of the present invention are generated by increasing the expression of one or more transcription factors selected from the group consisting of: PAX6, FOXG1, HMGA1/2, OTX2, ASCL1, POU3F2, NR2F1/2, NR6A1, MEIS2, NEUROD6, HES5, ATF4/5 and RXRG. In one embodiment, the light-sensitive rescue cells are generated by increasing the expression of one or more transcription factors selected from the group consisting of: PAX6, FOXG1, HMGA1/2, OTX2, ASCL1, POU3F2, NR2F1/2, NR6A1, MEIS2, NEUROD6, HES5, ATF4/5 and RXRG in enriched potential stem cells, early neural progenitor cells or late neural progenitor cells.

In certain embodiments, photorescive rescue cells (PRCs) have been engineered to include one or more therapeutic agents, such as small molecule drugs, aptamers or other nucleic acid agents, or recombinant proteins.

Genetically engineered light-sensitive rescue cells can also be used to target gene products to sites of degeneration. Such gene products may include survival-promoting factors that rescue native degenerating neurons, factors that can be used in an autocrine manner to promote the survival and differentiation of transplanted cells into site-specific neurons, or to deliver neurotransmitters to allow functional restoration. Ex vivo gene therapy, such as recombinant engineering of high-potential stem cells, progenitor cells, early neural progenitor cells, late neural progenitor cells, or photoreceptor rescue cells, can be effectively used as a neuroprotective strategy to prevent retinal cell loss in RP (retinitis pigmentosa), AMD, and glaucoma and diseases that cause retinal detachment by delivering growth factors and neurotrophic factors, such as FGF2, NGF, ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF), which have been shown to significantly slow the cell death process in models of retinal degeneration. Therapy using PRCs engineered to synthesize growth factors or combinations of growth factors could not only ensure sustained delivery of neuroprotectants but also rebuild the damaged retina.

The photosensitive rescue cells of the present invention can be administered in combination with one or more other therapeutic agents. As used herein, the phrases "co-administration" and "co-administration" refer to any form of administration of two or more different therapeutic entities in combination, so that the previously administered therapeutic agent (such as cells) is still effective in the body while the second agent is administered (for example, two therapeutic agents are effective in the patient's body at the same time, which may include a synergistic effect of the two agents). For example, different therapeutic agents can be administered using the same formulation, in which the cells are amenable to co-formulation; or they can be administered concomitantly or sequentially using separate formulations. Therefore, individuals receiving such treatments can benefit from the combination of transplanted cells and one or more different therapeutic agents.

One or more angiogenesis inhibitors may be administered in combination (i.e., in conjunction) with the cell preparation, preferably in a therapeutically effective amount for the prevention or treatment of ocular diseases (e.g., angiogenesis-related ocular diseases). Exemplary ocular diseases include macular degeneration (e.g., wet AMD or dry AMD), diabetic retinopathy, and choroidal neovascularization. Exemplary angiogenesis inhibitors include VEGF antagonists, such as inhibitors of VEGF and/or VEGF receptors (VEGFR, such as VEGFR1 (FLT1, FLT), VEGFR2 (KDR, FLK1, VEGFR, CD309), VEGFR3 (FLT4, PCL)), such as peptides, peptide mimetics, small molecules, chemicals, or nucleic acids, such as pegaptanib sodium ( sodium), aflibercept, bevasiranib, rapamycin, AGN-745, vitalanib, pazopanib, NT-502, NT-503 or PLG101, CPD791 (a di-Fab' polyethylene glycol (PEG) conjugate that inhibits VEGFR-2), an anti-VEGF antibody or a functional fragment thereof (such as bevacizumab (AVASTIN®) or ranibizumab (LUCENTIS®)), or an anti-VEGF receptor antibody (such as IMC-1121(B) (a monoclonal antibody against VEGFR-2), or IMC-18F1 (an antibody against the extracellular binding domain of VEGFR-1)). Other exemplary inhibitors of VEGF activity include fragments or domains of VEGFR receptors, examples of which are VEGF traps (Aflibercept), fusion proteins of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 with the Fc fragment of IgG1. Another exemplary VEGFR inhibitor is AZD-2171 (Cediranib), which inhibits VEGF receptors 1 and 2. Other exemplary VEGF antagonists include tyrosine kinase inhibitors (TKIs), including TKIs reported to inhibit VEGFR-1 and/or VEGFR-2, such as Sorafenib (Nexavar), SU5416 (Semaxinib), SU11248/Sunitinib (Sutent), and Vandetanib (ZD 6474). Other exemplary VEGF antagonists include Ly317615 (Enzastaurin), which is believed to target downstream kinases (protein kinase C) involved in VEGFR signaling. Other exemplary angiogenesis inhibitors include inhibitors of α5β1 integrin activity, including anti-α5β1 integrin antibodies or functional fragments thereof (such as volociximab); peptides, peptide mimetics, small molecules, chemicals or nucleic acids, such as 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetamido)-2-(alkyl-amino)-propionic acid, (S)-2- [(2,4,6-Trimethylphenyl)sulfonyl]amino-3-[7-benzyloxycarbonyl-8-(2-pyridylaminomethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-en-3-yl]carbonylaminopropionic acid, EMD478761, or RC*D(ThioP)C* (Arg-Cys-Asp-thioproline-Cys; asterisk indicates cyclization via a cysteine residue through a disulfide bond). Other exemplary angiogenesis inhibitors include 2-methoxyestradiol, αVβ3 inhibitor, angiopoietin 2, angiogenesis inhibitory steroids and heparin, angiostatin, angiostatin-related molecules, anti-α5β1 integrin antibodies, anti-histatin S antibodies, antithrombin III fragments, bevacizumab, calreticulin, canstatin, carboxyamidotriazole, cartilage-derived angiogenesis inhibitor, CDAI, CM1 01. CXCL10, endostatin, IFN-α, IFN-β, IFN-γ, IL-12, IL-18, IL-4, linomide, maspin, matrix metalloproteinase inhibitors, Meth-1, Meth-2, osteopontin, pegaptanib, platelet factor-4, prolactin, prothrombin-related proteins, prothrombin (kringle domain-2) domain-2), ranibizumab, restin, soluble NRP-1, soluble VEGFR-1, SPARC, SU5416, suramin, tecogalan, tetrathioate, thalidomide, lenalidomide, thrombospondin, TIMP, TNP-470, TSP-1, TSP-2, angiostatin, VEGFR antagonist, VEGI, volociximab (also known as M200), fiber binding protein fragments such as anastellin (see Yi and Ruoslahti, Proc Natl Acad Sci USA. 2001 Jan 16;98(2):620-4), or any combination thereof. The amount of the angiogenesis inhibitor is preferably sufficient to prevent or treat proliferative (neovascular) eye diseases, such as choroidal neovascular membrane (CNV) associated with wet AMD and other diseases of the retina. Other exemplary angiogenesis inhibitors include: Lenvatinib (E7080), Motesanib (AMG 706), Pazopanib (Votrient), and IL-6 antagonists, such as anti-IL-6 antibodies. Other exemplary angiogenesis inhibitors include fragments, mimetics, chimeras, fusions, analogs and/or domains of any of the foregoing. Other exemplary angiogenesis inhibitors include combinations of any of the foregoing. In an exemplary embodiment, the photosensitive rescue cell composition comprises an anti-VEGF antibody, such as bevacizumab, such as between about 0.1 mg and about 6.0 mg (e.g., between about 1.25 mg and about 2.5 mg) of bevacizumab per injection into the eye. In other exemplary embodiments, the photosensitive rescue cell composition comprises one or more VEGF activity inhibitors and one or more α5β1 integrin activity inhibitors.

One or more anti-inflammatory agents may be administered in combination with the photosensitive rescue cell composition. Exemplary anti-inflammatory agents include: glucocorticoids, nonsteroidal anti-inflammatory drugs, aspirin, ibuprofen, naproxen, cyclooxygenase (COX) enzyme inhibitors, aldosterone, beclometasone, betamethasone, corticosteroids, cortisol, corticosterone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, fluocinolone acetonide, fluocinol ... acetonide (e.g. ILUVIEN®), glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone.

One or more antioxidants, antioxidant cofactors and/or other factors that promote increased antioxidant activity may be administered in combination with the photosensitive rescue cell composition, examples of which may include OT-551 (Othera), vitamin C, vitamin E, beta-carotene, zinc (e.g., zinc oxide) and/or copper (e.g., copper oxide).

One or more macular xanthophyll (such as lutein and/or zeaxanthin) may be administered in combination with the photorescive cell composition.

One or more long-chain omega-3 fatty acids, such as docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA), may be administered in combination with the light-sensitive rescue cell composition.

One or more amyloid inhibitors, such as fenretinide, Arc-1905, Copaxone (glatiramer acetate, Teva), RN6G (PF-4382923, Pfizer) (humanized monoclonal antibodies against ABeta40 and ABeta42), GSK933776 (GlaxoSmithKline) (anti-amyloid antibody), can be administered in combination with the photorescue cell composition.

One or more ciliary neurotrophic factor (CNTF) agonists (e.g., CNTF deliverable using an intraocular device, such as NT-501 (Neurotech)) can be administered in combination with the photoreceptor rescue cell composition.

One or more inhibitors of RPE65, such as ACU-4429 (Aculea), can be administered in combination with the photorescive cell composition.

One or more agents that target A2E and/or lipofuscin accumulation (such as fenretinide and ACU-4429) can be administered in combination with the light-sensitive rescue cell composition.

One or more downregulators or inhibitors of photoreceptor cell function and/or metabolism, such as fenretinide and ACU-4429, can be administered in combination with the photoreceptor rescue cell composition.

One or more α2-adrenaline agonists, such as brimonidine tartrate, can be administered in combination with the photosensitive rescue cell composition.

One or more selective serotonin 1A agonists, such as Tandospirone (AL-8309B), can be administered in combination with the photorescive cell composition.

One or more factors that target C-5, the membrane attack complex (C5b-9), and/or any other drusen component may be administered in combination with the photorescue cell composition, examples of which include complement factor D, inhibitors of C-3, C-3a, C5 and C5a, and/or factor H agonists, such as ARC 1905 (Ophthotec) (an anti-C5 aptamer that selectively inhibits C5), POT-4 (Potentia) (a compstatin derivative that inhibits C3), complement factor H, Eculizumab (Soliris, Alexion) (a humanized IgG antibody that inhibits C5), and/or FCFD4514S (Genentech, San Francisco) (a monoclonal antibody against complement factor D).

One or more immunosuppressive agents, such as Sirolimus (rapamycin), can be administered in combination with the photosensitive rescue cell composition.

One or more agents for preventing or treating lipofuscin accumulation, such as piracetam, centrophenoxine, acetyl-L-carnitine, Ginkgo Biloba or an extract or preparation thereof, and/or DMAE (dimethylethanolamine), may be administered in combination with the photosensitive rescue cell composition.

In the case of one or more agents (e.g., angiogenesis inhibitors, antioxidants, antioxidant cofactors, other factors that contribute to increased antioxidant activity, macular lutein, long-chain omega-3 fatty acids, amylin inhibitors, CNTF agonists, RPE65 inhibitors, factors that target A2E and/or lipofuscin accumulation, downregulators or inhibitors of photoreceptor function and/or metabolism, alpha2-adrenaline stimulating receptor agonists, selective serotonin 1A agonists, factors that target C-5, the attack complex (C5b-9) and/or any other component of drusen, immunosuppressants, preventive or An agent for treating lipofuscin accumulation is administered in combination with a photosensitive rescue cell composition, and the agent can be administered concurrently with, before, and/or after the photosensitive rescue cell composition. For example, the agent can be administered to the patient's eye during a procedure in which the photosensitive rescue cell composition is introduced into the patient's eye. Administration of the agent can begin before the cells are administered to the patient's eye and/or continue after the cells are administered to the patient's eye. For example, the agent can be provided in a solution, a suspension, a sustained release form, and/or in a sustained delivery system (e.g., Allergan Novadur™ delivery system, NT-501, or another intraocular device or sustained release system).

In certain embodiments, cells can be engineered to include recombinant expression constructs that, when expressed by cells in vivo, produce recombinant forms of the agents described herein. In the case of antibodies, this includes bi-chain monoclonal antibodies, as well as antigen-determining fragments thereof, such as Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and fragments comprising VL or VH domains, as well as fibronectin scaffolds and other antibody CDR mimetics. Engineered cells may include expression constructs encoding recombinant peptides and proteins, as well as constructs that, when transcribed, form transcripts that produce RNA interferers (such as siRNA, hairpin RNA, or the like), aptamers, baits (which bind to transcription factors and inhibit native gene expression), antisense strands, or the like. Recombinant genes may be operably linked to transcriptional regulatory elements, such as promoters and/or enhancers, that are active in the transplanted cells (such as constitutively active or photoreceptor cell active elements) or that are regulated by small molecules.

Exemplary recombinant agents expressed by the transplanted cells include anti-angiogenic agents, such as those that reduce the development of choroidal neovascularization (wet AMD). These include agents that inhibit VEGF-mediated ocular angiogenesis, such as anti-VEGF antibodies and VEGF receptor traps. Such proteins include antibodies and antibody analogs (such as single chain antibodies, monofunctional antibodies, antigen binding sites and their analogs), such as ranibizumab, VEGF traps, such as Aflibercept, which is a soluble protein that includes the ligand binding domain of the VEGF receptor, which binds to VEGF or VEGF receptor and blocks receptor activation.

For some patients, activation of the alternative complement pathway is implicated in disease progression, particularly in the case of dry AMD. Another class of exemplary recombinant agents expressed by the transplanted cells includes complement inhibitors, such as complement factor D, factor C5, and/or factor C3 inhibitors. For illustration purposes only, these may be RNA agents or recombinant antibodies.

Drusen deposits in dry AMD resemble amyloid deposits. Accordingly, transplanted cells can be engineered to express anti-β-amyloid agents. These include recombinant antibodies, β-secretase inhibitors, and their analogs.

The transplanted cells may also be engineered to express one or more anti-inflammatory agents, such as pro-inflammatory interleukins (such as IL-1, IL-2, IL-3 and TNF-α) or antagonists/inhibitors of anti-inflammatory interleukins (such as IL-37). Antagonists/inhibitors of pro-inflammatory interleukins include recombinant antibodies, receptor traps, aptamers, etc. In one specific example, the transplanted cells may be engineered to express recombinant lipocortin, a potent anti-inflammatory protein.

In the method of the present invention, the cells to be transplanted are transferred to the recipient in any physiologically acceptable formulation (including isotonic formulations prepared under sufficient sterile conditions for administration to humans). For general principles of pharmaceutical formulations, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996. The choice of cell formulation and any accompanying components in the composition will be adjusted according to the route and device used for administration. The cells can be introduced by injection, catheter or the like. Alternatively, the cells can be frozen at liquid nitrogen temperature and stored for a longer period of time, which can be used when thawed. If frozen, cells are typically stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

The pharmaceutical preparations of the present invention are packaged in suitable containers together with written instructions as needed for the desired purpose. Such formulations may include a mixture of retinal differentiation factors and/or nutritional factors in a form suitable for combination with PRC. Such compositions may further include a suitable buffer and/or excipient suitable for transfer into animals. Such compositions may further include cells to be transplanted. Pharmaceutical preparations

PRC can be formulated with a pharmaceutically acceptable carrier. For example, PRC can be administered alone or as a component of a pharmaceutical formulation. The compounds of the present invention can be formulated in any convenient way for administration. Pharmaceutical preparations suitable for administration may include a combination of PRC and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., balanced salt solutions (BSS)), dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes or suspending agents or thickening agents. An exemplary pharmaceutical formulation comprises PRC in combination with ALCON® BSS PLUS® (a balanced salt solution containing per mL in water: 7.14 mg sodium chloride, 0.38 mg potassium chloride, 0.154 mg calcium chloride dihydrate, 0.2 mg magnesium chloride hexahydrate, 0.42 mg sodium hydrogen phosphate, 2.1 mg sodium bicarbonate, 0.92 mg dextrose, 0.184 mg glutathione disulfide (oxidized glutathione), hydrochloric acid and/or sodium hydroxide (to adjust the pH to about 7.4)).

When administered, the pharmaceutical preparations used in the present disclosure may be in a pyrogen-free, physiologically acceptable form.

The preparation containing PRC used in the method described herein can be transplanted in a suspension, gel, colloid, slurry or mixture. In addition, the preparation can be appropriately encapsulated in the vitreous humor or injected into the vitreous humor in a viscous form for delivery to the retinal or choroidal injury site. In addition, at the time of injection, the cryopreserved PRC can be resuspended in a commercially available balanced salt solution to achieve the desired osmotic pressure and concentration, thereby being administered by subretinal injection. The preparation can be administered to the central peri-macular area that has not yet been completely diseased, which can promote the attachment and/or survival of the administered cells.

PRCs can be frozen (cryopreserved) as described herein. After thawing, such cells can have a viability of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% of the cells collected after thawing are alive, or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% of the number of cells initially frozen are collected in a viable state after thawing). In some cases, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% of the cells in the composition are alive before and after cryopreservation and thawing. In some cases, after thawing, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85% of the cells in the composition are alive. In some cases, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about From about 60% to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, from about 60% to about 65%, from about 65% to about 85%, from about 65% to about 80%, from about 65% to about 75%, from about 65% to about 70%, from about 70% to about 85%, from about 70% to about 80%, from about 70% to about 75%, from about 75% to about 85%, from about 75% to about 80%, or from about 80% to about 85%. In some cases, after cryopreservation and thawing, at least about 50% of the cells in the composition are alive. In some cases, after cryopreservation and thawing, at least about 55% of the cells in the composition are alive. In some cases, at least about 60% of the cells in the composition are alive after cryopreservation and thawing. In some cases, at least about 65% of the cells in the composition are alive after cryopreservation and thawing. In some cases, at least about 70% of the cells in the composition are alive after cryopreservation and thawing. In some cases, at least about 75% of the cells in the composition are alive after cryopreservation and thawing. In some cases, the cell viability is about 80% before and after thawing. In some cases, at least 90% or at least 95% or about 95% of the frozen cells are recovered. The cells can be frozen as single cells or as aggregates. For example, cells can be frozen as neurospheres.

The PRC of the present disclosure can be delivered by intraocular injection in a pharmaceutically acceptable ophthalmic formulation. For example, when the formulation is administered by intravitreal injection, the solution can be concentrated so that the minimum volume can be delivered. The concentration used for injection can be any amount that is effective and non-toxic, depending on the factors described herein. The pharmaceutical preparation of PRC for treating a patient can be formulated at a dose of at least about 10 ⁴ cells/ml. The PRC preparation for treating a patient is formulated at a dose of at least about 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ PRC cells/ml. For example, the photosensitive rescue cells can be formulated in a pharmaceutically acceptable carrier or formulation.

The pharmaceutical preparations of PRC described herein may contain at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000 PRC. The pharmaceutical preparation of PRC may comprise at least about 1×10 ⁴ , at least about 2×10 ⁴ , at least about 3×10 ⁴ , at least about 4×10 ⁴ , at least about 5×10 ⁴ , at least about 6×10 ⁴ , at least about 7×10 ⁴ , at least about 8×10 ⁴ , at least about 9×10 ⁴ , at least about 1×10 ⁵ , at least about 2×10 ⁵ , at least about 3×10 ⁵ , at least about 4×10 ⁵ , at least about 5×10 ⁵ , at least about 6×10 ⁵ , at least about 7×10 ⁵ , at least about 8×10 ⁵ , at least about 9×10 ⁵ , at least about 1×10 ⁶ , at least about 2×10 ⁶ , at least about 3×10 ⁶ , at least about 4×10 ⁶ , at least about 5×10 ⁶ , at least about 6×10 ⁶ , at least about 7×10 ⁶ , at least about 8×10 ⁶ , at least about 9×10 ⁶ , at least about 1×10 ⁷ , at least about 2×10 ⁷ , at least about 3×10 ⁷ , at least about 4×10 ⁷ , at least about 5×10 ⁷ , at least about 6×10 ⁷ , at least about 7×10 ⁷ , at least about 8×10 ⁷ , at least about 9×10 ⁷ , at least about 1×10 ⁸ , at least about 2×10 ⁸ , at least about 3×10 ⁸ , at least about 4×10 ⁸ , at least about 5×10 ⁸ , at least about 6×10 ⁸ , at least about 7×10 ⁸ , at least about 8×10 ⁸ , at least about 9×10 ⁸ , at least about 1×10 ^{9 , at least about 2×10 9 ,} at least about 3×10 ⁹ , at least about 4×10 ⁹ , at least about 5×10 ⁹ , at least about 6×10 ⁹ , at least about 7×10 ⁹ , at least about 8×10 ⁹ , at least about 9×10 ⁹ , at least about 1×10 ¹⁰ , at least about 2×10 ¹⁰ , at least about 3×10 ¹⁰ , at least about 4×10 ¹⁰ , at least about 5×10 ¹⁰ , at least about 6×10 ¹⁰ , at least about 7×10 ¹⁰ , at least about 8×10 ¹⁰ , or at least about 9×10 ¹⁰ PRCs. The pharmaceutical preparation of PRCs may contain at least about 1×10 ² to about 1×10 ³ , about 1×10 ² to about 1×10 ⁴ , about 1×10 ⁴ to about 1×10 ⁵ , or about 1×10 ³ to about 1×10 ⁶ PRCs. The pharmaceutical preparation of PRCs may contain at least about 10,000, at least about 20,000, at least about 25,000, at least about 50,000, at least about 75,000, at least about 100,000, at least about 125,000, at least about 150,000, at least about 175,000, at least about 180,000, at least about 185,000, at least about 190,000, or at least about 200,000 PRCs. For example, the pharmaceutical preparation of PRCs may contain at least about 20,000 to about 200,000 PRCs in a volume of at least about 50 to about 200 μL. Additionally, the pharmaceutical formulation of PRCs may contain about 50,000 PRCs in a volume of about 150 μL, about 200,000 PRCs in a volume of about 150 μL, or at least about 180,000 PRCs in a volume of at least about 150 μL.

In the aforementioned pharmaceutical preparations and compositions, the number of PRCs or the concentration of PRCs can be determined by counting live cells and excluding non-live cells. For example, non-live PRCs can be detected by not excluding key dyes (such as conycin blue) or using functional assays (such as the ability to adhere to the culture medium, phagocytosis, etc.). In addition, the number of PRCs or the concentration of PRCs can be determined by counting cells expressing one or more PRC markers and/or excluding cells expressing one or more markers, which indicate cell types other than PRCs.

The PRC can be formulated for delivery in a pharmaceutically acceptable ophthalmic vehicle so as to maintain the formulation in contact with the ocular surface for a sufficient period of time to allow cellular penetration into the affected area of the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary body, lens, choroid, retina, sclera, supracortial space, conjunctiva, subconjunctival space, suprascleral space, intracorneal space, corneal epithelial space, pars plana, surgically induced avascular area, or macula.

PRCs may be contained within the cell lamellae. For example, a cell sheet comprising PRCs can be prepared by culturing PRCs on a substrate from which the intact cell sheet can be peeled off, such as a surface grafted with a thermoresponsive polymer such as thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm), followed by attachment and proliferation of the cells at the culture temperature, and then upon temperature shift, changes in surface characteristics causing the cultured cell sheet to detach (e.g., by cooling to below the lower critical solution temperature (LCST) (see da Silva et al., Trends Biotechnol. 2007 Dec; 25(12):577-83; Hsiue et al., Transplantation. 2006 Feb 15; 81(3):473-6; Ide, T. et al. (2006); Biomaterials 27, 607-614, Sumide, T. et al. (2005), FASEB J. 20, 392-394; Nishida, K. et al. (2004), Transplantation 77, 379-385; and Nishida, K. et al. (2004), N. Engl. J. Med. 351, 1187-1196, each of which is incorporated herein by reference in its entirety). The cell sheet can be attached to a substrate suitable for transplantation, such as a substrate that dissolves in vivo when the sheet is transplanted into a host organism, for example, by culturing cells on a substrate suitable for transplantation or releasing cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. Exemplary substrates may include gelatin (see Hsiue et al., supra). Alternative substrates that may be suitable for transplantation include fibrin-based matrices and other matrices. The cell sheets may be used to manufacture medicaments for the prevention or treatment of retinal degenerative diseases. The PRC sheets may be formulated for introduction into the eye of an individual in need thereof. For example, the cell sheets may be introduced into an eye in need thereof by subfoveal membranectomy, by transplantation of the PRC cell sheets, or may be used to manufacture medicaments for transplantation following subfoveal membranectomy.

The volume of the preparation administered according to the methods described herein also depends on factors such as the mode of administration, the number of PRCs, the age and weight of the patient, and the type and severity of the disease being treated. If administered by injection, the volume of the pharmaceutical preparation of the PRCs of the present disclosure may be at least about 1 mL, about 1.5 mL, about 2 mL, about 2.5 mL, about 3 mL, about 4 mL, or 5 mL. The volume may be at least about 1 mL to about 2 mL. For example, if administered by injection, the volume of the pharmaceutical preparation of PRC of the present disclosure may be at least about 1 μL, about 2 μL, about 3 μL, about 4 μL, about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL, about 10 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about 15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL, about 20 μL, about 21 μL, about 22 μL, about 23 μL, about 24 μL, about 25 μL, about 26 μL, about 27 μL, about 28 μL, about 29 μL, about 30 μL, about 31 μL, about 32 μL, about 33 μL, about 34 μL, about 35 μL, about 36 μL, about 37 μL, about 38 μL, about 39 μL, about 40 μL, about 41 μL, about 42 μL, about 43 μL, about 44 μL, about 45 μL, about 46 μL, about 47 μL, about 48 μL, about 49 μL, about 50 μL, about 51 μL, about 52 μL, about 53 μL, about 54 μL, about 55 μL, about 56 μL, about 57 μL, about 58 μL, about 59 μL, about 60 μL, about 61 μL, about 62 μL, about 63 μL, about 64 μL μL, about 38 μL, about 39 μL, about 40 μL, about 41 μL, about 42 μL, about 43 μL, about 44 μL, about 45 μL, about 46 μL, about 47 μL, about 48 μL, about 49 μL, about 50 μL, about 51 μL, about 52 μL, about 53 μL, about 54 μL, about 55 μL, about 56 μL, about 57 μL, about 58 μL, about 59 μL, about 60 μL, about 61 μL, about 62 μL, about 63 μL, about 64 μL, about 65 μL, about 66 μL, about 67 μL, about 68 μL, about 69 μL, about 70 μL, about 71 μL, about 72 μL, about 73 μL, about 74 μL, about 75 μL, about 76 μL, about 77 μL, about 78 μL, about 79 μL, about 80 μL, about 81 μL, about 82 μL, about 83 μL, about 84 μL, about 85 μL, about 86 μL, about 87 μL, about 88 μL, about 89 μL, about 90 μL, about 91 μL, about 92 μL, about 93 μL, about 94 μL, about 95 μL, about 96 μL, about 97 μL, about 98 μL, about 99 μL, about 100 μL, about 101 μL, about 102 μL, about 103 μL, about 104 μL, about 105 μL, about 106 μL, about 107 μL, about 108 μL, about 109 μL, about 100 μL, about 111 μL, about 112 μL, about 113 μL, about 114 μL, about 115 μL, about 116 μL, about 117 μL, about 118 μL, about 119 μL, about 120 μL, about 121 μL, about 122 μL, about 123 μL, about 124 μL, about 125 μL, about 126 μL, about 127 μL, about 128 μL, about 129 μL, about 130 μL, about 131 μL, about 132 μL, about 133 μL, about 134 μL, about 135 μL, about 136 μL, about 137 μL, about 138 μL, about 139 μL, about 140 μL, about 141 μL, about 142 μL, about 143 μL, about 144 μL, about 145 μL, about 146 μL, about 147 μL, about 148 μL, about 149 μL, about 150 μL, about 151 μL, about 152 μL, about 153 μL, about 154 μL, about 155 μL, about 156 μL, about 157 μL, about 158 μL, about 159 μL, about 160 μL, about 161 μL, about 162 μL, about 163 μL, about 164 μL, about 165 μL, about 166 μL, about 167 μL, about 168 μL, about 169 μL, about 170 μL, about 171 μL, about 172 μL, about 173 μL, about 174 μL, about 175 μL, about 176 μL, about 177 μL, about 178 μL, about 179 μL, about 180 μL, about 181 μL, about 182 μL, about 183 μL, about 184 μL, about 185 μL, about 186 For example, the volume of the formulation of the present disclosure may be at least about 10 μL to about 50 μL, about 20 μL to about 50 μL, about 25 μL to about 50 μL, or about 1 μL to about 200 μL. The volume of the formulations of the present disclosure may be at least about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 100 μL, about 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, or about 200 μL or more.

For example, the formulation may comprise at least about 1×10 ³ , about 2×10 ³ , about 3×10 ³ , about 4×10 ³ , about 5×10 ³ , about 6×10 ³ , about 7×10 ³ , about 8×10 ³ , about 9×10 ³ , about 1×10 ⁴ , about 2×10 ⁴ , about 3×10 ⁴ , about 4×10 ⁴ , about 5×10 ⁴ , about 6×10 ⁴ , about 7×10 ⁴ , about 8×10 ⁴ , or about 9×10 ⁴ PRCs/μL. For example, the formulation may contain at least about 1×10 ³ to about 9×10 ⁴ , about 2×10 ³ to about 9×10 ⁴ , about 3×10 ³ to about 9×10 ⁴ , about 4×10 ³ to about 9×10 ⁴ , about 5×10 ³ to about 9×10 ⁴ , about 6×10 ³ to about 9×10 ⁴ , about 7×10 ³ to about 9×10 ⁴ , about 8×10 ³ to about 9×10 ⁴ , about 9×10 ³ to about 9×10 ⁴ , about 1×10 ⁴ to about 9×10 ⁴ , about 2×10 ⁴ to about 9×10 ⁴ , about 3×10 ⁴ to about 9×10 ⁴ , about 4×10 ⁴ to about 9×10 ⁴ , about 5×10 ⁴ to about 9×10 ⁴ 4 , about 6×10 ³ to about 9×10 ⁴ , about 7×10 ⁴ to about 9×10 ⁴ , about 8×10 ⁴ to about 9×10 ⁴ , about 1×10 ³ to about 8×10 ⁴ , about 2×10 ³ to about 8×10 ⁴ , about 3×10 ³ to about 8×10 ⁴ , about 4×10 ³ to about 8×10 ⁴ , about 5×10 ³ to about 8×10 ⁴ , about 6×10 ³ to about 8×10 ⁴ , about 7×10 ³ to about 8×10 ⁴ , about 8×10 ³ to about 8×10 ⁴ , about 9×10 ³ to about 8×10 ⁴ , about 1×10 ⁴ to about 8×10 ⁴ , about 2×10 ⁴ to about 8×10 ⁴ , about 3×10 ⁴ to about 8×10 ⁴ , about 4×10 ⁴ to about 8×10 ⁴ , about 5×10 ⁴ to about 8×10 ⁴ , about 6×10 ⁴ to about 8×10 ⁴ , about 7×10 ⁴ to about 8×10 ⁴ , about 1×10 ³ to about 7×10 ⁴ , about 2×10 ³ to about 7×10 ⁴ , about 3×10 ³ to about 7×10 ⁴ , about 4×10 ³ to about 7×10 ⁴ , about 5×10 ³ to about 7×10 ⁴ , about 6×10 ³ to about 7×10 ⁴ , about 7×10 ³ to about 7×10 ⁴ , about 8×10 ³ to about 7×10 ⁴ , about 9×10 ³ to about 7×10 ⁴ , about 1×10 ⁴ to about 7×10 ⁴ 4 , about 2×10 ⁴ to about 7×10 ⁴ , about 3×10 ⁴ to about 7×10 ⁴ , about 4×10 ⁴ to about 7×10 ⁴ , about 5×10 ⁴ to about 7×10 ⁴ , about 6×10 ⁴ to about 7×10 ⁴ , about 1×10 ³ to about 6×10 ⁴ , about 2×10 ³ to about 6×10 ⁴ , about 3×10 ³ to about 6×10 ⁴ , about 4×10 ³ to about 6×10 ⁴ , about 5×10 ³ to about 6×10 ⁴ , about 6×10 ³ to about 6×10 ⁴ , about 7×10 ³ to about 6×10 ⁴ , about 8×10 ³ to about 6×10 ⁴ , about 9×10 ³ to about 6×10 ⁴ , about 1×10 ⁴ to about 6×10 ⁴ , about 2×10 ⁴ to about 6×10 ⁴ , about 3×10 ⁴ to about 6×10 ⁴ , about 4×10 ⁴ to about 6×10 ⁴ , about 5×10 ⁴ to about 6×10 ⁴ , about 1×10 ³ to about 5×10 ⁴ , about 2×10 ³ to about 5×10 ⁴ , about 3×10 ³ to about 5×10 ⁴ , about 4×10 ³ to about 5×10 ⁴ , about 5×10 ³ to about 5×10 ⁴ , about 6×10 ³ to about 5×10 ⁴ , about 7×10 ³ to about 5×10 ⁴ , about 8×10 ³ to about 5×10 ⁴ , about 9×10 ³ to about 5×10 ⁴ , about 1×10 ⁴ to about 5×10 ⁴ 4 , about 2×10 ⁴ to about 5×10 ⁴ , about 3×10 ⁴ to about 5×10 ⁴ , about 4×10 ⁴ to about 5×10 ⁴ , about 1×10 ³ to about 4×10 ⁴ , about 2×10 ³ to about 4×10 ⁴ , about 3×10 ³ to about 4×10 ⁴ , about 4×10 ³ to about 4×10 ⁴ , about 5×10 ³ to about 4×10 ⁴ , about 6×10 ³ to about 4×10 ⁴ , about 7×10 ³ to about 4×10 ⁴ , about 8×10 ³ to about 4×10 ⁴ , about 9×10 ³ to about 4×10 ⁴ , about 1×10 ⁴ to about 4×10 ⁴ , about 2×10 ⁴ to about 4×10 ⁴ , about 3×10 ⁴ to about 4×10 ⁴ , about 1×10 ³ to about 3×10 ⁴ , about 2×10 ³ to about 3×10 ⁴ , about 3×10 ³ to about 3×10 ⁴ , about 4×10 ³ to about 3×10 ⁴ , about 5×10 ³ to about 3×10 ⁴ , about 6×10 ³ to about 3×10 ⁴ , about 7×10 ³ to about 3×10 ⁴ , about 8×10 ³ to about 3×10 ⁴ , about 9×10 ³ to about 3×10 ⁴ , about 1×10 ⁴ to about 3×10 ⁴ , about 2×10 ⁴ to about 3×10 ⁴ , about 1×10 ³ to about 2×10 ⁴ , about 2×10 ³ to about 2×10 ⁴ , about 3×10 ³ to about 2×10 ⁴ 4 , about 4×10 ³ to about 2×10 ⁴ , about 5×10 ³ to about 2×10 ⁴ , about 6×10 ³ to about 2×10 ⁴ , about 7×10 ³ to about 2×10 ⁴ , about 8×10 ³ to about 2×10 ⁴ , about 9×10 ³ to about 2×10 ⁴ , about 1×10 ⁴ to about 2×10 ⁴ , about 1×10 ³ to about 1×10 ⁴ , about 2×10 ³ to about 1×10 ⁴ , about 3×10 ³ to about 1×10 ⁴ , about 4×10 ³ to about 1×10 ⁴ , about 5×10 ³ to about 1×10 ⁴ , about 6×10 ³ to about 1×10 ⁴ , about 7×10 ³ to about 1×10 ⁴ , about 8×10 ³ to about 1×10 ⁴ , about 9×10 ³ to about 1×10 ⁴ , about 1×10 ³ to about 9×10 ³ , about 2×10 ³ to about 9×10 ³ , about 3×10 ³ to about 9×10 ³ , about 4×10 ³ to about 9×10 ³ , about 5×10 ³ to about 9×10 ³ , about 6×10 ³ to about 9×10 ³ , about 7×10 ³ to about 9×10 ³ , about 8×10 ³ to about 9×10 ³ , about 1×10 ³ to about 8×10 ³ , about 2×10 ³ to about 8×10 ³ , about 3×10 ³ to about 8×10 ³ , about 4×10 ³ to about 8×10 ³ , about 5×10 ³ to about 8×10 ³ , about 6×10 ³ to about 8×10 ³ , about 7×10 ³ to about 8×10 ³ , about 1×10 ³ to about 7×10 ³ , about 2×10 ³ to about 7×10 ³ , about 3×10 ³ to about 7×10 ³ , about 4×10 ³ to about 7×10 ³ , about 5×10 ³ to about 7×10 ³ , about 6×10 ³ to about 7×10 ³ , about 1×10 ³ to about 6×10 ³ , about 2×10 ³ to about 6×10 ³ , about 3×10 3 to about 6×10 ³ , about 4×10 ³ to about 6×10 ³ , about 5×10 ³ to about 6×10 ³ , about 1×10 ³ to about 6×10 ³ , about 2×10 ³ to about 6×10 3 ³ to about 6×10 ³ , about 3×10 ³ to about 6×10 ³ , about 4×10 ³ to about 6×10 ³ , about 5×10 ³ to about 6×10 ³ , about 1×10 ³ to about 5×10 ³ , about 2×10 ³ to about 5×10 ³ , about 3×10 ³ to about 5×10 ³ , about 4×10 ³ to about 5×10 ³ , about 1×10 ³ to about 4×10 ³ , about 2×10 ³ to about 4×10 ³ , about 3×10 ³ to about 4×10 ³ , about 1×10 ³ to about 3×10 ³ , about 2×10 ³ to about 3×10 ³ , or about 1×10 ³ to about 2×10 ³ PRCs/μL. The formulation may contain about 2000 PRCs/μL, such as about 100,000 PRCs/50 μL or about 180,000 PRCs/90 μL.

The method of treating retinal degeneration may further comprise administering an immunosuppressant. Immunosuppressants that may be used include, but are not limited to, anti-lymphocyte globulin (ALG) polyclonal antibodies, anti-thymocyte globulin (ATG) polyclonal antibodies, azathioprine, BASILIXIMAB® (anti-IL-2Rα receptor antibody), cyclosporin (cyclosporin A), DACLIZUMAB® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody), sirolimus, and tacrolimus. The immunosuppressant may be administered at least about 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg/kg. When an immunosuppressant is used, it can be administered systemically or locally, and it can be administered before, simultaneously with, or after the administration of PRC cells. Immunosuppressive therapy can continue for weeks, months, years, or indefinitely after PRC administration. For example, 5 mg/kg of cyclosporine can be administered to a patient for 6 weeks after PRC administration.

Methods for treating retinal degeneration may include administering a single dose of PRC. In addition, the treatment methods described herein may include a course of treatment in which PRC is administered multiple times over a period of time. Exemplary courses of treatment may include weekly, biweekly, monthly, quarterly, semi-annually, or annually. Alternatively, treatment may be performed in stages, with multiple doses administered initially (e.g., daily doses administered in the first week), and fewer and less frequent doses required thereafter.

If administered by intraocular injection, the PRCs may be delivered one or more times periodically during the patient's life. For example, the PRCs may be delivered once a year, once every 6 to 12 months, once every 3 to 6 months, once every 1 to 3 months, or once every 1 to 4 weeks. Alternatively, certain conditions or disorders may require more frequent administration. If administered by an implant or device, the PRCs may be administered once or one or more times periodically during the patient's life, as required by the particular patient and the disorder or condition being treated. Similarly, consider treatment regimens that change over time. For example, more frequent treatments (e.g., daily or weekly treatments) may be required at the beginning. Over time, as your condition improves, you may need less frequent treatments or no further treatment at all.

Intraocular administration includes injecting an aqueous solution, an isotonic solution and/or a saline solution into the subretinal space, thereby forming an anterior blisters; and removing the aqueous solution, followed by administering a population of photoreceptor rescue cells into the same subretinal space in the form of the aqueous solution. Before the cells are administered, a subretinal blisters may be formed, for example by injecting saline or another suitable liquid (anterior blisters), which may then be removed before the cells are administered. However, the cells may also be administered without forming an anterior blisters. The cells may be administered into the blisters at the location of the temporal lobe fovea. For example, the blisters may extend within the arcade vessels as desired. The blisters may be positioned so that they do not deviate from the central fovea of the macula.

The methods described herein may further comprise the step of monitoring the efficacy of the treatment or prevention by measuring the individual's retinal galvanogram response, visual acuity threshold, or brightness threshold. The methods may also comprise monitoring the efficacy of the treatment or prevention by monitoring the immunogenicity of cells in the eye or the migration of cells.

PRC can be used to make a medicament that can treat retinal degeneration. The present disclosure also encompasses the use of a formulation containing PRC for treating blindness. For example, a formulation containing human PRC can be used to treat retinal degeneration associated with a variety of vision-altering conditions that lead to photoreceptor cell damage and blindness, such as diabetic retinopathy, macular degeneration (including age-related macular degeneration, such as wet age-related macular degeneration and dry age-related macular degeneration), retinitis pigmentosa and Stargardt's disease (yellow spots on the eye), night blindness, and color blindness. The formulation may contain at least about 5,000 to 500,000 PRCs (e.g., 100,000 PRCs), which may be administered to the retina to treat retinal degeneration associated with a variety of vision-altering conditions that lead to photoreceptor cell damage and blindness, such as diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease.

The PRCs provided herein can be derived from mammals. However, it should be noted that human cells can be used in human patients as well as animal models or animal patients. For example, human cells can be tested in mouse, rat, cat, dog or non-human primate retinal degeneration models. In addition, human cells can be used therapeutically to treat animals in need, such as in veterinary medicine. Examples of veterinary subjects or patients include, but are not limited to, dogs, cats and other companion animals, and economically valuable animals such as livestock and horses.

The present disclosure further provides novel cryoprotective formulations that require at least a cryoprotectant, albumin, and optionally a sugar. The cryoprotectant may be DMSO, glycerol, ethylene glycol, trehalose, or taurine. In some specific examples, the cryoprotectant is DMSO. The albumin may be human albumin. The sugar may be glucose. The formulation may further comprise a buffer or a buffered saline solution, such as, but not limited to, phosphate buffered saline (PBS). The pH of the formulation may be about physiological pH (e.g., in the range of 6 to 8). The cryoprotectant may be present in the range of about 1% to about 10% volume/volume (v/v). The cryoprotectant may be present in the range of about 2% to about 10% volume/volume (v/v). The hypothermic protectant may be present in the range of about 3% to about 10% volume/volume (v/v). The hypothermic protectant may be present in the range of about 4% to about 10% volume/volume (v/v), such as 4%, 5%, 6%, 7%, 8%, 9% or 10%. Albumin may be present in the range of about 2% to about 3% (w/v). Albumin may be present in the range of about 2% to about 10% (w/v), about 2% to about 8% (w/v), about 2% to about 7.5% (w/v), about 3.5% to about 7.5% (w/v), about 4% to about 7.5% (w/v), about 4% to about 8% (w/v), about 4% to about 8.5% (w/v), about 4% to about 9% (w/v) or about 4% to about 10% (w/v). Sugar may be present in the range of about 0% to about 1.5% (w/v).

In some embodiments, the cryopreservation formulation comprises about 4% to about 10% DMSO (v/v), about 2% to about 3% albumin (w/v), about 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. The formulation may consist essentially of about 4% to about 10% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. The formulation may consist of about 4% to about 10% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline solution may be phosphate buffered saline (PBS).

In some embodiments, the cryopreservation formulation comprises about 4% to about 6% DMSO (v/v), about 2% to about 3% albumin (w/v), about 0.08% to about 0.1% glucose (w/v), and a buffer or a buffered saline. In some embodiments, the cryopreservation formulation consists essentially of about 4% to about 6% DMSO (v/v), about 2% to about 3% albumin (w/v), about 0.08% to about 0.1% glucose (w/v), and a buffer or a buffered saline. In some embodiments, the cryopreservation formulation is composed of about 4% to about 6% DMSO (v/v), about 2% to about 3% albumin (w/v), about 0.08% to 0.1% glucose (w/v), and a buffer or a buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

In some embodiments, the cryopreservation formulation comprises about 4% to about 10% DMSO (v/v), about 2% to about 7.5% albumin (w/v), about 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. The formulation may consist essentially of about 4% to about 10% DMSO (v/v), about 2% to about 7.5% albumin (w/v), about 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. The formulation may consist of about 4% to about 10% DMSO (v/v), about 2% to about 7.5% albumin (w/v), about 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline solution may be phosphate buffered saline (PBS).

In some embodiments, the cryopreservation formulation comprises about 4% to about 6% DMSO (v/v), about 2% to about 7.5% albumin (w/v), about 0.08% to about 0.1% glucose (w/v), and a buffer or a buffered saline. In some embodiments, the cryopreservation formulation consists essentially of about 4% to about 6% DMSO (v/v), about 2% to about 7.5% albumin (w/v), about 0.08% to about 0.1% glucose (w/v), and a buffer or a buffered saline. In some embodiments, the cryopreservation formulation is composed of about 4% to about 6% DMSO (v/v), about 2% to about 7.5% albumin (w/v), about 0.08% to about 0.1% glucose (w/v), and a buffer or a buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

In some embodiments, a particular formulation is associated with a reduced incidence of multinucleated photorescrutin cells with more than 4 nuclei per cell after freezing and thawing. For example, the formulation comprises a high content of DMSO (e.g., 5%) and optionally also contains ethylene glycol (e.g., 5%).

In some embodiments, the hypothermic protectant is a cell-permeable hypothermic protectant, such as DMSO, glycerol, or ethylene glycol, which have been found to be associated with a reduced incidence of multinucleated RPE cells.

Still other formulations include about 5% DMSO (v/v), about 2.5% albumin (w/v), about 0.09% glucose (w/v), and a buffer or a buffered saline solution. Other formulations include about 2.5% albumin (w/v), about 0.09% glucose (w/v), about 5% (v/v) glycerol, about 200 mM taurine, and a buffer or a buffered saline solution. Other formulations include 2.5% albumin (w/v), about 0.09% glucose (w/v), about 5% (v/v) glycerol, about 100 mM trehalose, and a buffer or a buffered saline solution.

Still other formulations include about 5% DMSO (v/v), about 2.5% albumin (w/v), about 0.6% glucose (w/v), and a buffer or a buffered saline solution. Other formulations include about 2.5% albumin (w/v), about 0.6% glucose (w/v), about 5% (v/v) glycerol, about 200 mM taurine, and a buffer or a buffered saline solution. Other formulations include 2.5% albumin (w/v), about 0.6% glucose (w/v), about 5% (v/v) glycerol, about 100 mM trehalose, and a buffer or a buffered saline solution.

Still other formulations include about 5% DMSO (v/v), about 7.5% albumin (w/v), about 0.6% glucose (w/v), and a buffer or a buffered saline solution. Other formulations include about 7.5% albumin (w/v), about 0.6% glucose (w/v), about 5% (v/v) glycerol, about 200 mM taurine, and a buffer or a buffered saline solution. Other formulations include 7.5% albumin (w/v), about 0.6% glucose (w/v), about 5% (v/v) glycerol, about 100 mM trehalose, and a buffer or a buffered saline solution.

In some embodiments, the cryopreservation formulation comprises one or more of betaine, albumin, ethylene glycol, and pluronic.

In some embodiments, the cryopreserved cell preparation may include cells of interest (e.g., photosensitive rescue cells), 165 µg DMSO, 27 µg glucose, 750 µg recombinant human albumin, 110 µg sodium chloride, 26.6 µg sodium phosphate, 2.10 µg calcium chloride, 4.19 µg potassium chloride, 4.19 µg potassium dihydrogen phosphate, 2.10 µg magnesium chloride-6H ₂ O, 168 µg sodium chloride, and 24.1 µg sodium dihydrogen phosphate.

After thawing and dilution in a diluent as provided herein, in some embodiments, the final cell preparation to be administered to a subject may include cells of interest (e.g., photoreceptor rescue cells), sodium hyaluronate, sodium chloride, potassium chloride, sodium hydrogen phosphate dodecahydrate, dextrose (anhydrous), calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, and sodium citrate dihydrate.

Cells can be cryopreserved at higher concentrations (or densities) than standard prior art. For example, cells can be cryopreserved at a concentration (or density) of 10 ⁴ cells/µL or higher, including about 2×10 ⁴ cells/µL, about 3×10 ⁴ cells/µL, about 4×10 ⁴ cells/µL, about 5×10 ⁴ cells/µL, about 6×10 ⁴ cells/µL, about 7×10 ⁴ cells/µL, about 8×10 ⁴ cells/µL, about 9×10 ⁴ cells/µL, or about 10 ⁵ cells/µL or higher. In some embodiments, the cell density is in the range of about 10 ⁴ cells/µL to about 6×10 ⁴ cells/µL, including 1.5×10 ⁴ cells/µL to about 5×10 ⁴ cells/µL, including about 2×10 ⁴ cells/µL to about 5×10 ⁴ cells/µL, and about 3×10 ⁴ to about 5×10 ⁴ cells/µL. In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells/vial. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells/vial.

Additionally, the volume of cryopreserved cell preparations can be reduced compared to standard prior art techniques. For example, cells can be cryopreserved in a volume ranging from about 10 μL to about 100 μL, in some embodiments, including a volume of about 10 μL to about 50 μL, including a volume of about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, or about 100 μL. Because the cells can be frozen in large numbers but still at a low volume, the cryopreserved preparation can then be diluted with a suitable diluent to achieve the desired volume and concentration, and then directly administered to an individual without performing a wash step. Standard prior art techniques typically freeze cells at lower concentrations in larger volumes (e.g., about 1 mL), and then require the thawed preparation to be washed, sometimes multiple times, to remove the cryoprotectant prior to administration to a subject.

In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer, and is used to cryopreserve photosensitive rescue cells. This formulation allows the survival rate of the photosensitive rescue cells after thawing to be at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, at least 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. About 5% or about 10% DMSO can be used. Cells can be frozen at a concentration of approximately 50×10 ³ cells/µL. The formulation improves cell survival after thawing compared to the commercially available cryoprotectant formulation CS10.

In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or a buffered saline. DMSO may be present at about 4% (v/v), about 5% (v/v), and about 6% (v/v). The albumin may be human albumin, including recombinant human albumin, and may be present at about 2% (w/v), or about 2.5% (w/v), or about 3% (w/v). Glucose may be absent, or it may be present at about 0.08% (w/v), or about 0.09% (w/v), or about 0.1% (w/v). The buffered saline may be a phosphate buffered saline. In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or buffered saline. The albumin may be present at about 2% (w/v), about 2.5% (w/v), or about 3% (w/v). The glucose may be present at about 0.08% (w/v), or about 0.09% (w/v), or about 0.10% (w/v).

In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or a buffered saline solution. DMSO may be present at about 4% (v/v), about 5% (v/v), and about 6% (v/v). The albumin may be human albumin, including recombinant human albumin, and may be present at about 2% (w/v), or about 2.5% (w/v), or about 3% (w/v), or about 4% (w/v), or about 5% (w/v), or about 6% (w/v), or about 7% (w/v), or about 7.5% (w/v), or about 8% (w/v), or about 9% (w/v), or about 10% (w/v). Glucose may be absent, or it may be present at about 0.08% (w/v) or about 0.09% (w/v) or about 0.1% (w/v). The buffered saline may be phosphate buffered saline.

In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v/v), about 2% to about 3% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or a buffered saline solution. The albumin may be present at about 2% (w/v), about 2.5% (w/v), or about 3% (w/v). In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v/v), about 2% to about 7.5% albumin (w/v), 0 to 1.5% glucose (w/v), and a buffer or a buffered saline solution. Albumin may be present at about 2% (w/v), about 5% (w/v), or about 7.5 % (w/v). Glucose may be present at about 0.08% (w/v), or about 0.09% (w/v), or about 0.10% (w/v).

In some embodiments, the formulations or preparations provided herein exhibit physiological pH and physiological osmotic pressure (also referred to as physiological osmotic pressure). Physiological pH refers to a pH that is non-cytotoxic and similar to the pH of cells or tissues in their natural environment. For most cells and tissues, physiological pH is a pH of about 6.8 to about 7.8, such as a pH of about 7 to about 7.7, a pH of about 7.2 to about 7.6, a pH of about 7.2 to about 7.4, or a pH of about 7.4 to about 7.5. Accordingly, in some embodiments, the formulations or preparations provided herein exhibit a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. Physiological osmotic pressure refers to an osmotic pressure that is non-cytotoxic and similar to the osmotic pressure of cells or tissues in their natural environment. For most cells and tissues, the physiological osmotic pressure is about 270 to about 345 mOsm/l, such as about 280 to about 330 mOsm/l, about 290 to about 325 mOsm/l, about 300 to about 315 mOsm/l. In some embodiments, the physiological osmotic pressure is about 300 mOsm/l, about 305 mOsm/l, about 310 mOsm/l, about 315 mOsm/l, about 320 mOsm/l, or about 325 mOsm/l. In some embodiments, the formulations or preparations provided herein exhibit a physiological pH and an osmotic pressure greater than the physiological osmotic pressure, e.g., about 350 to about 450 mOsm/l, e.g., about 360 to about 440 mOsm/l, about 370 to about 430 mOsm/l, about 380 to about 420 mOsm/l, or about 390 to about 410 mOsm/l. In some embodiments, the osmotic pressure greater than the physiological osmotic pressure is about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, about 445, or about 450 mOsm/l. In some embodiments, the formulations or formulations provided herein have an osmotic pressure of about 1250 mOsm/kg, such as about 1250 mOsm/kg, about 1000 mOsm/kg, about 900 mOsm/kg, about 800 mOsm/kg, about 700 mOsm/kg, about 600 mOsm/kg, about 500 mOsm/kg, about 400 mOsm/kg, or about 300 mOsm/kg. In some embodiments, the formulations or formulations provided herein have an osmotic pressure of about 300 to 450 mOsm/kg, about 300 to 400 mOsm/kg, about 300 to 450 mOsm/kg, about 350 to 450 mOsm/kg, or about 350 to 400 mOsm/kg.

In some embodiments, the cryopreservation formulation provided herein comprises (a) a cryopreservative; (b) albumin; (c) a buffer that maintains the solution at physiological pH; and (d) glucose. In some embodiments, the formulation comprises a cryopreservative. The cryopreservative is used to protect cells from damage during the freezing process. In some embodiments, the formulation comprises a cryopreservative selected from dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. In some embodiments, the cryopreservative is DMSO. In some embodiments, the formulation comprises about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% (volume/volume, v/v) DMSO. In some embodiments, the solution comprises about 4%-10%, about 4%-9.5%, about 4%-9%, about 4%-8.5%, about 4%-8%, about 4%-7.5%, about 4%-7%, about 4%-6.5%, about 4%-6%, about 4%-5.5%, about 4%-5%, about 4%-4.5%, about 4.5%-10%, about 4.5%-9.5%, about 4.5%-9%, about 4.5%-8.5%, about 4.5%-8%, about 4.5%-7.5%, about 4.5%-7%, about 4.5%-6.5%, about 4.5%-6%, about 4.5%-5.5%, about 4.5%-5%, about 5%-10%, about 5%-9.5%, about 5%-9%, about 5%-8.5%, about 5%-8%, about 5%-7.5%, about 5%-7%, about 5%-6.5%, about 5%-6%, about 5%-5.5%, about 5.5%-10%, about 5.5%-9.5%, about 5.5%-9%, about 5.5%-8.5%, about 5.5%-8%, about 5.5%-7.5%, About 5.5%-7%, about 5.5%-6.5%, about 5.5%-6%, about 6%-10%, about 6%-9.5%, about 6%-9%, about 6%-8.5%, about 6%-8%, about 6%-7.5%, about 6%-7%, about 6%-6.5%, about 6.5%-10%, about 6.5%-9.5%, about 6.5%-9%, about 6.5%-8.5%, about 6.5%-8%, about 6.5%-7.5%, about 6.5%-7%, about 7%-10%, about 7%-9.5%, About 7%-9%, about 7%-8.5%, about 7%-8%, about 7%-7.5%, about 7.5%-10%, about 7.5%-9.5%, about 7.5%-9%, about 7.5%-8.5%, about 7.5%-8%, about 8%-10%, about 8%-9.5%, about 8%-9%, about 8%-8.5%, about 8.5%-10%, about 8.5%-9.5%, about 8.5%-9%, about 9%-10%, about 9%-9.5%, or about 9.5%-10% (v/v) DMSO. In some embodiments, the formulation comprises about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%. , about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9% or about 10% (volume/volume, v/v) DMSO.

In some embodiments, the formulation comprises albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is recombinant human (rh) albumin. The term "recombinant albumin" may be used interchangeably with the term "rA" or "r-albumin". The term "recombinant human albumin" may be used interchangeably with the term "rHA", "rHSA", "rh-albumin". In some embodiments, the cryopreservation formulation comprises about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6.0%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about about 9.0%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, or about 10.0% albumin. In some embodiments, the cryopreservation formulation comprises about 2.0%-3.0%, about 2.0%-2.9%, about 2.0%-2.8%, about 2.0%-2.7%, about 2.0%-2.6%, about 2.0%-2.5%, about 2.0%-2.4%, about 2.0%-2.3%, about 2.0%-2.2%, about 2.0%-2.1%, about 2.1%-3.0%, about 2.1%-2.9%, about 2.1%- 2.8%, about 2.1%-2.7%, about 2.1%-2.6%, about 2.1%-2.5%, about 2.1%-2.4%, about 2.1%-2.3%, about 2.1%-2.2%, about 2.2%-3.0%, about 2.2%-2.9%, about 2.2%-2.8%, about 2.2%-2.7%, about 2.2%-2.6%, about 2.2%-2.5%, about 2.2%-2.4%, about 2.2%-2.3 %, about 2.3%-3.0%, about 2.3%-2.9%, about 2.3%-2.8%, about 2.3%-2.7%, about 2.3%-2.6%, about 2.3%-2.5%, about 2.3%-2.4%, about 2.4%-3.0%, about 2.4%-2.9%, about 2.4%-2.8%, about 2.4%-2.7%, about 2.4%-2.6%, about 2.4%-2.5%, about 2.5%-3.0%, about 2.5%-2.9%, about 2.5%-2.8%, about 2.5%-2.7%, about 2.5%-2.6%, about 2.6%-3.0%, about 2.6%-2.9%, about 2.6%-2.8%, about 2.6%-2.7%, about 2.7%-3.0%, about 2.7%-2.9%, about 2.7%-2.8%, about 2.8%-3.0%, about 2.8%-2.9%, or about 2.9%-3.0% albumin.

In some embodiments, the cryopreservation formulation comprises a buffer. A buffer refers to an agent that can maintain the pH of a solution, formulation or formulation by neutralizing the added acid or base to make it relatively stable. Typically, the buffer comprises a weakly conjugated acid-base pair, that is, a weak acid and its conjugated base, or a weak base and its conjugated acid. In some embodiments, the buffer contained in the cryopreservation formulation provided herein is a water-based saline solution, which comprises sodium dihydrogen phosphate (Na ₂ HPO ₄ ) and sodium chloride (NaCl). In some embodiments, the buffer contained in the formulation provided herein further comprises potassium chloride (KCl) and potassium dihydrogen phosphate (KH ₂ PO ₄ ). In some embodiments, the formulation comprises a buffered saline, such as phosphate-buffered saline (PBS). In some embodiments, the buffer is Dulbecco's phosphate-buffered saline (DPBS).

In some embodiments, the buffer comprises divalent cations. Suitable divalent cations include, but are not limited to, for example, Ca2+, Mg2+, Zn2+, Fe2+, Mn2+, Cr2+, Cu2+, Ba2+, and Sr2+. In some embodiments, the divalent cations comprise calcium. In some embodiments, the divalent cations comprise magnesium. In some embodiments, the divalent cations comprise two or more different divalent cations, such as calcium and magnesium. In some embodiments, the buffer comprises calcium. In some embodiments, the buffer comprises a pharmaceutically acceptable calcium salt. In some embodiments, the buffer comprises magnesium. In some embodiments, the buffer comprises a pharmaceutically acceptable magnesium salt. In some embodiments of the solutions provided herein, the buffer comprises calcium chloride. In some embodiments, the buffer comprises calcium chloride dihydrate. In some embodiments, the buffer comprises magnesium chloride. In some embodiments, the buffer comprises magnesium chloride hexahydrate.

In some embodiments, the cryopreservation formulations provided herein include glucose. In some embodiments, the cryopreservation formulations include about 0%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.5%, 4%, 4.5% or 5% glucose. In some embodiments, the solution comprises about 0%-1.5%, 0%-1.4%, 0%-1.3%, 0%-1.2%, 0%-1.1%, 0%-1.0%, 0%-0.90%, 0%-0.80%, 0%-0.70%, 0%-0.60%, 0%-0.50%, 0%-0.40%, 0%-0.30%, 0%-0.20%, 0%-0.10%, 0%-0. 09%, 0%-0.08%, 0%-0.07%, 0%-0.06%, 0%-0.05%, 0%-0.04%, 0%-0.03%, 0%-0.02%, 0%-0.01%, 0.01%-1.5%, 0.01%-1.4%, 0.01%-1.3%, 0.01%-1.2%, 0.01%-1.1%, 0.01%-1.0%, 0.01%- 0.90%, 0.01%-0.80%, 0.01%-0.70%, 0.01%-0.60%, 0.01%-0.50%, 0.01%-0.40%, 0.01%-0.30%, 0.01%-0.20%, 0.01%-0.10%, 0.01%-0.09%, 0.01%-0.08%, 0.01%-0.07%, 0.01%-0.06 %, 0.01%-0.05%, 0.01%-0.04%, 0.01%-0.03%, 0.01%-0.02%, 0.02%-1.5%, 0.02%-1.4%, 0.02%-1.3%, 0.02%-1.2%, 0.02%-1.1%, 0.02%-1.0%, 0.02%-0.90%, 0.02%-0.80%, 0.02%-0. 70%, 0.02%-0.60%, 0.02%-0.50%, 0.02%-0.40%, 0.02%-0.30%, 0.02%-0.20%, 0.02%-0.10%, 0.02%-0.09%, 0.02%-0.08%, 0.02%-0.07%, 0.02%-0.06%, 0.02%-0.05%, 0.02%-0.04%, 0.02%-0.03%, 0.03%-1.5%, 0.03%-1.4%, 0.03%-1.3%, 0.03%-1.2%, 0.03%-1.1%, 0.03%-1.0%, 0.03%-0.90%, 0.03%-0.80%, 0.03%-0.70%, 0.03%-0.60%, 0.03%-0.50%, 0.03%-0.40 %, 0.03%-0.30%, 0.03%-0.20%, 0.03%-0.10%, 0.03%-0.09%, 0.03%-0.08%, 0.03%-0.07%, 0.03%-0.06%, 0.03%-0.05%, 0.03%-0.04%, 0.04%-1.5%, 0.04%-1.4%, 0.04%-1.3%, 0.04% -1.2%, 0.04%-1.1%, 0.04%-1.0%, 0.04%-0.90%, 0.04%-0.80%, 0.04%-0.70%, 0.04%-0.60%, 0.04%-0.50%, 0.04%-0.40%, 0.04%-0.30%, 0.04%-0.20%, 0.04%-0.10%, 0.04%-0.09%, 0 .04%-0.08%, 0.04%-0.07%, 0.04%-0.06%, 0.04%-0.05%, 0.05%-1.5%, 0.05%-1.4%, 0.05%-1.3%, 0.05%-1.2%, 0.05%-1.1%, 0.05%-1.0%, 0.05%-0.90%, 0.05%-0.80%, 0.05%-0.70% , 0.05%-0.60%, 0.05%-0.50%, 0.05%-0.40%, 0.05%-0.30%, 0.05%-0.20%, 0.05%-0.10%, 0.05%-0.09%, 0.05%-0.08%, 0.05%-0.07%, 0.05%-0.06%, 0.06%-1.5%, 0.06%-1.4%, 0.06% -1.3%, 0.06%-1.2%, 0.06%-1.1%, 0.06%-1.0%, 0.06%-0.90%, 0.06%-0.80%, 0.06%-0.70%, 0.06%-0.60%, 0.06%-0.50%, 0.06%-0.40%, 0.06%-0.30%, 0.06%-0.20%, 0.06%-0.10%, 0 .06%-0.09%, 0.06%-0.08%, 0.06%-0.07%, 0.07%-1.5%, 0.07%-1.4%, 0.07%-1.3%, 0.07%-1.2%, 0.07%-1.1%, 0.07%-1.0%, 0.07%-0.90%, 0.07%-0.80%, 0.07%-0.70%, 0.07%-0.60% , 0.07%-0.50%, 0.07%-0.40%, 0.07%-0.30%, 0.07%-0.20%, 0.07%-0.10%, 0.07%-0.09%, 0.07%-0.08%, 0.08%-1.5%, 0.08%-1.4%, 0.08%-1.3%, 0.08%-1.2%, 0.08%-1.1%, 0.08%-1. 0%, 0.08%-0.90%, 0.08%-0.80%, 0.08%-0.70%, 0.08%-0.60%, 0.08%-0.50%, 0.08%-0.40%, 0.08%-0.30%, 0.08%-0.20%, 0.08%-0.10%, 0.08%-0.09%, 0.09%-1.5%, 0.09%-1.4%, 0.0 9%-1.3%, 0.09%-1.2%, 0.09%-1.1%, 0.09%-1.0%, 0.09%-0.9%, 0.09%-0.80%, 0.09%-0.70%, 0.09%-0.60%, 0.09%-0.50%, 0.09%-0.40%, 0.09%-0.30%, 0.09%-0.20%, 0.09%-0.10%, 1.0%-1.5%, 1.0%-1.4%, 1.0%-1.3%, 1.0%-1.2%, 1.0%-1.1%, 1.1%-1.5%, 1.1%-1.4%, 1.1%-1.3%, 1.1%-1.2%, 1.2%-1.5%, 1.2%-1.4%, 1.2%-1.3%, 1.3%-1.5%, 1.3%-1.4% or 1.4%-1.5% v/v glucose.

In some embodiments, the formulation comprises about 4-10% (v/v) cell hypothermic protectant, about 2-3% (w/v) albumin, about 0-1.5% (w/v) glucose, and a buffer (optionally a buffered saline solution). In some embodiments, the formulation comprises about 4-10% (v/v) cell hypothermic protectant, about 2-3% (w/v) albumin, about 0.08-0.10% (w/v) glucose, and a buffer (optionally a buffered saline solution). In some embodiments, the formulation comprises about 4-6% (v/v) cell hypothermic protectant, about 2-3% (w/v) albumin, about 0.08-0.10% (w/v) glucose, and a buffer (optionally buffered saline).

In some embodiments, the formulation comprises about 4-10% (v/v) DMSO, about 2-3% (w/v) albumin, about 0-1.5% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-10% (v/v) DMSO, about 2-3% (w/v) albumin, about 0.08-0.10% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-6% (v/v) DMSO, about 2-3% (w/v) albumin, about 0.08-0.10% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.09% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.6% (w/v) glucose, and a buffer.

In some embodiments, the formulation comprises about 4-10% (v/v) DMSO, about 2-8% (w/v) albumin, about 0-1.5% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-6% (v/v) DMSO, about 2-8% (w/v) albumin, about 0-1.5% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.6% (w/v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v/v) DMSO, about 7.5% (w/v) albumin, about 0.6% (w/v) glucose, and a buffer.

In some embodiments, the formulations provided herein further comprise at least one other excipient. In some embodiments, the formulation further comprises taurocholine. In some embodiments, the formulation further comprises trehalose. In some embodiments, the formulation comprises a polymer excipient. In some embodiments, the polymer excipient is polydextrose. In some embodiments, the formulation does not comprise a polymer excipient. In some embodiments, the formulation does not comprise polydextrose. Cell preparation

Some aspects of the present disclosure provide cell preparations comprising cell populations or tissues in cryopreserved formulations as provided herein. In some embodiments, the cell preparations are cryopreserved. The cell preparations provided herein can be cryopreserved by cryopreservation at any suitable temperature known in the art. For example, in some embodiments, the cell preparations provided herein are cryopreserved at a temperature between -100°C and -200°C. In some embodiments, the cell preparations provided herein are cryopreserved at a temperature below -125°C. In some embodiments, the cell preparations provided herein are cryopreserved at a temperature below -135°C. In some embodiments, the cell preparations provided herein are cryopreserved at a temperature below -150°C.

The cryopreservation formulations and cell preparations provided herein can be used in combination with the photosensitive rescue cells of the present invention.

In some embodiments, the cell preparation is suitable for transplantation into an individual after thawing and dilution with a diluent. In some embodiments, the diluent is GS2 or GS2 plus (see WO2017031312, which is incorporated herein by reference in its entirety; and Table 5 below).

As used herein, GS2 medium refers to a medium used for cell recovery, storage, transport and/or administration to an individual. The medium was prepared as follows: 48.75 ml of 0.9% NaCl in water; 13.10 ml of Alcon Balanced Salt Solution (BSS ^® ) in water, 300 mOsm; and 3.75 ml of 5% dextrose (560 mOsm) in 0.9% NaCl (in water) were combined to obtain 65.6 mL of medium with a final concentration of 0.29% dextrose and an osmotic pressure of 315 mOsm.

Basal GS2 medium thus contains about 145 mM NaCl (about 0.85% NaCl), about 2 mM KCl (about 0.015% KCl), about 0.7 mM CaCl ₂ (calcium chloride) (about 0.01% CaCl ₂ dihydrate (calcium chloride dihydrate)), about 0.3 mM MgCl ₂ (magnesium chloride) (about 0.006% MgCl ₂ hexahydrate (magnesium chloride hexahydrate)), about 1 mM sodium citrate (about 0.035% sodium citrate dihydrate) and about 16 mM glucose (about 0.29% dextrose) in water.

GS2 plus medium thus contains about 145 mM NaCl (about 0.85% NaCl), about 2 mM KCl (about 0.015% KCl), about 0.7 mM CaCl ₂ (calcium chloride) (about 0.01% CaCl ₂ dihydrate (calcium chloride dihydrate)), about 0.3 mM MgCl ₂ (magnesium chloride) (about 0.006% MgCl 2 hexahydrate (magnesium chloride hexahydrate)), about 1 mM sodium citrate (about 0.035% sodium citrate dihydrate), about 16 mM glucose (about 0.29% dextrose) and about 3 mM sodium dihydrogen phosphate dihydrate in water.

In some embodiments, the diluent is a solution comprising about 0.1 to about 1.2 mM CaCl ₂ , about 0.05 to about 5 mM MgCl ₂ , about 1 to about 2.5 mM KCl, about 0.5 to about 2 mM sodium citrate, about 14 to about 17 mM dextrose, and about 125 to about 175 mM NaCl. In some embodiments, the diluent is a solution comprising about 0.9 mM CaCl ₂ , about 0.3 mM MgCl ₂ , about 2 mM KCl, about 1.2 mM sodium citrate, about 15 mM dextrose, and about 145 mM NaCl. The diluent may further comprise sodium acetate. The diluent may further comprise a polymer, such as hyaluronic acid or a solvate thereof, such as sodium hyaluronate, at a concentration of about 0.01 to about 0.05% (w/v), including about 0.05% (w/v), as desired. It may further comprise a buffer component, such as sodium dihydrogen phosphate heptahydrate and/or sodium dihydrogen phosphate monohydrate and/or sodium dihydrogen phosphate dihydrate.

In some embodiments, the diluent is a solution comprising about 0.008% to about 0.012% CaCl ₂ dihydrate, about 0.0048% to about 0.0072% MgCl ₂ hexahydrate, about 0.012% to about 0.018% KCl, about 0.028% to about 0.042% sodium citrate dihydrate, about 0.23% to about 0.35% dextrose, and about 0.68% to about 1.02% NaCl. In some embodiments, the diluent is a solution comprising about 0.01% CaCl ₂ dihydrate, about 0.006% MgCl ₂ hexahydrate, about 0.015% KCl, about 0.035% sodium citrate dihydrate, at least 0.25% dextrose, and about 0.85% NaCl. The diluent may further comprise sodium acetate. The diluent may further comprise a polymer, such as hyaluronic acid or a solvate thereof, such as sodium hyaluronate, at a concentration of about 0.01 to about 0.05% (w/v), including about 0.05% (w/v), as required. It may further comprise a buffer component, such as sodium dihydrogen phosphate heptahydrate and/or sodium dihydrogen phosphate monohydrate and/or sodium dihydrogen phosphate dihydrate.

In some embodiments, the diluent comprises about 0.27% glucose, about 0.84% sodium chloride, about 0.016% potassium chloride, about 0.01% calcium chloride, about 0.006% magnesium chloride, about 0.036% sodium citrate, and optionally sodium acetate (e.g., about 0.08%), optionally sodium hyaluronate (e.g., about 0.049%), and optionally sodium dihydrogen phosphate heptahydrate (e.g., about 0.0007%) and sodium dihydrogen phosphate monohydrate (e.g., about 0.0001%). In some embodiments, the diluent comprises about 15 mM glucose, about 144 mM sodium chloride, about 2.1 mM potassium chloride, about 0.9 mM calcium chloride, about 0.3 mM magnesium chloride, about 1.2 mM sodium citrate, optionally sodium acetate (e.g., about 6 mM), optionally sodium hyaluronate, and optionally sodium dihydrogen phosphate heptahydrate (e.g., about 0.027 mM) and sodium dihydrogen phosphate monohydrate (e.g., about 0.007 mM). Such diluents include GS2.

In some embodiments, the diluent comprises about 0.27% glucose, about 0.84% sodium chloride, about 0.016% potassium chloride, about 0.01% calcium chloride, about 0.006% magnesium chloride, about 0.036% sodium citrate, and optionally sodium acetate (e.g., about 0.08%), optionally sodium hyaluronate (e.g., about 0.049%), and optionally sodium dihydrogen phosphate dihydrate (e.g., about 0.047%). In some embodiments, the diluent comprises about 15 mM glucose, about 144 mM sodium chloride, about 2.1 mM potassium chloride, about 0.9 mM calcium chloride, about 0.3 mM magnesium chloride, about 1.2 mM sodium citrate, optionally sodium acetate (e.g., about 6 mM), optionally sodium hyaluronate, and optionally sodium dihydrogen phosphate dihydrate (e.g., about 3 mM). Such diluents include GS2 Plus.

If necessary, the GS2 medium may further comprise an amount of a viscoelastic polymer effective to reduce the shear stress of cells (e.g., a final concentration of about 0.005-5% w/v). In some specific examples, the viscoelastic polymer is hyaluronic acid or a salt or solvent thereof.

In a specific embodiment, GS2 and GS2 plus medium are as shown in Table 5 below: Table 5 percentage mM Components GS2 (from IND ) GS2 from MBR GS2 Plus MW GS2 (from IND ) GS2 from MBR GS2 Plus Sodium Hyaluronate 0.051 0.0490 0.0490 NA NA NA NA Dextrose hydrate 0.3 198.2 15.1 - - D(+)-Anhydrous Glucose 0.2729 0.2729 180.16 - 15.1 15.1 Sodium chloride (NaCl) 0.8412 0.8414 0.8414 58.44 144 144 144 Potassium chloride (KCl) 0.01595 0.0159 0.0159 74.551 2.14 2.13 2.13 Calcium chloride dihydrate (CaCl ₂ ) 0.01021 0.0102 0.0102 110.98 0.92 0.918 0.918 Magnesium chloride hexahydrate (MgCl ₂ ) 0.006374 0.0064 0.0064 203.3 0.314 0.313 0.313 Sodium acetate trihydrate 0.08296 0.0827 0.0827 136.08 6.1 6.08 6.08 Trisodium citrate dihydrate 0.03618 0.0361 0.0361 294.1 1.23 1.23 1.23 Disodium Hydrogen Phosphate Heptahydrate 0.000714 0.0007 268.07 0.0266 0.0256 - Sodium dihydrogen phosphate monohydrate 0.0001 0.0001 137.99 0.0073 0.0071 - Sodium dihydrogen phosphate dihydrate 0.0469 156.01 - 3

In some embodiments, the diluent is a solution comprising (a) a buffer that maintains the solution at physiological pH; and (b) at least 2 mM glucose; and (c) an osmotic active agent that maintains the solution at a physiological osmotic pressure. In some embodiments, the diluent is GS2 or GS2 plus. In some embodiments, the diluent is a solution comprising (a) a buffer that maintains the solution at physiological pH; (b) at least 2 mM glucose; and (c) an osmotic active agent that maintains the solution at an osmotic pressure higher than the physiological osmotic pressure, for example, an osmotic pressure in the range of 350 to 450 mOsm/kg or an osmotic pressure in the range of 1250 mOsm/kg to 300 mOsm/kg. In some embodiments, the diluent further comprises a divalent cation. In some embodiments, the divalent cation comprises calcium and/or magnesium. In some embodiments, the diluent comprises calcium. In some embodiments, the diluent further comprises magnesium. In some embodiments, the diluent comprises an acetate buffer and/or a citrate buffer. In some embodiments, the diluent is a solution that is a combination of two or more criteria or any number of criteria (e.g., pH, osmotic pressure, solute (buffer, glucose, osmotic active agent, magnesium, calcium, potassium, polymer), concentration, etc.). In some embodiments, the diluent is a solution comprising a buffer, glucose, and an osmotic active agent with or without the addition of a polymer, a solution comprising potassium and a solution not comprising potassium, and a solution comprising any combination of solutes at any concentration provided for the respective solutes. It will also be understood that the present disclosure encompasses solutions comprising the listed solutes and solutions consisting essentially of or consisting of the listed solutes and a solvent (e.g., water). Such alternatives are not listed here for the sake of brevity.

For example, in some embodiments, the diluent is a solution comprising or consisting essentially of calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and glucose (e.g., D-glucose) in water. In some embodiments, the diluent is a solution comprising or consisting essentially of calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (e.g., D-glucose) and potassium chloride in water. In some embodiments, the diluent is GS2 or GS2 plus, as described in WO2017/031312A1, the contents of which are incorporated herein by reference.

In some embodiments, the diluent is a solution comprising about 0.1 to about 1.2 mM CaCl ₂ , about 0.05 to about 5 mM MgCl ₂ , about 1 to about 2.5 mM KCl, about 0.5 to about 2 mM sodium citrate, about 15 to about 17 mM dextrose, and about 125 to about 175 mM NaCl. The diluent may further comprise a polymer, and the polymer may be present at a concentration of about 0.01 to about 0.05% (w/v). The polymer may be hyaluronic acid or a solvate thereof, such as sodium hyaluronate. In some embodiments, the diluent is a solution comprising about 0.9 mM CaCl ₂ , about 0.3 mM MgCl ₂ , about 2 mM KCl, about 1.2 mM sodium citrate, about 15 mM dextrose, and about 145 mM NaCl. The diluent may further comprise sodium acetate.

In some embodiments, the diluent is a solution comprising about 0.008% to about 0.012% _CaCl2 dihydrate, about 0.0048% to about 0.0072% _MgCl2 hexahydrate, about 0.012% to about 0.018% KCl, about 0.028% to about 0.042% sodium citrate dihydrate, about 0.23% to about 0.35% dextrose, and about 0.68% to about 1.02% NaCl. In some embodiments, the diluent is a solution comprising about 0.01% _CaCl2 dihydrate, about 0.006% _MgCl2 hexahydrate, about 0.015% KCl, about 0.035% sodium citrate dihydrate, at least 0.25% dextrose, and about 0.85% NaCl. The diluent may further comprise sodium acetate.

In some embodiments, the cell preparations provided herein comprising photorescive cells are formulated for administration to a subject after thawing and dilution, for example, by injection.

Exemplary cell or tissue preparations can be formulated to be suitable for treating human patients after thawing, e.g., pyrogen-free or substantially pyrogen-free, pathogen-free, sterile, and at physiological pH and osmotic pressure. In some embodiments, the preparations provided herein are formulated for injection into a specific site, e.g., in the case of an ophthalmic preparation for treating a retinal disease or disorder, into the vitreous humor for delivery to the retina or choroidal injury site via subretinal delivery or via suprachoral delivery.

The cell preparations provided by the present invention may include additional therapeutic agents, such as immunosuppressants, pro-angiogenic agents, or nutrients or growth factors that support the survival and/or engraftment of the cells in the preparation.

The volume of the cell preparation to be administered and the number of cells will depend on the specific application. Typically, for cell transplantation applications, it is necessary to reduce the volume administered as much as possible. Accordingly, the cell preparation can be formulated to deliver the smallest volume. The cell concentration injected can be any concentration that is effective and non-toxic. In some specific examples, the volume of the cell preparation to be administered is between 1 and 1000 μL. In some embodiments, the volume of the cell preparation to be administered is between 1 and 50 μl, or between 10 and 50 μl, or between 25 and 50 μl, or between 50 and 100 μl, or between 50 and 200 μl, or between 50 and 300 μl, or between 50 and 400 μl, or between 50 and 500 μl, or between 100 and 500 μl, or between 100 and 400 μl, or between 100 and 300 μl, or between 100 and 300 μl, or about 200 μl, or about 150 μl. In some embodiments, the volume of the cell preparation to be administered is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 950, or 1000 μl.

In some embodiments, the number of cells and/or the cell concentration in the cell preparations provided herein can be determined by counting live cells and excluding non-live cells. For example, non-live cells can be detected by not excluding key dyes (such as conifer blue) or using functional assays (such as the ability to adhere to the culture medium, phagocytosis, etc.). In addition, the number of cells or the cell concentration of the desired cell type can be determined as follows: cells expressing one or more cell markers that are specific to that cell type are counted and/or cells expressing one or more markers that indicate cell types other than the desired cell type are excluded. In some embodiments, the cell number and/or cell concentration in a cell preparation can be determined by manual counting using the ViCell Blu-2 program (which counts live and dead cells separately) and/or ViCell Blu Mammalian (default settings).

In some embodiments, the cell preparation to be administered comprises about at least 1×10 ⁴ , 2×10 ⁴ , 3×10 ⁴ , 4×10 ⁴ , 5×10 ⁴ , 6×10 ⁴ , 7×10 ⁴ , 8×10 ⁴ , 9×10 ⁴ , 1×10 ⁵ , 2×10 ⁵ , 3×10 ⁵ , 4×10 ⁵ , 5×10 ⁵ , 6×10 ⁵ , 7×10 ⁵ , 8×10 ⁵ , 9×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁹ 、1×10 ¹⁰ 、2×10 ¹⁰ 、3×10 ¹⁰ 、 ⁴ ×10 ¹⁰ 、 ⁵ ×10 ¹⁰ 、 ⁶ ×10 ^{10 、 7} ×10 ¹⁰ 、 ⁸ ×10 ^{10 、 9} × ^{10 10} 、 ^{1 × 10 10 、2×10 10 、 3} ×10 ¹⁰ 、4× ^{10 10 、 5} ×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ or 9×10 ¹⁰ cells or more.

In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells/vial before cryopreservation. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells/vial before cryopreservation. In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells/vial after cryopreservation, thawing, and dilution. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells/vial before cryopreservation, thawing, and dilution.

The above number of cells may be present in a cryopreserved cell preparation in a single cryovial. The cells may be present in about 10 to about 500 μL of a cryoprotectant formulation, including about 10 to about 200 μL of a cryoprotectant formulation, or about 10 to about 100 μL of a cryoprotectant formulation, or about 10 to about 50 μL of a cryoprotectant formulation, or about 20 to about 50 μL of a cryoprotectant formulation. In some embodiments, the photoreceptor rescue cell population is suitable for transplantation into an individual's eye. The cells thus prepared can be generated by directed differentiation of high-potential or multi-potential stem cells (including human induced high-potential stem cells (hiPSCs), human embryonic stem cells (hESCs) and somatic cells (including transdifferentiated cells and stem cells)).

In some specific examples, the cell preparation includes a photosensitive rescue cell population in the cryopreservation formulation provided herein. Suitable photosensitive rescue cells can be differentiated from high-potential stem cells (such as human embryonic stem cells or iPS cells) and are molecularly different from embryonic stem cells, iPS cells, adult-derived photosensitive rescue cells, and fetal-derived photosensitive rescue cells. In some specific examples, adult-derived photosensitive rescue cells and fetal-derived photosensitive rescue cells are used. In some specific examples, when ES cell-derived photosensitive rescue cells are used, the cell preparation does not contain detectable residual ES cells, so that the cell preparation provided herein does not constitute an unacceptable risk to the recipients of such preparations. In some embodiments, where iPS cell-derived photorescive cells are used, the cell preparations do not contain detectable amounts of residual iPS cells, such that the cell preparations provided herein do not pose an unacceptable risk to recipients of such preparations.

In some embodiments, a cell preparation comprising a cell population suitable for transplantation into an individual's eye is suitable for injection into the individual's eye. In some embodiments, such cell preparations can be used to treat retinal degenerative diseases or conditions, including but not limited to retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy or associated with a variety of vision-altering conditions that lead to photoreceptor cell damage and blindness, such as achoroidal malformations, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spots on the eye).

The volume of the pharmaceutical composition provided in some embodiments of the present disclosure depends on factors such as the mode of administration, the number of cells to be delivered, the age and weight of the patient, and the type and severity of the disease being treated. For example, if administered by injection, the volume of the pharmaceutical composition of cells of the present disclosure may be about 1 to 1000 μL. In some embodiments, the volume may be about 1 to 200 μL. For example, the volume of the composition of the present disclosure may be about 10 to 50, 20 to 50, 25 to 50, or 1 to 200 μL. The volume of the compositions of the present disclosure may be about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μL or more.

In some embodiments, the cell concentration in the cryopreservation formulation (or cryopreserved cell preparation) is about 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 cells/µL. In some embodiments, the cell concentration in the cryopreservation formulation (or cryopreservation cell preparation) is about 10,000-100,000 cells/µL, 10,000-90,000 cells/µL, 10,000-80,000 cells/µL, 10,000-70,000 cells/µL, 10 ,000-60,000 cells/µL, 10,000-50,000 cells/µL, 10,000-40,000 cells/µL, 10,000-30,000 cells/µL, 10,000-20,000 cells/µL, 20,000-100,000 cells/µL 、20,000-90,000 cells/µL、20,000-80,000 cells/µL、20,000-70,000 cells/µL、20,000-60,000 cells/µL、20,000-50,000 cells/µL、20,000-40,000 cells/ µL, 20,000-30,000 cells/µL, 30,000-100,000 cells/µL, 30,000-90,000 cells/µL, 30,000-80,000 cells/µL, 30,000-70,000 cells/µL, 30,000-60,000 cells/µL, 30,000-50,000 cells/µL, 30,000-40,000 cells/µL, 40,000-100,000 cells/µL, 40,000-90,000 cells/µL, 40,000-80,000 cells/µL, 40,000-70,0 00 cells/µL, 40,000-60,000 cells/µL, 40,000-50,000 cells/µL, 50,000-100,000 cells/µL, 50,000-90,000 cells/µL, 50,000-80,000 cells/µL, 50,000-7 0,000 cells/µL, 50,000-60,000 cells/µL, 60,000-100,000 cells/µL, 60,000-90,000 cells/µL, 60,000-80,000 cells/µL, 60,000-70,000 cells/µL, 70,00 In some embodiments, the concentration of the cell population in the cell preparation is about 300 cells to 10,000 cells/µL. In some embodiments, the concentration of the cell population in the cell preparation is about 3,000 cells to 5,000 cells/µL or 3,000 cells to 4,000 cells/µL.

In some embodiments, the pharmaceutical composition or cell preparation is undiluted (e.g., not diluted prior to administration to a subject).

In some embodiments, the formulation supports the survival of cells in the cell population during storage of the formulation. In some embodiments, the formulation supports the survival of cells during storage for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or longer.

In some embodiments, the preparation is stored at -100 to -200°C, preferably at less than -135°C for about 1 to 10 years, about 1 to 9 years, about 1 to 8 years, about 1 to 7 years, about 1 to 6 years, about 1 to 5 years, about 1 to 4 years, about 1 to 3 years, or about 1 to 2 years or less, and at least 30% of the cells in the cell population are alive. In some embodiments, the preparation is stored at -100 to -200°C, preferably at less than -135°C for about 1 to 10 years, about 1 to 9 years, about 1 to 8 years, about 1 to 7 years, about 1 to 6 years, about 1 to 5 years, about 1 to 4 years, about 1 to 3 years, or about 1 to 2 years or less, and at least 40% of the cells in the cell population are alive. In some embodiments, the preparation is stored at -100 to -200°C, preferably at less than -135°C for about 1 to 10 years, about 1 to 9 years, about 1 to 8 years, about 1 to 7 years, about 1 to 6 years, about 1 to 5 years, about 1 to 4 years, about 1 to 3 years, or about 1 to 2 years or less, and at least 50% of the cells in the cell population are alive. In some embodiments, the preparation is stored at -100 to -200°C, preferably at less than -135°C for about 1 to 10 years, about 1 to 9 years, about 1 to 8 years, about 1 to 7 years, about 1 to 6 years, about 1 to 5 years, about 1 to 4 years, about 1 to 3 years, or about 1 to 2 years or less, and at least 55% of the cells in the cell population are alive. In some embodiments, after the preparation is stored at -100 to -200°C, preferably at less than -135°C, for about 1 to 10 years, about 1 to 9 years, about 1 to 8 years, about 1 to 7 years, about 1 to 6 years, about 1 to 5 years, about 1 to 4 years, about 1 to 3 years, or about 1 to 2 years or less, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the cells in the cell population are alive. In some embodiments, the preparation supports the maintenance of the vaccination efficiency of the cell population during the storage period of the preparation. In some embodiments, the preparation is stored at -100 to -200°C, preferably less than -135°C for about 1 to 10 years, about 1 to 9 years, about 1 to 8 years, about 1 to 7 years, about 1 to 6 years, about 1 to 5 years, about 1 to 4 years, about 1 to 3 years, or about 1 to 2 years or less, and the cell population exhibits at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of its original vaccination efficiency, wherein the original vaccination efficiency refers to the vaccination efficiency of the cell population at the beginning of the storage period. In some embodiments, the preparation is in a storage container. In some embodiments, the preparation is in a syringe or a cryovial.

The cell preparations provided herein are suitable for administration to individuals after minimal processing (e.g., dilution) after thawing. In some embodiments, the preparations are mainly composed of cells, cell groups or tissues and cryopreserved formulations as provided herein. In some embodiments, the preparations include one or more pharmaceutically active ingredients, such as preservatives, antioxidants, free radical scavengers, immunosuppressants, angiogenic factors, anti-angiogenic factors, growth hormones, or cell nutrients or substrates that support cell growth, survival, and implantation.

The following are examples that illustrate the present invention and should not be considered to limit the scope of the present invention.

Cellular properties and gene expression profiles of PRC cells, ESCs, and retinal pigment epithelium (RPE) cells were determined by cluster analysis and quantification of selected neuronal genes. Cluster analysis of ESC/RPE/PRC

Single cell suspensions processed according to the manufacturer's instructions were used to preform cluster analysis of ESC/RPE/PRC using the 10X Next GEM Chip G Single Cell Kit (10X Genomics #PN-1000120 or #PN-1000127) and the 10X Chromium Next GEM Single Cell 3'GEM, Library & Gel Bead Kit (10X Genomics #PN-1000121 or #PN-1000128) on a 10X Chromium machine ( Figure 1A ). Labeled cells in the library preparation were processed according to the kit instructions for the Illumina Single Indext Plate T Set A (Illumina #PN-2000240). The libraries were then shipped to Genewiz (now Azenta) for Illumina-based paired-end sequencing. Raw sequence data were mapped to the GRCh38 human genome using CellRanger-5.0.1. Mapped sequence data from scRNAseq were processed with Seurat and filtered based on quality control metrics, and underwent dimensionality reduction for visualization. Cells were first plotted in 2D UMAP and colored according to sample. ESC: undifferentiated research-grade J1-ESC and Kd-ESC. RPE: J1-RPE at P3+4 months. PRC: J1-PRC (P4), Kd-PRC (P4), GB2R-PRC-2019 (P4), GB2R-PRC-PR1 (P4), GB2R-PRC-CN2 (P4), GB2R-PRC-CN3 (P4).

PRC batches (e.g., PRC compositions) include GB2R-PRC-2019 (or GB2R-2019), GB2R-PRC-CN1 (or GB2R-CN1), GB2R-PRC-CN2 (or GB2R-CN2), GB2R-PRC-CN3 (or GB2R-CN3), GB2R-PRC-PR1 (or GB2R-PR1), and GMP-MBC-GB2R.

The expression of neuronal genes in ESCs, RPE, and PRCs was quantified by qRNA validation ( Figure 1B ). For RNA extraction and cDNA synthesis, wells were washed with PBS and 350 μl of LT buffer with 1:100 B-ME was added to the wells. Cells were disrupted by pipetting 20 to 30 times, and samples were then transferred to 2.0 mL Eppendorf tubes and stored at -80°C until further use. RNA extraction was performed using QIAcube (Qiagen) and RNeasy Plus Mini Kit (Qiagen, catalog number 74134) using the manufacturer's recommendations. RNA concentration was measured using Nanodrop 2000 (Thermofisher, catalog number ND2000CLAPTOP). cDNA synthesis was performed using the Invitrogen™ SuperScript™ IV First Strand Synthesis System using ezDNase™ enzyme (Thermofisher, catalog number 11766500) according to the manufacturer's protocol. Briefly, 1 μg of total RNA was incubated with ezDNase buffer and ezDNase in a GeneAmp® PCR System 2700 for 2 minutes at 37°C. SuperScript™ IV VILO™ Master Mix with reverse transcriptase (RT) and water was then added according to the manufacturer's protocol. The sample was gently mixed and incubated at 25°C for 10 minutes to anneal the primer, followed by incubation at 50°C for 10 minutes to reverse transcribe the RNA, and an enzyme inactivation step was performed at 85°C for 5 minutes. All reactions were performed in a GeneAmp® PCR System 2700. Room temperature reactions were diluted 1:20 and stored at -20°C until further use. PCR reactions were used in combination with Taqman fast advanced master mix (Thermofisher, Cat. No. A44360), nuclease-free water and 5 ng cDNA/reaction - individual taqman expression assays and custom PCR plates. PCR reactions were performed in an Applied Biosystems™ Quant studio 7 Flex PCR machine (Thermofisher, Cat. No. 4485701) with a denaturation step at 95°C for 20 seconds after 40 cycles of: annealing at 95°C for 1 second and extension at 60°C for 20 seconds. See Table 6 .

Table 6. Analysis IDs for individual Taqman probes and custom PCR plates . Individual Taqman Probes PCR Custom Plate Gene Gene PAX6 STMN2 RAX DCX NES MAP2 SOX2 C1ORF61 OCT4 LIN00461 LHX2 DLX2 SIX3 DLX5 SIX6 DLX1 RCVRN C11ORF96 RHO MART OPN1MW GAD2 OPN1SW SCGN ASCL1 ERBB4 CRX CALB2 RORB ELAVL4 OTX2 PRKCA NR2E3 GAD1 NRL NEUROD2 NEUROD6 TUJ1/TUBB3 SLA SOX11 NFIB NFIA EMX2 OCT4 BRN3A MATH5 NANOG CHX10 GAPDH C1ORF43 Bioinformatics analysis : error propagation in qPCR validation of selected neuronal genes

The relative qPCR expression of cell identity markers in GB2R PRCs compared to their expression in hESCs was calculated as follows. Assuming that the expression of the gene of interest (experimental condition, GB2R PRCs) is defined as TE = E(TE) + dTE and the expression of the gene of interest (control condition, hESCs) is defined as TC = E(TC) + dTC, the fold change between the expression representing the experimental condition and the expression representing the control condition is obtained according to the expression formula FC = 2-ddCt + dFC, where ddCt = E(TE) - E(TC) + dTE - dTC. The base 10 logarithm of the fold change is then calculated, revealing log10 FC ≈ log10 E(FC) + ln2/ln10 (d(dCtc)- d(dCtE)). The relative expression of hESCs was therefore defined as log10 E(FC) and its error bar was defined as s2logFC ≈ (ln2/ln10)2 (s2dCtC + s2dCtE - 2cov(dCtc, dCtE)) according to the instructions, where s2dCtC is the squared standard deviation of the dCt of the gene of interest (GB2R PRC) under the experimental condition (all technical/biological replicates) and s2dCtE is the squared standard deviation of the dCt of the gene of interest (hESC) under the control condition (all technical/biological replicates). Single cell analysis of PRC-P4

Single cell suspensions from each sample were processed using the 10X Next GEM Chip G Single Cell Kit (10X Genomics #PN-1000120 or #PN-1000127) and 10X Chromium Next GEM Single Cell 3' GEM, Library, and Gel Bead Kit (10X Genomics #PN-1000121 or #PN-1000128) on a 10X Chromium machine according to the manufacturer's instructions ( Figure 1C ). Labeled cells in the library preparation were processed according to the kit instructions for the Illumina Single Indext Plate T Set A (Illumina #PN-2000240). The libraries were then shipped to Genewiz (now Azenta) for Illumina-based paired-end sequencing. The raw sequence data were mapped to the GRCh38 human genome using CellRanger-5.0.1. The mapped sequence data of scRNAseq were filtered according to quality control metrics, underwent dimensionality reduction for visualization, and clustered using the common nearest neighbor method combined with the Louvain algorithm. Differential representation was thus performed on clusters, and the genes with the strongest differential representation were compared between clusters and several public data sources. Using many human datasets ( see Table 2 ), clusters were assigned "best guessed" cell attributes based on differentially expressed genes and expression patterns in development and reviewed datasets. Cells were first mapped in two-dimensional UMAP and colored according to the assigned cell type. The markers driving the "best guess" of the assigned cell type are shown in Table 8 .

Mapped sequence data for scRNA-seq were filtered according to quality control metrics, underwent dimensionality reduction for visualization, and clustered using the common nearest neighbor method combined with the Louvain algorithm. Differential performance analysis was performed on the clusters, and the most differentially expressed genes (DEGs) were compared between the clusters and several public data sources. See Table 7. Table 7 : Reference datasets for scRNAseq analysis Cell type Dataset used Human retina www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE142526 Human hippocampus www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119212 Human midbrain www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76381 Human skin www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE132672 Table 8 : Markers driving cellular properties in scRNAseq analysis Cell type Markers for identification Range of cell types in PRC All PRC groups FOXG1, MAP2 86.7% to 92.1% bi-positive range (98.5% single positive for Map2) Inhibitory neurons DLX5, TUBB3, SCGN, ERBB4, CALB2 38.9%-48.6% Excitatory neurons NEUROD2, NEUROD6, SLA, NELL2, SATB2 0.7%-8.2% Hybrid Neuron MEIS2, PBX3, GRIA2, CACNA1C 17.9%-36.1% Progenitor cells VIM, MKI67, CLU, GLI3 20.8%-34.1% Astrocytes GFAP, LUCAT1, MIR99AHG, FBXL7 0.9%-1.4% qPCR attribute analysis

Cells were harvested using RNAprotect (Qiagen) following the manufacturer’s instructions for qRNA profiling ( Fig. 1D ). RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, catalog no. 74134), and RNA concentration was then determined using NanoDrop (Thermo Fisher). cDNA was prepared at a concentration of 1 ng/μl in nuclease-free water using the SuperScript IV VILO (Thermo catalog no. 11766050) protocol. RNA expression of candidate genes was tested by qPCR using TaqMan probes (Thermo Fisher). 1 ng of total cDNA was used per qPCR reaction well. The final reaction mixture per well consisted of cDNA, Fast Advanced Master Mix (Thermo Fisher), nuclease-free water, and the respective TaqMan probe. The total reaction volume per well was 12 μl. qPCR was run using the comparative Ct program on a Quant Studio 7 flex instrument. The qPCR cycle parameters were as follows: "Fast", hold phase at 95°C (00.20); 40 cycles of PCR phases at 95°C (00.01) (step 1) and 60°C (00.20) (step 2). GAPDH was used as a reference gene. The mRNA expression values of the target gene normalized to the reference gene were obtained by calculating ΔCt (Ct value of the target gene - Ct value of the reference gene), and then 2-ΔCt values were derived.

Tables 9 to 12 show the cell types identified in the PRC preparations, the markers used to identify the cell types, and the expression of various markers. FIG. 1E shows the expression of eye movement area progenitor cell markers, rod/cone photoreceptor cell markers, and neuron markers in the inhibitory neurons, excitatory neurons, selective neurons, progenitor cells, and astrocytes shown in FIG. 1C. Table 9 shows cell markers classified according to cell type markers and their expression (via the PRC composition manufacturing scheme disclosed herein). Table 10 shows single cell sequencing results, which provide the percentage of single cells expressing cell markers in the PRC composition disclosed herein. Table 11 shows the transcripts per million (TPM) in bulk RNA-seq data of PRC compositions expressing cell markers as disclosed herein . Figures 1F to 1T show the expression of cell markers in the PRC compositions listed in Table 10 at day 2 (D2), day 12 (D12), day 19 (D19), day 37 (D37/P0), day 55 (D55/P1), day 72 (D72/P2), day 90 (D90/P3) and day 107 (D107/P4). Table 12 shows cells that have been classified based on the expression of astrocyte, excitatory neuron, inhibitory neuron, selective neuron or progenitor cell markers and the percentage of those cells expressing a particular marker (as determined by single cell sequencing). Table 9 Cell type markers Markers used to identify this cell type Performance Eye movement area progenitor cell marker Pax6 10,000x increase by RNAseq vs. hESCs at d19-21; 85%+ by flow cytometry at d19-21; high expression in sc-qPCR at p4 (vs. no expression in J1 ESCs), 60x increase in bulk qPCR at p4 vs. GMP1 iPSCs Rx1 Trace/absent at p4 (by sc and bulk qPCR), similar to J1 hESCs and GMP1 iPSCs; not assessed at the protein level at d19-21 (only 14.8 TPM by RNAseq) LxD At d19-21, according to flow cytometry, it was 89%; in sc and bulk PCR, it was highly expressed in p4 J1 PRC and GMP1 PRC d19-21 Six3 149.2 transcripts per million (TPM) RNAseq d19-21 Six6 Trace/absent at the genetic level (qPCR/RNAseq) d19-21 Time Tbx3 Trace/absent at the genetic level (qPCR/RNAseq) d19-21 Nestin At d19-21, it exceeded hESC by 3.39X (via PCR) d19-21 Sox2 Protein level: 78% (d19-21, by flow cytometry) and 33% (p3, by flow cytometry); Gene level: 1.87X higher than hESC (by PCR, d19 to 21) Rod/cone photoreceptor cell markers MASH1 Highly represented in scRNAseq relative to hESCs at p4 and 12x increased relative to GMPi-iPSCs at p4, 364 TPM by RNAseq at p3 (not compared to ESCs) CRX Absence, similar to J1 hESC/GMP1 iPSC at p4 by sc and bulk qPCR; Absence, similar to J1 hESC/GMP1 iPSC at p3 by RNAseq R sc-qPCR: some detected in very few cells, but lower than J1 ESC; bulk qPCR: similar to GMP1-iPSC; very low by ICC at p3 Tb2 At p3, it was lower, at 14.9 transcripts/million as measured by RNAseq (non-hESC comparison) NR2E3 Absent in sc and bulk qPCR, similar to J1 ESC and GMP1-iPSC NRL Absent in sc and bulk qPCR, similar to J1 ESC and GMP1-iPSC Rhodopsin ICC at p4: When using the 4D2 antibody, the expression is not higher than the background. ICC at p4: When using the 1D4 antibody, the signal is detected not only in neural stem cells but also in GRCs, so the signal is non-specific staining (not actually rhodopsin). No expression in vivo Opsin sc-qPCR: Opn1mw - similar to J1 ESCs, moderate signal detected; bulk qPCR: opn1mw - 10x increase compared to GMP1-iPSCs PDE6B sc-qPCR: small population, lower expression than J1 ESC; bulk qPCR: 40% of GMP1-iPSCs at p4 Recovery protein At p3, it was absent by RNAseq; at p4, it was absent by sc and bulk qPCR. ARR3 sc-qPCR: lacking, similar to J1 ESC bulk qPCR: lacking, relative to GMP1-iPSC Cnb sc-qPCR: lacking, similar to J1 ESC bulk qPCR: lacking, relative to GMP1-iPSC Gnat1 sc-qPCR: lacking, similar to J1 ESC bulk qPCR: lacking, relative to GMP1-iPSC Gnat2 sc-qPCR: lacking, similar to J1 ESC bulk qPCR: lacking, relative to GMP1-iPSC Neuron markers Tuj1 (mature) At p0, it increased by 61x compared to hESC. At p4, sc-qPCR showed high expression, slightly exceeding the already highly expressed J1 ESC; bulk qPCR: increased by about 5x compared to GMP1-iPSC NFIA At p4, according to ICC, it is 60%; sc-qPCR: highly expressed relative to J1 ESCs Bulk qPCR: increased by about 215x relative to GMP1-iPSCs DCX sc-qPCR: highly expressed relative to J1 ESC at p3 Bulk-qPCR: increased by about 535x relative to GMP1-iPSC at p3 NFIB sc-qPCR: highly expressed relative to J1 ESC at p3 Bulk-qPCR: increased by about 190x relative to GMP1-iPSC at p3 OTX2 At p4, 86% by flow cytometry; downregulated relative to J1 ESCs by sc-qPCR; deficient relative to GMP1-iPSCs by bulk qPCR; and hESCs below 30.9 TPM by RNAseq Retinal neural progenitor cell markers CHX10 Only 2.1 transcripts per million (TPM) at p0, which is 73x more than hESC at p0. Trace by RNAseq at p3, although still 73X more than hESC; earlier datasets show trace/absent at p4 (sc and bulk qPCR). ICC data from d19 to 21 show trace/absent at the protein level Potential stem cell markers SSEA4 Negative Oct4 Negative PRC attribute tag MAP2, FoxG1 By flow cytometry for MAP2, it was 88%; by flow cytometry for FoxG1, it was 95%. MAP2 measured by bulk qPCR was increased 100x relative to iPSCs Other neuronal markers Elavl3 sc-qPCR: highly expressed compared to J1 ESC; bulk qPCR: greatly increased compared to GMP1-iPSC Elavl4 sc-qPCR: highly expressed compared to J1 ESC; bulk qPCR: greatly increased compared to GMP1-iPSC Slc1a2 sc-qPCR: highly expressed compared to J1 ESC; bulk qPCR: greatly increased compared to GMP1-iPSC Slc1a3 sc-qPCR: High expression, similar to that of J1 ESCs Bulk qPCR: Approximately 7x increase compared to GMP1-iPSCs Hcn1 sc-qPCR: lacking, similar to J1 ESCs; bulk qPCR: lacking, relative to GMP1-iPSCs Hes5 sc-qPCR: increased expression relative to J1 ESCs Bulk-qPCR: increased by approximately 180x relative to GMP1-iPSCs Functional markers of neuronal electrophysiological activity Voltage-gated sodium current (Nav) Artificial patch clamp: subpopulation expresses moderate amounts. (Photoreceptor cells do not express Nav) M-type potassium current Artificial patch clamp: cells in the PRC population with Nav also have larger M-currents A-type potassium current Artificial patch clamp: Expressions enriched in populations expressing Nav Superpolarized Induced Current (Ih) Artificial patch clamp: Not observed (photoreceptor cells have Ih mediated by HCN1 channels) Table 10 : Percentage of cells expressing cell markers Marker Type Gene GB2R-2019 GB2R-CN2 GB2R-CN3 GB2R-PR1 average value PRC Mark FOXG1 78.8 57.4 72.6 81.3 72.525 MAP2 94.8 77.8 89.9 92.3 88.7 STMN2 70.1 72 81.6 82.7 76.6 DCX 88.3 75.9 85.9 88 84.525 LINC00461 93.1 75.6 87.1 91.1 86.725 NEUROD2 2 3.1 3 18.9 6.75 GAD1 61.4 44.1 64.4 44.4 53.575 NFIA 69.4 69.1 71.1 85.8 73.85 Inhibitory neuron markers DLX5 68.7 54.9 65.6 61.1 62.575 TUBB3 92.4 78.3 86 90 86.675 SCGN 61.1 49.5 50.3 54.7 53.9 ERBB4 67.8 66.9 71 63.9 67.4 CALB2 62 51.4 42.7 54.2 52.575 Excitatory neuron markers NEUROD2 2 3.1 3 18.9 6.75 NEUROD6 0.8 4.2 2.4 23.4 7.7 SLA 1.2 2.9 1.9 16.2 5.55 NELL2 35.2 20.6 27.2 30.1 28.275 SATB2 3.3 4 3 11.3 5.4 Progenitor cell markers VIM 68.4 46.3 43.5 59.9 54.525 MKI67 11.7 7.3 7 8.5 8.625 CLU 50.3 31.3 26.5 46.5 38.65 GLI3 21.9 15.3 16.9 28.3 20.6 Astrocyte markers GFAP 13.1 3.3 1.7 3.2 5.325 LUCAT1 12.8 7.6 10 14.7 11.275 MIR99AHG 87.8 75.8 81.2 87.6 83.1 FBXL7 54.6 38.6 34.9 45.5 43.4 Selective neuron labeling MEIS2 81.6 50.4 77.6 62.2 67.95 PBX3 70.3 49.7 74.5 48.1 60.65 GRIA2 31.3 23.1 46.3 39.7 35.1 CACNA1C 53 39.4 59.5 54.2 51.525 Eye movement area progenitor cell marker PAX6 50.6 30.5 41.6 46.6 42.325 LHX2 9 7.2 7 24.6 11.95 SIX3 6.6 2.2 10 2.8 5.4 NES 32.9 19.8 30.2 30.4 28.325 SOX2 78 60.8 72.8 70.4 70.5 Rod/cone photoreceptor cell markers ASCL1 26.9 29.4 29.7 41.3 31.825 RORB 12.4 11.8 20.4 twenty two 16.65 NR2E3 2.9 2.1 3.7 3 2.925 NRL 6.6 2.9 8 5.8 5.825 Neuron markers TUBB3 92.4 78.3 86 90 86.675 NFIA 69.4 69.1 71.1 85.8 73.85 NFIB 94.9 83.4 89.6 94.4 90.575 OTX2 5.7 2.2 2.8 4.1 3.7 ELAVL3 84.8 60.9 76.3 82.5 76.125 ELAVL4 51 51.8 60.4 71.4 58.65 SLC1A2 71 51.9 66 72.9 65.45 SLC1A3 17.7 11.8 11.2 23.1 15.95 HCN1 1.5 3.3 3.9 4 3.175 HES5 19.4 13.7 10.5 25 17.15 Negative marker CRX 0.3 0.1 0.3 0.3 0.25 RHO 0 0 0 0 0 OPN1SW 1.1 0.7 1.2 1.8 1.2 PDE6B 1 1.7 1.7 3.3 1.925 RCVRN 0.5 0.2 0.2 0.3 0.3 ARR3 0.8 0.9 0.9 1 0.9 CNGB1 0.3 0.1 0.2 0.5 0.275 GNAT1 0 0 0.3 0.1 0.1 GNAT2 0.2 0.1 0.1 0.2 0.15 VSX2 0.2 0 0.2 0.3 0.175 POU5F1 0.2 0.3 0.4 0.5 0.35 OCT4 #N/A #N/A #N/A #N/A SSEA4 #N/A #N/A #N/A #N/A RAX 0.1 0.1 0 0.1 0.075 SIX6 0 0 0 0 0 TBX3 0.1 0 0.2 0.3 0.15 Table 11 : RNA Bulk Sequencing ( TPM ) ​ ​ ​ Marker Type Gene GB2R-2019 GB2R-CN1 GB2R-CN2 GB2R-CN3 GB2R-PR1 average value PRC Mark FOXG1 131.915 59.99 149.915 161.895 169.505 134.644 MAP2 509.965 290.29 494.075 611.475 561.91 493.543 STMN2 223.885 193.79 464.255 555.88 544.98 396.558 DCX 485.58 259.43 781.595 847.215 701.575 615.079 LINC00461 89.065 53.43 92.27 88.665 85.38 81.762 NEUROD2 0.555 0.03 1.105 0.465 8.19 2.069 GAD1 16.895 12.45 27.21 40.525 16.495 22.715 NFIA 116.92 33.56 79.41 64.925 86.905 76.344 Inhibitory neuron markers DLX5 88.92 50.78 137.29 131.595 82.535 98.224 TUBB3 307.12 162.86 428.605 378.475 375.25 330.462 SCGN 122.955 74.94 172.31 120.075 103.74 118.804 ERBB4 36.235 18.24 93.655 73.075 57.945 55.83 CALB2 89.615 53.85 196.435 76.895 94.915 102.342 Excitatory neuron markers NEUROD2 0.555 0.03 1.105 0.465 8.19 2.069 NEUROD6 5.45 1.2 19.85 12.525 203.175 48.44 SLA 4.39 0.35 5.425 3.78 50.21 12.831 NELL2 27.21 4.86 27.59 25.695 128.885 42.848 SATB2 2.49 0.73 2.445 2.85 20.405 5.784 Progenitor cell markers VIM 861.375 262.82 320.97 270.5 294.65 402.063 MKI67 33.66 11.89 19.225 21.155 21.825 21.551 CLU 365.23 46.78 101.825 63.205 89.87 133.382 GLI3 43.93 11.95 23.48 23.345 37.635 28.068 Astrocyte markers GFAP 122.39 0.48 14.305 3.93 16.89 31.599 LUCAT1 N/A N/A N/A N/A N/A N/A MIR99AHG 23.81 6.68 12.76 10.535 16.065 13.97 FBXL7 23.785 7.72 13.91 11.94 14.385 14.348 Selective neuron labeling MEIS2 138.325 15.28 61.52 173.24 67.2 91.113 PBX3 45.6 5.49 29.995 88.11 12.635 36.366 GRIA2 19.73 4.48 11.35 29.025 27.295 18.376 CACNA1C 4.605 1.14 3.665 6.68 3.57 3.932 Eye movement area progenitor cell marker PAX6 99.025 36.67 77.07 73.885 87.035 74.737 LHX2 15.465 8.54 10.81 9.78 35.71 16.061 SIX3 10.17 2.48 5.865 19.94 3.85 8.461 NES 26.805 7.39 12.94 11.805 10.9 13.968 SOX2 196.375 96.53 173.06 156.77 139.38 152.423 Rod/cone photoreceptor cell markers ASCL1 111.61 68.31 127.745 98.52 122.375 105.712 RORB 2.675 3.03 5.005 6.125 5.28 4.423 NR2E3 0.415 0.17 0.35 0.395 0.505 0.367 NRL 0.715 0.28 0.585 1.36 0.545 0.697 Neuron markers TUBB3 307.12 162.86 428.605 378.475 375.25 330.462 NFIA 116.92 33.56 79.41 64.925 86.905 76.344 NFIB 486.145 187.76 473.24 405.535 605.53 431.642 OTX2 22.915 8.13 14 13.415 16.94 15.08 ELAVL3 60.595 22.61 74.855 78.615 79.42 63.219 ELAVL4 38.49 28.91 80.165 84.065 83.235 62.973 SLC1A2 87.305 22.2 59.16 62.325 68.415 59.881 SLC1A3 176.995 108.35 73.53 78.325 79.625 103.365 HCN1 0.13 0.14 0.49 0.345 0.54 0.329 HES5 25.83 15.38 37.59 18.065 45.155 28.404 Negative marker CRX 0.25 0 0.075 0 0.025 0.07 RHO 0 0.02 0.015 0.025 0 0.012 OPN1SW 0.05 0 0.085 0.065 0.17 0.074 PDE6B 0.045 0.07 0.165 0.155 0.155 0.118 RCVRN 0.1 0 0.085 0.02 0 0.041 ARR3 0.16 0 0 0 0 0.032 CNGB1 0.025 0.03 0.04 0.015 0.04 0.03 GNAT1 0.02 0 0 0.02 0.09 0.026 GNAT2 0.38 0 0 0.055 0 0.087 VSX2 0.105 0.02 0.065 0.015 0.185 0.078 POU5F1 1.41 5.42 2.42 3.545 3.93 3.345 OCT4 #N/A #N/A #N/A #N/A #N/A SSEA4 #N/A #N/A #N/A #N/A #N/A RAX 0.145 0.02 0.09 0.1 0.255 0.122 SIX6 0.2 0.02 0.045 0.015 0.035 0.063 TBX3 0.075 0.04 0.02 0.09 0.09 0.063 Table 12 : Percentage of cells with marker expression > 0 Mark Astrocytes Excitatory neurons Inhibitory neurons Selective Neuron Progenitor cells ARR3 1.7 0.2 1.1 1.0 0.4 ASCL1 12.1 20.2 42.4 9.6 41.9 CNGB1 0.0 1.1 0.2 0.2 0.4 CRX 06 0.5 0.3 0.2 0.3 GNAT1 0.0 0.4 0.1 0.1 0.1 GNAT 2 0.6 0.0 0.1 0.2 0.1 LHX2 48.6 56.8 2.7 2.9 29.3 NES 32.4 8.1 32.6 16.6 41.0 NR2E3 6.4 2.3 3.9 2.7 1.9 NRL 7.5 4.6 4.9 10.0 4.2 OPN1SW 4.6 0.5 1.1 1.0 1.6 PAX6 56.6 13.0 46.5 41.8 45.2 PDE6B 4.0 3.0 2.0 1.5 1.8 RAX 0.0 0.0 0.0 0.0 0.1 RCVRN 0.0 0.0 0.5 0.0 0.4 RHO 0.0 0.0 0.0 0.0 0.0 RORB 60.1 24.1 10.7 15.0 26.8 SIX3 9.2 0.4 1.6 11.7 7.1 SIX6 0.0 0.0 0.0 0.0 0.1 SOX2 66.5 12.8 88.5 62.5 63.7 TBX3 0.6 0.2 0.2 0.2 0.1 DCX 37.6 81.7 98.7 92.9 58.3 ELAVL3 61.3 70.8 92.1 82.0 51.4 ELAVL4 17.3 60.3 84.5 46.6 32.8 FOXG1 80.9 69.1 90.4 68.1 54.4 HCN1 1.2 0.9 5.7 1.8 0.8 HESS 42.2 12.8 10.1 6.1 41.3 MAP2 93.6 86.3 97.8 95.5 71.1 NFIA 100.0 71.2 86.5 51.8 77.5 NFIB 98.8 90.7 98.5 91.2 79.5 OTX2 30.6 1.8 0.5 0.4 12.2 POU5F1 1.2 0.2 0.2 0.4 0.5 SLC1A2 75.1 65.9 76.6 68.2 49.2 SLC1A3 96.5 35.0 3.8 4.6 43.0 SSEA4 NA NA NA NA NA TUBB3 66.5 84.0 96.7 88.7 73.0 VSX2 0.0 0.2 0.1 0.3 0.2 PRC Bright Vision Image

Bright field images were acquired at 20x, 10x, and 4x magnification using a Keyence microscope (BZ-X710) on different days of PRC differentiation. The images shown are representative images of GB2R-derived PRCs acquired at P4 (d107) ( Figure 2A ). Scorecard analysis of PRC differentiation

For the scorecard analysis, cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Thermofisher, Catalog No. 4368813) according to the manufacturer’s protocol ( Figure 2B ). TaqMan hPSC Scorecard Assay (Thermofisher, Catalog No. A15870) was used following the manufacturer’s protocol using TaqMan Standard Master Mix (Thermofisher, Catalog No. 4369016). PCR reactions were performed in Applied Biosystems™ Quant studio 7 Flex using the manufacturer’s recommended cycling parameters. The trilineage potential of PRCs was quantified using the hPSC Scorecard Assay software (Thermofisher). ICC performance in PRC-P4

At P4, immunocytochemistry (ICC) staining of PRCs was performed using black Ibidi µ-dishes 24-well (Ibidi, catalog number 82406) ( Figure 2C ). Cells were washed 3 times with PBS -/-, fixed with 4% PFA and washed 3 times with PBS -/-. After removing PBS, cells were treated with permeabilization solution containing 0.2% Triton-X for 10 minutes. Fixed cells were washed with PBS (containing calcium and magnesium) and subsequently blocked with PBS (containing calcium and magnesium) containing 5% goat serum (+/- 5% donkey serum) for 1 hour at room temperature. Primary antibodies (Pax6 (Millipore ab#2237), Otx2 (Thermo, #MA5-15854), STMNN2 (ProteinTech catalog #10586-1-AP), CALB2 (Sigma catalog #MAB51568), SCGN (Sigma catalog #HPA006641), and DCX (Santa Cruz catalog #SC-271390) were diluted in blocking solution and incubated overnight at 4°C. The cells were incubated with 0.05% Samples were washed three times in PBS with Tween (PBST) for 5 to 10 minutes. Secondary antibodies were diluted in blocking solution and incubated for 1 hour at room temperature. Samples were then washed with PBST for 15 minutes and then washed twice for 5 minutes in PBS containing calcium and magnesium. ProlongGold (Thermofisher, catalog number P36931) containing DAPI was added to the samples. Imaging was performed using a Leica DMi8 microscope using LAS X software. PRC labeling

Successful PRC markers were identified by analyzing the total transcripts of PRCs during different phases. The total transcripts of GB2R PRCs during different phases of their regimen were combined into a single dataset, and transcription factors were focused on as the main drivers of GB2R PRC differentiation and maturation processes. PCA-based dimensionality reduction techniques were applied, and the identified dominant components revealed the differentiation and maturation trajectories of GB2R PRCs in the latent space of the dominant components. A linear model describing the variation in gene expression along the maturation trajectory was constructed and transcription factors whose expression varied systematically and monotonically along the differentiation trajectory were identified. Transcription factors with the greatest variation in expression along the differentiation/maturation trajectory of PRC GB2R were identified as successful PRC markers. Figure 2D shows that the expression of NEUROD2 increased at P1, while FOXG1 and HMGA1 remained largely the same as at P0.

Flow cytometry was performed to determine the percentage of cells expressing FOXG1, MAP2, and not SSEA4 ( Fig. 2E ). ESC and PRC-P4 cells were thawed in 15 ml tubes and the cell density was adjusted to 1×10 ⁶ /ml and processed as described below. Percentage of cells expressing purity markers: Mean % (GB2R-PRC-CN3 FCP, N = 3). FOXG1: 95.6 (94.2-96.7); MAP2: 88.4 (84.7-91.4); and SSEA4: 0.44 (0.15-0.88).

Bulk RNA-seq analysis was performed on PRC-P3 and PRC-P4 ( Fig. 2F ). Nineteen genes with differential expression were identified between PRC-P3 and PRC-P4. AGT, ACBLN2, CDH7, DNAH11, and EGR1 increased in expression at P4 compared to P3. FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, and UNC13C decreased in expression at P4 compared to P3.

Live / dead cell staining : Cells were centrifuged at 300 g for 5 minutes at room temperature and after aspiration, 1 ml PBS containing a 1:10000 diluted fixable red dead cell stain kit was added and cells were incubated at 4°C for 30 minutes. 5 ml PBS was added and samples were centrifuged at 300 g for 5 minutes at room temperature. Samples were aspirated and cells were resuspended in HBSS (Thermo Fisher Scientific, catalog number 14025092) + 2% FBS to 3×10 ⁶ /ml.

SSEA4 staining : Transfer 100 µl (3 × 10 ⁵ cells) of the above cell suspension to a "U" bottom 96-well plate. Add 1 µl of anti-SSEA4-FITC antibody (Miltenyi Biotech, 130-122-918) to each well, mix well and incubate cells at 4°C for 30 minutes. Wash cells twice: 1st wash with 200 µl HBSS + 2% FBS per well, followed by centrifugation at 2000 rpm for 1 minute at 4°C; 2nd wash with 200 µl PBS per well, followed by centrifugation at 2000 rpm for 1 minute at 4°C.

Fixation and Permeabilization : Add 100 µl of Fixation/Permeabilization Buffer (Transcription Factor Buffer Set, BD Bioscience, Catalog No. 562574) to each well, mix well, and incubate cells at 4°C for 30 minutes. Wash cells twice: wash with 200 µl of Fixation/Permeabilization Buffer per well each time, followed by centrifugation at 2000 rpm for 1 minute at 4°C.

FOXG1/MAP2 staining : Resuspend cells with 50 µl of Fix/Permeabilization Buffer per well, add 50 µl of 2 µg/ml anti-FOXG1 (Abcam, catalog number ab196868) or 2 µg/ml anti-MAP2 (Cell Signaling, catalog number 4542S) antibody per well, mix well and incubate at 4°C for 30 minutes. Wash cells twice: wash with 200 µl of Fix/Permeabilization Buffer per well each time, then centrifuge at 2000 rpm for 1 minute at 4°C. Add 100 µl of fixation/permeabilization buffer containing 1:250 diluted anti-rabbit-IgG-PE antibody (Thermo Fisher Scientific, catalog number A10542) to each well, mix well and incubate at 4°C for 30 minutes; wash cells twice, each time with 200 µl of fixation/permeabilization buffer per well, followed by centrifugation at 2000 rpm for 1 minute at 4°C. Flow cytometry analysis was performed using a Sony cell analyzer SA3800. Resuspend cells with 200 µl of HBSS+2% FBS per well, mix well and analyze samples using a Sony cell analyzer SA3800. 10,000 gated events were acquired per well and data were analyzed using FlowJo software (version 10.8.1). Positive signals against IgG control were confirmed for each cell type. The percentage of PRC-P4 positivity in populations with higher signal intensity than ESC was calculated. Stimulation of neuroprotective factors

PRC cells were stimulated by oxidative stress using tertiary butyl hydroperoxide (TBHP), and secreted factors were detected using Luminex analysis ( Figure 3A ). NRF2-mediated oxidative stress response pathways in the retina of hNPC-treated RCS rats were activated in vitro and in vivo as shown in Jones et al., 2019, Proteomics, 19, e1800213.

PRC-P4 cells were thawed on compliant matrices (PDL, fibronectin and laminin-521) at a density of 220K to 250K cells per well of 24W plates (well surface area - 1.9 ^cm2 ). The next day (D1), TBHP dilutions (10 μM to 50 μM) were prepared in PRC medium. The concentration of TBHP solutions is extremely high (approximately 5.1 M or 5100 mM, mention the lot number for the exact concentration) and 1:10 serial dilutions were used to reduce the target concentration. The final TBHP dilution was done in PRC medium. As a vehicle control, the decane solution was diluted in PRC medium after the TBHP dilution step (up to 50 μM TBHP dilution). Cells were treated with TBHP for 2 days and conditioned medium was collected from each well at D3 post-inoculation. Conditioned medium was centrifuged at 4000 g for 10 minutes to remove debris. Conditioned medium was aliquoted and stored at -80°C for Luminex/ELISA. Luminex analysis was performed according to the manufacturer's instructions (Life technologies Procartaplex 6 Plex Luminex assay, assay ID: MX323GF, species: human, targets (neural): CNTF, GFAP, MIF, NCAM-1, S100B, Tau (all)). The mean fluorescence intensity (MFI) of each sample and standard was obtained using a Millipore Milliplex analyzer in conjunction with Luminex MAP technology. The secretion levels of various interleukins in the test samples were compared using the MFI of the test samples. To calculate the concentration, a standard curve of known concentrations versus the reading (MFI) at various concentrations was plotted. Curve fitting and data analysis were performed using Sigmoidal 4PL (4-parameter logic) in GraphPad Prism. The concentration of the test sample was extrapolated on the standard curve.

Results were compared to factors secreted by PRC cells in the absence of oxidative stress ( Fig. 3B ). PRC-P4 cells were thawed and seeded at a density of 120K cells per well of 24W plates on compliant matrices (PDL, fibronectin, and laminin-521) (well surface area - 1.9 ^cm2 ). Fresh medium was fed to PRCs one day after feeding or after collecting the inoculated conditioned medium 2 days after feeding or 3 days after inoculation. Conditioned medium was centrifuged at 4000 g for 10 minutes to remove debris. Conditioned medium was aliquoted and stored at -80°C for Luminex analysis. Cells from one to two wells were counted using acurase. Record cell counts at the collection step. Luminex analysis was performed according to the manufacturer's instructions (Life technologies Procartaplex 6 Plex Luminex assay, assay ID: MX323GF, species: human, targets (neural): CNTF, GFAP, MIF, NCAM-1, S100B, Tau (all)). The mean fluorescence intensity (MFI) of each sample and standard was obtained using the Millipore Milliplex analyzer in conjunction with Luminex MAP technology. The secretion levels of various interleukins in the test samples were compared using the MFI of the test samples. To calculate the concentration, a standard curve of known concentrations relative to the readings (MFI) of various concentrations was plotted. Curve fitting and data analysis were performed using Sigmoidal 4PL (4 parameter logic) on GraphPad Prism. Extrapolate the test sample concentration on the standard curve.

Subretinal transplantation of PRC inhibited microglial infiltration into the ONL ( Fig. 3C ). In RCS retinas, microglia migrate to the outer retina in response to oxidative stress. Confocal imaging of retinal cross-sections showed reduced infiltration of Iba1+ (Wako 019-19741) microglia into the outer retina in the presence of transplanted PRC compared with non-transplanted areas, where microglia fully filled the subretinal space. The outer retina is bounded by the outer tuft layer (OPL) at the anterior end and enclosed by the RPE layer at the posterior end, as indicated by the yellow dashed line. In addition, co-staining of HuNu+ PRC with phosphorylated (or activated) nuclear factor-erythroid factor 2-related factor (Nrf2) (anti-pNrf2 antibody, Invitrogen PA5-67520) and superoxide dismutase 2 (SOD2) (anti-SOD2, Abcam Ab110300), one of its downstream regulators, revealed the possible role of Nrf2-mediated pathway in regulating the response of transplanted PRC to oxidative stress. The stained retinal sections were imaged on a Leica SP8 using confocal z-stack analysis.

Microglial cell line SIM-A9 was stimulated with LPS and inhibited with L-carnitine. 60K SIM-A9 cells were seeded per well of 48W culture plates (well surface area - 0.95 ^cm2 ) ( Figure 3D ). The next day (D1 after seeding), L-carnitine solutions with concentrations of 30 mM, 10 mM, 2 mM, and 0 mM were prepared in PRC medium (NDM) (vehicle - water). The L-carnitine solutions were diluted to half of the original concentration by adding an equal volume of SIM-A9 medium to the L-carnitine solutions. The final concentrations of the L-carnitine solutions were 15 mM, 5 mM, 1 mM, and 0 mM. SIM-A9 cells were treated with L-carnitine solution for 48 hours.

48 hours after L-carnitine treatment (D3 after inoculation), 10 μg/mL LPS solution was prepared from 5 mg/mL LPS stock solution in SIM-A9 medium. Carnitine solutions with concentrations of 30 mM, 10 mM, 2 mM, and 0 mM were prepared in PRC medium (NDM) (vehicle - water). L-carnitine solutions were diluted to half of the original concentration by adding an equal volume of SIM-A9 medium containing 10 μg/ml LPS to the L-carnitine solutions. The final concentrations of L-carnitine solutions were 15 mM, 5 mM, 1 mM, and 0 mM, and the final concentration of LPS in each L-carnitine solution was 5 μg/ml. SIM-A9 cells were treated with L-carnitine solution containing LPS for 24 hours. Conditioned medium was collected from SIM-A9 wells. Conditioned medium was centrifuged at 4000 g for 10 minutes to remove debris. Conditioned medium was aliquoted and then stored at -80°C for TNF-α ELISA. ELISA analysis was performed following the manufacturer's instructions (Mouse TNF α ELISA Kit; Abcam ab208348). A Spectramax-M5 plate reader from Molecular Devices was used to obtain the optical density (OD) of each sample and standard. A standard curve of known concentrations relative to the readings (OD) of various concentrations was plotted. Sigmoidal 4PL (4 parameter logic) was used for curve fitting and data analysis on GraphPad Prism. The concentration of the test sample was extrapolated on the standard curve. After collecting the conditioned medium from the SIM-A9 well, the cells in each well were collected and placed in RNA protection solution, and RNA extraction, cDNA synthesis, and RNA expression analysis were performed by qPCR for IL1b and NOS2 using the methods explained in the "RNA expression analysis by qPCR" section.

After hNSC/NPC transplantation (rd1 mice, RCS rats), reduced microglial cell activation was observed. After co-culture with hNSC, microglial cell activation was found to be inhibited. Inhibition of microglial cell infiltration was observed at the PRC transplantation site. (Li et al., 2016, Cytotherapy, 18, 771-784; Jones et al., 2016, MolVis, 22, 472-490). Phagocytosis analysis

The ability of PRC to phagocytose pHrodo E. coli particles was assessed ( Fig. 3E ). PRC-P4 cells were thawed and seeded at 2.5 × 10 ⁵ cells per well in a 24-well plate on PDL + fibronectin + laminin-521 (PDL: Advanced biomatrix, catalog number 5049-50 ml; human fibronectin: Akron Biotech, catalog number AK9715-0005; laminin-521: Biolamina, catalog number LN521). The next day, 0.5 ml of cells were re-injected per well. Prepare pHrodo bioparticles on day 2 after seeding cells as follows: Add 2 mL of neural differentiation medium (NDM) to one vial of pHrodo bioparticles to prepare a 1 mg bioparticle/mL suspension. Vortex the bioparticle vial at 3,200 rpm for 45 to 50 minutes at 4°C to verify the absence of aggregates. On day 2 after seeding cells, add 200 µl NDM + 300 µl (300 µg) pHrodo bioparticles per well. Incubate pHrodo bioparticles and cells at 37°C or 13°C (lower temperature control) for 20 to 28 hours. For collection and flow cytometric analysis, wash cells at least 3 times with 200 µl PBS per well. Cells were lysed using StemPro Aquapase (Thermo Fisher Scientific, Catalog No. A1110501) by adding 300 µl per well and incubating at 37°C for 7 minutes. Single cell suspensions were generated by pipetting the cell suspensions. 300 µl NDM was added per well and the collected cell suspensions were transferred to "U" bottom polystyrene tubes. Cell suspensions were centrifuged at 160 g for 5 minutes at room temperature. Supernatant was aspirated and 300 µl HBSS + 2% FBS + 1 µg/ml propidium iodide (Thermo Fisher Scientific, Catalog No. P3566) was added. Tubes were vortexed for approximately three (3) seconds and samples were analyzed using a Sony cell analyzer SA3800. 10,000 gated events were acquired for each tube, and data were analyzed by FlowJo (version 10.8.1). The % of PRC-P4 incorporated into pHrodo bioparticles was calculated by comparison with two negative controls: no bioparticle control (37°C, no pHrodo bioparticles) or cold control (13°C, with pHrodo bioparticles). PRC internalizes rod outer segment ( ROS ) fragments in the retina of RCS rats

Royal College of Surgeons (RCS) rats received subretinal injections of 100,000 GB2R-PRCs or GS2 vehicle at P23 ( Fig. 3F ). Rats were anesthetized with ketamine (75 mg/kg) and dexmedetomidine (0.25 mg/kg) intraperitoneally (IP) at approximately P23. The pupils were dilated with 1% tropicamide (Bausch & Lomb, Rochester, NY) or 1% atropine (Bausch & Lomb) or 2.5% phenylephrine hydrochloride (Bausch & Lomb, Inc.). Nonabsorbable sutures (4-0) (Ethicon, Somerville, NJ) were placed in place to hold the eye in an upright orientation. An iridotomy was performed in the superior dorsal region of the temporal lobe of the eye using a 25 5/8 G metal needle. 2 µl of the cell suspension was drawn into a sterile glass pipette (internal diameter 50 to 150 µm) through a plastic tube filled with GS attached to a 25 µl Hamilton syringe. Cells or GS (2 μL volume) were injected into the subretinal space through the iridotomy site. To reduce intraocular pressure and limit the outflow of cells, the cornea was punctured using a 30 G metal needle. The fundus was examined immediately after the injection for retinal damage or vascular pain. The wound was closed using non-absorbable surgical sutures (10-0) (Ethicon). The sutures around the eyeball were removed and the eyelids were then placed in their normal position. Finally, ophthalmic erythromycin ointment (Bausch & Lomb) was applied topically. The animals recovered from anesthesia on a warming pad (37°C). During the 2 weeks following the subretinal procedure, the animals received daily injections of dexamethasone (American Regent, Shirley, NY) (1.6 mg/kg, intraperitoneally (ip)). In addition, the animals received cyclosporine-A (Bedford Laboratories, Bedford, OH) (210 mg/L) in their drinking water throughout the experiment.

Animals were sacrificed at p35 for immunocytochemistry (ICC) and confocal microscopy analysis ( Fig. 3F , left). Subretinal transplanted PRCs were identified by double positive ICC staining for HuNu (Millipore, MAB1281) and GFAP (Abcam, ab4674). Rhodopsin+ staining (Millipore, MABN15) identified rod outer segment fragments that had been internalized by PRCs in the subretinal space. Retinal cryosections were ICC stained essentially as described and imaged on a Leica SP8 confocal microscope. Animals were sacrificed at P60 for transmission electron microscopy ("EM") analysis. EM confirmed the presence of multilayered structures, identified as rod outer segment fragments, localized in the cytoplasm of PRCs transplanted in the subretinal space ( Fig. 3F , right). Tissue processing and imaging were performed according to standard procedures and images were captured on a Phillips CM10. Example 2 : In vivo characterization of PRCs Subretinal injection

Rats at approximately P25 were anesthetized with ketamine (75 mg/kg) and dexmedetomidine (0.25 mg/kg) intraperitoneally (IP). The pupils were dilated with 1% tropicamide (Bausch & Lomb, Rochester, NY) or 1% atropine (Bausch & Lomb) or 2.5% phenylephrine hydrochloride (Bausch & Lomb, Inc.). Nonabsorbable sutures (4-0) (Ethicon, Somerville, NJ) were placed in place to maintain the eye in a forward orientation. An iridotomy was made in the superior dorsal region of the temporal lobe of the eye using a 25 5/8 G metal needle. 2 µl of the cell suspension was drawn into a sterile glass pipette (internal diameter 50 to 150 µm) through a plastic tube filled with GS attached to a 25 µl Hamilton syringe. Cells or GS (2 μL volume) were injected into the subretinal space through the iridotomy site. To reduce intraocular pressure and limit the outflow of cells, the cornea was punctured using a 30 G metal needle. Immediately after the injection, the fundus was examined for retinal damage or vascular pain. The wound was closed using non-absorbable surgical sutures (10-0) (Ethicon). The sutures around the eyeball were removed and then the eyelid was placed in its normal position. Finally, ophthalmic erythromycin ointment (Bausch & Lomb) was applied topically. Animals recovered from anesthesia on a warming pad (37°C). During the 2 weeks following the subretinal procedure, animals received daily injections of dexamethasone (American Regent, Shirley, NY) (1.6 mg/kg, ip ) . In addition , animals received cyclosporine-A (Bedford Laboratories, Bedford , OH) (210 mg/L) in their drinking water throughout the experiment.

Animals were tested for OMR at P60, P90, P120, P210, and P270 using the OptoMotry test apparatus (Cerebral Mechanics, Inc., Lethbridge, Canada) (Prusky, 2004). The OptoMotry apparatus uses four computer monitors arranged in a square to project a virtual three-dimensional (3-D) space of a rotating cylinder lined with a vertical sine wave grating. Rats were placed unrestrained on the center of the square on the platform where they tracked the grating with reflective head movements. The spatial frequency of the grating was clamped at the viewing position by re-centering the 'rotating cylinder' on the animal's head. The acuity threshold was quantified by increasing the spatial frequency of the grating using a psychophysical stepwise progression until the sub-response was lost. Electroretinography ( ERG )

ERGs were performed on animals at P60, P90, P120, and P210. Animals were prepared for ERG recordings under dim red light after dark adaptation overnight. After an injection of anesthesia using a mixture of ketamine (150 mg/kg, ip) and xylazine (10 mg/kg, ip), the head was secured in a stereotaxic device and body temperature was maintained at 38°C during the experiment using a thermostatic blanket. The pupils were dilated using 2.5% topical phenylephrine plus 1% atropine and a drop of 0.9% saline was applied to the cornea to prevent dehydration and allow electrical contact with the recording electrodes. Single flash presentations were performed with a standard duration of 10 s and responses were recorded using an Espion E3 electroretinography system (Diagnosys, LLC). Tissue preparation

The animals were euthanized and the eyes were then removed, immersed in 2% paraformaldehyde for one hour, soaked overnight with consecutive intermediate incubation steps, 10% and/or 20% and/or 30% sucrose, and 30% sucrose. The next day, the eyes were embedded in OCT and cut in a cryostat (10 µm) onto Superfrost+ slides and stored at -80°C. Immunocytochemistry ( ICC )

Remove sections from -80°C and dry at room temperature (RT) for approximately 10 minutes, then wash in 1x PBS at RT for 5 minutes. Mark the border around the section using a liquid blocking super pap pen (Fisher Scientific, catalog number NC9827128). Slides are then incubated in blocking buffer (1X PBS containing 5% horse serum and 0.3% Triton-X) at 4°C or RT for 1 hour. Blocking buffer is removed and primary antibody solution is added overnight at 4°C in a humidified chamber. Slides are then washed with 1x PBS and incubated with secondary antibody solution and 4',6-dimethylamidino-2-phenylindole (DAPI) (1:1000) at 4°C for 1 hour. After washing, coverslips were mounted using Prolog Gold Anti-Fluorescence Mounting Fluorescence (Invitrogen, Catalog No. P36930) and allowed to cure for approximately 24 hours at room temperature. Morphological Determination of Transplant Relative to ONL Retention

Morphometric analysis was performed using section tissue from P120 RCS rats injected with lot number FY2019 (GB2R-PRC-2019-P4, 100,000 cells/eye). Morphometrics were performed using a complete series of section slides with full tracking of transplantation and photoreceptor outer nuclear layer (ONL), with no local selection or selection biased toward transplantation at the time of imaging. Morphometrics were performed using N=6 evenly distributed slides covering the complete series of sections from each eye: AS193R - select every 10th slide; AS193L - select every 10th slide; AS194R - select every 15th slide (to reflect the larger number of section slides in the series while maintaining the same N=6 slides for analysis); AS194L - select every 10th slide. The sampling depth through the eye was approximately equal to 2400 µm (AS193R, AS193L, AS194L) and 3600 µm (AS194R). Immunohistochemistry was performed as described above using an antibody selective for STEM121 (human cytoplasmic marker; Takara), and tissues were counterstained with DAPI. Two eyes per slide were imaged using a Leica DMi8 imaging unit with motorized stage and tiling, resulting in 12 images per eye for each RCS eye. Images were exported as tiff images scaled with an embedded scale bar, and full-scale plots of DAPI-labeled ONL and STEM121-labeled subretinal PRC transplants were then generated using ImageJ analysis software. Only the thin layer of ONL that was transplanted and maintained under the retina was measured; scattered individual photoreceptors were not tracked. Ocular transplantation was plotted relative to ONL area using GraphPad Prism8 statistical software. Immunohistochemistry

Immunohistochemical imaging was performed using a Leica SP8 confocal microscope, operating with the Leica LasX software suite. Image manipulation (re-grading, cropping, color correction) was performed using the National Institutes of Health (NIH) ImageJ image processing and analysis software. Images were assembled for presentation using Microsoft PowerPoint. Subretinal injection of rd10 mice

Prior to surgery, the administration device (a microsyringe (Hamilton 600 series) equipped with a 34 ga flat-tip needle (Hamilton #207434)) was pre-filled with cell suspension or vehicle. Test animals (P14 Rd10 mice) were administered a ketamine/xylazine mixture. When the test animal achieved deep anesthesia (indicated by slowed breathing and lack of response to toe pinch), it was placed on its side under a dissecting microscope with the eye undergoing the procedure positioned upward. The skin above and below the eye was pulled back, causing the front of the eye to droop (proptosis). Betadine eye drops (5%, sterile, ophthalmic grade) were administered to the eye to locally disinfect the globe, and artificial tears may be applied to flush excess betadine from the eye. Proparacaine eye drops were applied to render the eye and surrounding tissues numb, and phenylephrine (2.5%) and tropicamide eye drops were applied to dilate the pupil. Between eye drop/artificial tear applications, excess solution was removed using a fabric-tipped swab. For subretinal injections, a hole was cut in the conjunctiva to expose the sclera. Using a 30 ga beveled needle, a guide hole was created over the limbal region. Under visual guidance, a 34 ga needle microsyringe containing PRC suspension or vehicle buffer was inserted into the hole and the injection material was pushed into the eye by manually depressing the plunger. After injection, the needle was slowly removed from the eye and the animal was placed on the opposite side to perform the injection procedure on the second eye. OCT imaging was performed immediately after the injection procedure to assess the success of the injection. After imaging, ophthalmic erythromycin (0.5%) ointment was applied to the surgical site for antimicrobial and lubricating properties. For mice that were successfully injected, Antisedan (atipamezole, 2 mg/kg) was administered intraperitoneally to aid the recovery process. Subcutaneous saline may be administered for moisturizing. Finally, to prevent hypothermia, animals were moved to a heated cage until sternal recumbency and then returned to their home cages. ONL and cone length retention morphometry and rod synapse counts

Morphometric analysis and rod synapse counts were performed using tissue sections from P28 rd10 mice injected with FY2019 batch PRC (GB2R-PRC-2019 (P4), 100,000 cells/eye). Morphometric measurements were performed using area tracking of the photoreceptor outer nuclear layer (ONL) from subretinal PRC transplants and eye regions distal to the transplant site toward the central and peripheral retina. Morphometric measurements were performed using N=4 evenly spaced slides spanning the sequence of sections that included transplants from each of three eyes. The sampling depth through each eye was approximately equal to 480 µm. Immunohistochemistry was performed using antibodies selective for STEM121 (human cytoplasmic marker; Takara), and tissues were counterstained with DAPI. Two eyes were imaged per slide, and eight images were generated per eye for each rd10 eye. Images were exported as tiff images scaled with an embedded scale bar and ONL area traces were generated for DAPI-labeled ONL using ImageJ analysis software. ONL areas were plotted and compared using GraphPad Prism8 statistical software. For cone length analysis, serial sections were stained and imaged with antibodies selective for cone arrestin (Millipore) and STEM1281 (human nuclear antigen; Millipore) as described for morphometric measurements. The axial length of labeled cones from the outer segment to the axonal pedicle was measured at the subretinal PRC transplant site and distal to the transplant site toward the central and peripheral retina and compared using GraphPad Prism8 statistical software. For rod synapse analysis, sections were prepared as described for morphometric measurements and stained with antibodies selective for cone arrestin (MilliporeSigma) and ribeye/CtBP2 (BD Biosciences). Ribeye-positive/cone arrestin-negative synapses (rod synapses) were counted at the subretinal PRC transplant site and distal to the transplant site toward the central and peripheral retina and compared using GraphPad Prism8 statistical software. Subretinal injection of PRC-EVs and optomotor response ( OMR ) in RCS rats

RCS rats received subretinal injections of PRC-EV or GS2 (vehicle) from P23 to P25 (postnatal days 23 and 25, respectively). PRC-EV was administered at a dose of 9.42× ¹⁰⁸ EVs per eye. Spatial frequency was measured by recording optokinetic responses at P60, P90, and P120 (postnatal days 60, 90, and 120). Animals were sacrificed at P90 and P120 for histological analysis. Imaging and ONL quantification of PRC-EV injected eyes .

Cryostatic sections of the retina from P90 and P120 PRC-EV injected eyes were stained with the nuclear stain DAPI (4,6-dicarboxamidino-2-phenylindole) to visualize the retinal outer nuclear layer (ONL). The stained retinal sections were imaged using a Leica DMi8 epifluorescence microscope. ONL thickness was determined in a total of 3 to 5 regions of interest (ROIs) per retina by line drawing measurements of the ONL in both central and peripheral areas. Subretinal injection of PRC-EV and optomotor response ( OMR ) testing in RCS rats

RCS rats received subretinal injections of PRC-EV or GS2 (vehicle) on postnatal days 23 to 25 (P23 to P25). PRC-EV was administered at a dose of 9.42 × 10 ⁸ EVs per eye. To assess visual function after PRC-EV injection, spatial frequency was measured by recording optokinetic responses at P60, P90, and P120. Animals were sacrificed at P90 and P120 for histological analysis. Imaging and outer nuclear layer ( ONL ) quantification of PRC-EV injected eyes

Retinal cryosections were stained with the nuclear marker 4,6-dicarboxamidino-2-phenylindole (DAPI) to visualize the ONL, and the stained sections were imaged using a Leica DMi8 epifluorescence microscope. ONL thickness was determined for a total of 3 to 5 regions of interest (ROIs) per retina by line drawing measurements of the ONL in central and peripheral areas. Total ONL thickness was taken as the average ONL length of each ROI. Results: RCS (homozygous) rats

RCS (homozygous) rats were injected subretinally with 16.5 to 200k PRCs (outline depicted in Figure 4A ) into one eye. Control eyes (matched eyes) were injected with vehicle or not. Injections were performed at P25. OMR and ERG tests were performed at P60, P90, and P120. Histology of injected and control eyes was performed at P35, P60, and P120.

OMR and ERG analysis of RCS rats injected with 100,000 GB2R-PRC-P4-FDA PRC cells per eye at P25 ( Fig. 4B ). Statistical analysis was performed using two-way ANOVA coupled with Tukey's multiple comparison test (MCT), where test articles were compared to uninjected eyes and eyes injected with GS2 vehicle. Cell viability was 78.4% and was determined manually.

Representative images show that PRC transplantation significantly attenuated outer nuclear layer (ONL) degeneration over a 3-month period after transplantation ( Fig. 4C ). Confocal images showed that PRC transplantation was highly associated with ONL preservation. In each age group, although an overall gradual decrease in ONL thickness with age was observed in the “non-transplanted” area, the ONL in the transplanted area was significantly preserved compared with the non-transplanted area. Stained retinal sections were imaged using a Leica MDi8 epi-fluorescence microscope.

Quantification of ONL thickness showed that PRC transplantation was associated with significant maintenance at P35, P60, and P120 ( Figure 4D ). Retinal cryosections through PRC transplants were selected for ONL quantification and stained with HuNu (Millipore, MAB1281) to localize the transplanted PRC and DAPI to identify the ONL. ONL thickness was determined by line drawing measurements through the ONL for a total of 3 to 4 regions of interest (ROIs) per retina in both central and peripheral areas. ONL quantification was performed in a blinded manner using only the DAPI channel. Regions of interest were then identified as "transplanted" or "not transplanted" based on the presence of HuNu+ PRCs under the retina.

Plot of the area of GB2R-PRC-2019 (P4) implanted PRC relative to the area of photoreceptor ONL in the P120 RCS rat retinal degeneration model ( Fig. 4E ). Statistical analysis was performed using Pearson's correlation coefficient.

Immunohistochemistry for neurofibromatosis protein (rabbit anti-GFAP; ABCAM) was performed in P120 RCS rats ( Fig. 5A ). GFAP was expressed in Müller glia (MG) and optic nerve fiber astrocytes of the host rat retina (white arrows) and in PRCs implanted into the subretinal space. Increased GFAP expression in MG and astrocytes MG hypertrophy and expanded distribution of GFAP expression radially to MG cell bodies are characteristic of disease-related reactive neurofibrosis and can be found distal to the transplant site (yellow arrows, white arrows). Upregulation and increased distribution of GFAP, MG hypertrophy, and outer nuclear layer (ONL) degeneration were reduced or absent at the subretinal PRC transplant site.

TUNEL quantification (TUNEL labeling mix, Sigma catalog #11767291910, TUNEL enzyme Sigma catalog #11767305001) was performed at P35, during which time peak apoptotic activity was observed in the RCS retina ( Figure 5B ). PRCs implanted into the subretinal space were identified based on Ku80+ staining (Abcam, ab80592) of adjacent sections (see below). Few, if any, TUNEL+ nuclei were observed in the PRC implanted areas, indicating little apoptotic activity at the implanted sites. However, non-implanted areas contained significant TUNEL+ nuclei in the ONL, indicating widespread photoreceptor cell death at P35. Quantification of TUNEL+ nuclei in the ONL showed a significant attenuation of apoptotic activity at the implanted sites relative to non-implanted areas. Retinal sections through PRC grafts were selected for TUNEL quantification and stained with Ku80 to determine the location of implanted PRCs and DAPI to identify the ONL. A total of 3 to 4 regions of interest (ROIs) in the central and peripheral areas of each retina were imaged. TUNEL quantification was performed in a blinded manner using only the DAPI channel. ROIs were then identified as "transplanted" or "no transplanted" based on the presence of subretinal Ku80+ PRCs.

Extracellular vesicles (EVs) isolated from PRCs maintained the OMR response in RCS rats until P90 ( Fig. 5C ). EVs were isolated from PRC-conditioned medium and injected subretinaally into RCS rats at a dose of 9.42*10 ⁸ EVs per eye. Other RCS rats received subretinal injections of PRCs (100,000 cells/eye) or vehicle. Injections were performed at age P25. OMR analysis was performed at P60, 90, 120 and compared with non-injected (NI) animals of the same age. A single injection of PRC-EVs was able to maintain the OMR until P90, while a single subretinal injection of PRC cells maintained the OMR until P120, compared with NI and vehicle-injected animals. This suggests that PRC-derived EVs can at least partially reproduce the effects observed with subretinal injection of PRC cells and supports the secretion of paracrine-acting factors as part of the PRC mechanism of action.

A single subretinal injection of PRC-EVs maintained ONL thickness until P90 ( Fig. 5D ). Retinal ONL thickness was quantified in PRC-EV-injected RCS rats relative to uninjected eyes. Retinal cryosections were stained with DAPI to visualize the ONL. Stained retinal sections were imaged using a Leica DMi8 epifluorescence microscope. ONL thickness through the ONL was determined in central and peripheral regions by line drawing measurements as described herein.

Visual function was assessed by recording optomotor responses in PRC-EV-injected rats at P60, P90, and P120 ( Figure 5I ). Visual acuity was significantly preserved at P60 and P90, but this preservation was not maintained until P120. To further investigate the morphological preservation of photoreceptor cells after PRC-EV injection, ONL thickness was measured in P90 ( Figures 5E , 5F , and 5G ) and P120 ( Figure 5H ) rats. ONL analysis of EV-treated rats showed that a single injection of PRC-EVs conferred morphological preservation of the ONL until P90, but not until P120 ( Figure 5J ), corresponding to the lack of OMR preservation in the latter age group. While PRC-EV treatment maintained visual function and preserved external retinal morphology until P90, subretinal cell transplantation of PRC preserved ONL thickness until P120 ( Fig. 5K ), demonstrating that ONL morphology of subretinal transplanted cells was preserved longer than that of single EV injection treatment.

Spatial frequency was measured by recording optomotor responses in PRC-EV injected and PRC transplanted rats at P60, P90, and P120. OMR responses remained similar between EV and cell treatments and were significantly preserved until P90. However, in EV injected rats, preservation of OMR was lost by P120. ONL analysis of EV treated rats showed that a single injection of EVs resulted in significant preservation of ONL thickness until P90, but not until P120, corresponding to the lack of OMR preservation in the latter age group.

In the area of PRC implantation, ONL preservation was clearly observed as visualized by toluidine blue stain, while the non-implanted area showed severely degenerated ONL ( Fig. 6A ). P25-year-old rats were injected subretina with 100,000 GB2R PRCs or vehicle and then sacrificed at P60. Retinal tissues were formalin-fixed, paraffin-embedded, and cross-sections were stained with toluidine blue (Polysciences, 1234) to visualize the ONL. P60 eyes were processed for TEM analysis and Phillips CM10 imaging. All tissue processing and imaging were performed by the UMass Electron Microscopy Core.

Transmission electron microscopy (TEM) analysis confirmed that the ONL in the PRC-implanted area was significantly preserved compared with the ONL in the non-implanted area ( Fig. 6B ).

TEM ultrastructural analysis showed that the overall morphology of photoreceptor cells as well as finer structures such as connecting hairs of the inner segments of the rods was preserved in the presence of subretinal PRCs ( Fig. 6C ). Finally, PRC transplantation minimized the debris area in the subretinal space. In the absence of subretinal PRCs, rod outer segment debris filled most of the subretinal space because the outer segments were not cleared after abscission. Results: rd10 mice (homozygous)

On postnatal day 14 (P14), 100k cells were transplanted into the subretinal space of the right eye of rd10 mice, and GS2 vehicle was injected into the left eye as a control. OMR was performed at P21, P28, P35, P42, and P49 ( Fig. 7A ).

OMR analysis revealed the presence of tracking in both eyes at P21 and P28 ( Fig. 7B ). Cell-injected eyes showed tracking capabilities at P35, P42, and P49 (compared to blind control eyes).

Morphometric analysis of the preservation of the outer nuclear layer (ONL) of photoreceptor cells in P28rd10 mice ( Fig. 7C ). ONL preservation, quantified by ONL area (mm2), was identified at the PRC subretinal transplant site compared to adjacent central and peripheral retina without transplantation.

Analysis of cone length in P28 rd10 mice ( Fig. 7D ). The length of cone arrestin-labeled cone photoreceptor cells was significantly increased at the PRC subretinal transplant site compared to adjacent areas of the central and peripheral retina without subretinal transplantation. Confocal imaging showed CtBP2/ribeye protein-labeled rod contacts at the transplant site and in central and peripheral retinal locations without transplantation ( Fig. 7E ).

Quantification of rod synapses in P28 rd10 mice ( Fig. 7F ). The number of ribeye-positive/cone-arrestin-negative rod synapses was significantly increased at the PRC-transplanted sites compared with the non-transplanted central and peripheral retina, indicating that rod photoreceptor cells in the rd10 mouse retina exhibit PRC-associated preservation.

At P35, the OMR of multiple PRC batches with survival rates less than 70% were compared ( Figure 8A ). PRC survival rates of 66.05%, 68.5%, and 68.8% showed similar maintenance effects. PRC-injected eyes showed superior OMR efficacy compared to control eyes.

Anatomical preservation was observed in multiple PRC batches with survival rates less than 70% ( Figure 8B ). Histological analysis confirmed the OMR data. At P35, injected PRCs with a survival rate of 66.05% preserved cone anatomy as assessed by ONL thickness, axon length, and morphology. At P50, injected PRCs with a survival rate of 68.5% preserved ONL thickness and density of ribeye+synapses. At P70, injected PRCs with a survival rate of 68.8% also preserved ONL thickness and density of ribeye+synapses.

On postnatal day 14 (P14), 100k cells were transplanted into the subretinal space of the right eye of rd10 mice, and GS2 vehicle was injected into the left eye as a control ( Figure 9A ). At P50, eyes were collected for IHC staining. DAPI staining showed that only one row of photoreceptor cells was present in the non-transplanted retina, while multiple rows of photoreceptor cells were observed in the transplanted retina. Quantification of data from 5 animals and 3 sections from each animal showed that the ONL thickness and area in the transplanted retina were significantly higher than the ONL thickness and area in the non-transplanted retina (6 μm vs. 18 μm; 5000 ^μm2 vs. 10000 ^μm2 ).

Cone morphology was detected using ICC images stained with arrestin (Millipore AB15282 1:500) ( Fig. 9B , left). In the non-transplanted retina, arrestin was only expressed in the cell bodies, outer segments, and axons of the cones. In the transplanted retina, the cones were preserved by maintaining their normal morphology, and arrestin was expressed in the outer segments, cell bodies, axons, and peduncles. Quantitative data showed that the axons of cones in the transplanted retina were significantly longer than those in the non-transplanted retina (8 μm vs. 4 μm). Ribeye protein (also known as ct-BP2, BD Transduction Labs 612044, 1:500) is a marker for presynaptic structures located at the ends of photoreceptor axons, which indicates synaptic connections between photoreceptor cells and horizontal cells ( Fig. 9B , right). Quantitative data showed that ribeye protein spots were more abundant in transplanted retina than in non-transplanted retina (40 vs. 20/100 μm retina).

Figure 9C shows a schematic diagram of drug administration in P23H rats. P23H rats are a model of somatic dominant retinitis pigmentosa (RP) in which a mutant mouse rhodopsin ( Rho ) transgene is incorporated into wild-type Sprague Dawley rats. One eye of P23 hemizygous rats was injected subretina with 100K cells at P25. The counterpart eye was treated with vehicle or no injection as a control. OMR and ERG were performed on both eyes at P60, P90, and P120, and histological analysis was performed at P120.

OMR analysis of P23H hemizygous rats injected with 100,000 GB2R-PRC-P4-FDA PRC cells per eye at P25 revealed a statistically significant preservation of the OMR response compared to uninjected controls at P60, P90, and P120 and compared to vehicle-injected controls at P90 ( FIG. 9D ). Statistical analysis was performed using two-way ANOVA coupled with Dukey's multiple comparison test (MCT). Example 3 : Pharmacokinetic Methods

Subretinal injections and immunohistochemistry (IHC) were performed as described above. Comparative subretinal transplant morphometrics

For each of RCS rats, P23H homozygous rats, P23H hemizygous rats, and rd10 mice injected with 100,000 FY19 GB2R-PRC-2019 (P4) PRC cells per eye, evenly distributed slides spanning a series of sections showing subretinal transplantation were stained with antibodies selective for human cytoplasmic antigens (STEM121; Takara) and counterstained with DAPI. Eyes were imaged using a Leica DMi8 imaging set with motorized stage and tiling. Images of slides showing the greatest extent of subretinal transplantation (maximum subretinal coverage length relative to retinal length) were identified and used to calculate the relative maximum subretinal transplant coverage length. N=4; N=3; N=3 and N=5 eyes (RCS, rd10, P23H homozygous and P23H hemizygous eyes, respectively) were used for calculations. Maximum subretinal graft length was measured as the total length of the overlying retina. Data were plotted to show the coverage of subretinal graft relative to retinal length. In addition, the ratio of maximum subretinal graft relative to retinal length was calculated and plotted. One-way analysis of variance combined with Tukey's multiple comparison test was used to compare the differences in maximum graft ratios between models. Results

Schematic depiction of the method for quantification of HuNu+ cells ( Figure 10A ). RCS rats aged P23 to P25 were given subretinal injections of 100,000 GB2R PRCs or GS2 vehicle. Animals were sacrificed at P120 to obtain retinal tissue cryosectioning and histological analysis. Every 10th retinal section through the graft was selected for quantification, in which HuNu+ PRCs were quantified. Cell numbers were interpolated between sections and the total was calculated for all grafts. Total PRC grafts were calculated starting from n = 4.

At P120 or 3 months after transplantation, the number of engrafted PRCs was quantified in 4 transplanted animals ( Fig. 10B ). Quantification of total HuNu+ PRCs transplanted subretinally showed an average of 80,000 cells engrafted per eye, which translated to an overall 80% engraftment rate compared to an initial injection of 100,000 cells per injection.

Differential PRC engraftment in retinal degeneration models with different disease onset and severity after treatment ( Fig. 10C ). The specific batches and doses of PRC used in this analysis are shown in the top boxes of the figure. Bar graphs showing relative subretinal engraftment: RCS rat and P23H hemizygous rat retinal degeneration models demonstrated similar engraftment of GB2R-PRC, with maximum engraftment found to cover 54 and 41% of the subretinal space, respectively. The rd10 mouse and P23H homozygous rat retinal degeneration models showed significantly lower engraftment: 19% (rd10 mouse) and 2% (P23H homozygous rat) of the subretinal space were shown to have the maximum length of PRC engraftment. Relative retinal length graft coverage plot: Relative ratio calculations provide coverage relative to different sizes of mouse and rat eyes, while the maximum length plot shows absolute graft length. Compared to RCS and P23H hemizygous rats, rd10 mice and P23H rats showed reduced grafts both relative and absolute. Example 4 : In vivo safety analysis

PRCs had limited proliferation, did not display potentiation markers and no safety abnormalities were observed in PRC-treated RCS. By P120, proliferation of subretinal PRCs was downregulated ( Figure 11A ). Subretinal transplanted HuNu (Millipore, MAB1281) and Ki67 (Abcam ab15580) double positive cells were quantified in retinal cross-sections from P35, P60 (not shown) and P120 animals. A total of three animals were used for each time point. Confocal images show Ki67 and HuNu double positive cells in the subretinal space at P35. However, by P120, no proliferative (Ki67+) PRCs were observed. Quantification revealed that Ki67 was significantly downregulated in HuNu double positive PRCs by P120 or 3 months after transplantation (bar graph in the lower left panel).

Lack of Oct4 (Abcam ab27985) expression in implanted HuNu+ PRCs showed a lack of rich potential at all time points studied after transplantation (P35, P60, and P120) ( Figure 11B ). A total of three animals were used for each time point. Cultured GMP1 iPSCs were used as a positive control for Oct4 expression, where subcellular compartment localization was performed using WGA-647 (Thermo Scientific W32466) and DAPI staining (small images in the lower middle panel).

An overview of general welfare, ocular examinations, and histopathology reports from a board-certified veterinary histopathologist indicated that subretinal transplanted PRCs were generally well tolerated with no adverse effects. P270 RCS rats (8 months post-transplantation (Tx)) 1) maintained stable weights during the last month of life, similar to untreated RCS rats; 2) no welfare issues were observed; 3) no PRC-related abnormalities were observed by ophthalmoscopy; and 4) no PRC-Tx-related observations macroscopically or microscopically. P50 rd10 mice (36 days post-DP Tx) 1) maintained stable growth during the study; 2) no welfare issues were observed; and 3) no abnormalities were observed by ophthalmoscopy when transplanted with cryopreserved PRCs. Conclusions

The effects of PRC on 3 specific targets in the degenerating retina may be responsible for the efficacy of PRC in delaying vision loss: (1) maintaining the health of photoreceptor cells, which is important for initiation of the visual phototransduction cascade; (2) limiting excessive microglial activity, which would otherwise exacerbate the degenerative process; and (3) reducing the accumulation of degenerative debris via phagocytosis, which would otherwise contribute to further photoreceptor cell loss.

Methods of action relevant to target biology include (1) paracrine-mediated neuroprotection of photoreceptor cells; data on PRC-secreted EVs suggest that neuroprotection is the primary contributor to PRC efficacy, while other mechanisms may play a supporting role, such as: (2) PRC-associated reductions in neuroglial responsiveness and expression of antioxidant factors; and (3) PRC phagocytosis of denatured debris.

The target indication/patients for treatment with PRC are patients diagnosed with retinitis pigmentosa, regardless of genetic mutation, with a BCVA of 20/100 or worse. PRC is expected to maintain or slow the loss of visual acuity as assessed by BCVA or other visual tests (TBD). For example, slowing of retinal degeneration as assessed by fundus spontaneous fluorescence, spectral domain (SD)-OCT, and/or micro-field examination.

In RP, genetic mutations in the rod photoreceptor cells lead to degeneration of the peripheral retina, which subsequently leads to secondary degeneration of cones in the central retina (Humphries, 1992). Activation of microglia is thought to be a protective response to injury/degeneration, however it often becomes exacerbated/dysregulated in chronic retinal degeneration and leads to this cascade of photoreceptor cell loss, as does the accumulation of degenerative debris. Progressive loss of photoreceptor cells weakens the visual phototransduction pathway, leading to vision loss, therefore the ability of the PRC to maintain photoreceptor cell health and function is thought to be critical. Evidence suggests that various neuroprotective molecules can support the health and function of photoreceptor cells, helping to slow retinal degeneration and vision loss (Kutluer 2020). Inhibition of microglial proliferation and expression of antioxidant molecules can also help slow the degenerative process by limiting stress and additional loss of photoreceptor cells (Murakami 2020; Peng, 2014; Rashid 2019; Newton 2020). Phagocytosis of debris from dead photoreceptor cells can also help clear toxic signals, thereby limiting secondary damage to neighboring photoreceptor cells (Newton 2020). PRCs target all of these biological processes. Example 5 : PRC Manufacturing

Schematic diagrams of alternative PRC production methods are shown in Figures 12A and 12B . hESCs from GMP-GB2R-MCB were thawed (day -10), counted and seeded in culture vessels coated with iMatrix (laminin-511, Matrixome) and cultured in StemFit medium (Ajinomoto) supplemented with 100 ng/mL bFGF (Peprotech) and 10 µM ROCK inhibitor (Y-27632; Fujifilm/Wako) without feeder cells. After four days of daily medium changes (StemFit+bFGF without ROCK inhibitor), hESCs were harvested using cell lysis buffer (Gibco) and replated using the above culture conditions. After an additional 4 days of culture (day -2), hESCs were harvested as described above, % viability and live cell counts were obtained, and hESCs were seeded at 3,000 cells/cm2 in iMatrix-coated T75 flasks in StemFit+bFGF medium supplemented with ROCK inhibitor (Y-27632) for PRC differentiation. After inoculation on day -2, the cultures were fed: (1) on day -1 - StemFit + bFGF (without ROCK inhibitor); (2) on days 0 to 3 (daily) - rescue induction medium (RIM: DMEM/F12 + B27 + N2 + non-essential amino acids (all from Gibco) + glucose (Sigma) + insulin (Akron Biotech) + Noggin (Gibco)); and (3) on days 4 to 19 (every 2 to 3 days) - neural differentiation medium + Noggin (NDM+: neural basal medium + B27 + N2 + non-essential amino acids + glucose + glutamax (Gibco) + Noggin).

On day 19, cultures were "upgraded" to suspension culture (2D to 3D) by incubation with a combination of liberase and thermolysin (Roche Custom Labs) and plated on ultra-low attachment T75 flasks. 3D cultures were maintained for 3 to 4 days using noggin-free NDM (NDM-) to allow neural spheroid ("sphere") formation. Then, spheres in suspension were seeded (3D to 2D) onto poly-D-lysine (Advanced Biomatrix), virus-killed human fibronectin (Akron Biotech), and laminin-521 (Biolamina) coated culture vessels to start passage 0 (P0d0) and cultured in 2D conditions with NDM- (fed every 2 to 3 days) for 14 days (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2, and P3, 3D culture for 3 to 4 days and 2D culture for 14 days) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Akuzyme (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. The P3 spheres were then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to vapor phase liquid nitrogen for storage. The cryopreserved P3 spheres are referred to as cell stock solution (CS).

Examples of methods for "lifting" cells into suspension include, but are not limited to, those shown in Table 13. Table 13 Time point method 1 Method 2 Method 3 Days Inheritance days 19 Cell scraper Release enzyme + Thermolysin Akubin + ROCK inhibitor 37 P0d14 Cell scraper Release enzyme + Thermolysin Akuzyme 54 P1d14 Cell scraper Release enzyme + Thermolysin Akuzyme 72 P2d14 Cell scraper Release enzyme + Thermolysin Akuzyme 90 P3d14 Akuzyme Akuzyme Akuzyme 107 P4d14 Akuzyme Akuzyme Akuzyme

To produce the composition of the present invention, the CS vials were thawed in a 37°C water bath, resuspended in NDM- and transferred to ultra-low attachment T75 flasks and cultured in 3D suspension for 2 to 3 days, then seeded on poly-D-lysine/fibronectin/laminin-521 coated T75 flasks and cultured in 2D conditions with NDM- for 14 days to complete the 4th passage. At P4d14, cells were harvested using acurase, resuspended in NDM-, wet-triturated and filtered through a 40 µm cell filter to obtain a single cell suspension constituting the PRC composition of the present invention, which can then be formulated with a cryopreservative.

The PRC manufacturing protocol can be modified to allow for scale-up. For example, the flasks used can be changed from T75 flasks to T225 flasks or cell stacks (e.g., Corning® CellSTACK®).

In addition, the PRC manufacturing scheme may include an intermediate low temperature storage step between P0 and P1, between P1 and P2, between P2 and P3, and/or between P3 and P4. In some specific examples, the PRC manufacturing scheme may exclude the intermediate low temperature storage step.

The intermediate cryopreservation step may include 5% DMSO instead of 10% DMSO. In some embodiments, the intermediate cryopreservation step may include about 1% DMSO, about 2% DMSO, about 3% DMSO, about 4% DMSO, about 5% DMSO, about 6% DMSO, about 7% DMSO, about 8% DMSO, about 9% DMSO, about 10% DMSO, about 11% DMSO, about 12% DMSO, about 14% DMSO, or about 15% DMSO.

The intermediate cryopreservation step may include cryopreservation formulations as described herein. The PRC production protocol may include thermolysin and liberase and acurase at D19, followed by overnight incubation with Rock inhibitor. The raised cells or cell clusters may be plated in Aggrewells™ to allow for the development of uniformly sized spheres. On day 19, the cultures were "raised" to suspension cultures (2D to 3D) by incubation with acurase (Innovative Cell Technologies) and plated on ultra-low attachment T75 flasks using NDM (NDM-) medium without Noggin supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako). After 24 hours, the growth medium was replaced with NDM-free Y-27632 and 3D cultures were maintained for another 2 to 3 days using NDM- to allow for the formation of neural spheroids ("spheres"). Spheres in suspension were then seeded (3D to 2D) onto poly-D-lysine (Advanced Biomatrix), virus-inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) coated culture vessels to initiate passage 0 (P0d0) and cultured under 2D conditions using NDM- (fed every 2 to 3 days) for 14 days (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2 and P3, 3-4 days of 3D culture and 14 days of 2D culture) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Akuzyme (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. P3 spheres were then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to vapor phase liquid nitrogen for storage. Cryopreserved P3 spheres are called cell stock (CS).

In some embodiments, the PRC production protocol uses Aggrewells. According to this method, on day 19, the culture is "upgraded" to suspension culture (2D to 3D) by incubation with acurase (Innovative Cell Technologies) and plated on Aggrewell plates (Stemcell Technologies) using NDM (NDM-) medium without Noggin, which is supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako) to allow neural spheres ("spheres") to form. After 24 hours, cells are collected from the Aggrewell plates and transferred to ultra-low attachment T75 flasks, where 3D cultures are maintained for an additional 2 to 3 days using NDM- without Y-27632. Then, spheres in suspension were seeded (3D to 2D) onto poly-D-lysine (Advanced Biomatrix), virus-killed human fibronectin (Akron Biotech), and laminin-521 (Biolamina) coated culture vessels to start passage 0 (P0d0) and cultured in 2D conditions with NDM- (fed every 2 to 3 days) for 14 days (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2, and P3, 3D culture for 3 to 4 days and 2D culture for 14 days) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Akuzyme (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. The P3 spheres are then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to vapor phase liquid nitrogen for storage. The cryopreserved P3 spheres are referred to as cell stock solution (CS). The PRC composition can be pretreated with sucrose before being formulated with a cryoprotectant, for example, at D14 and P4.

Finally, the cryopreservation step of the final PRC formulation may include poloxamer 188 (a non-ionic block linear copolymer) and/or sucrose. Example 6 : Formulations and formulated PRC dosages

The cell culture was cryopreserved and prepared as a composition at the manufacturing site. The cells were harvested and separated into a single cell suspension using acurase on day 14 of passage 4 (approximately day 107, as shown in FIG. 12C ). The harvested cells were then subjected to a wash step to separate live cells from any dead cells and debris generated during the harvest step. The cells were then mixed with cryopreservation buffer and dispensed into the final product container. The final composition was then frozen using a controlled rate freezer.

The frozen composition is then shipped to clinical sites where the PRC composition is rapidly thawed and diluted with GS2 diluent for subretinal injection (GS2+). The diluted PRC composition is then mixed, then loaded into a syringe and delivered to the patient via subretinal injection.

FIG. 13 is a schematic diagram showing the process of manufacturing, recovering and injecting formulated photosensitive rescue cells.

After thawing at P4, the viability of PRCs tested in different formulations was measured ( Figures 14A and 14B ). In this experiment, PRCs were cultured until P4d14, then harvested and isolated into single cells. The cell suspension was then washed using OptiPrepTM (STEMCELL Technologies, Inc., Vancouver, Canada) density gradient to separate live and dead cells. Live cells were harvested, washed in growth medium and resuspended in 0.5% rHA/DPBS. The cells were then distributed in a 1:2 volume ratio into buffers containing different cryopreservatives, where each formulation contained a final concentration of 2.5% rHA and 0.6% glucose in DPBS containing Ca/Mg, as well as different concentrations of permeable and non-permeable cryopreservatives, as shown in Figure 14B . For comparison, PRC cells were also formulated in commercially available CryoStor® CS10 cell freezing medium (STEMCELL Technologies, Inc., Vancouver, Canada) containing 10% DMSO. Cells were rapidly thawed at 37°C and diluted in GS2+ diluent buffer, and post-thaw cell viability was assessed. Post-thaw survival was assessed using the Cone Blue cell count method. The formulation containing 5% DMSO showed similar survival to the formulation of the present invention containing 10% DMSO (see Figure 14B ), while both formulations showed higher survival than the commercially available CryoStor® CS10. The formulation with the highest potency was selected for further analysis.

As a follow-up experiment, 6 different formulations were selected for further analysis. Cells were prepared using a similar procedure and post-thaw analysis was again assessed using conch blue exclusion. During this experiment, post-thaw viability was higher in 10% ethylene glycol + 0.2 M sucrose than in the 5% DMSO formulation.

The OMR responses of animals injected subretinally with 100,000 or 200,000 GB2R PRCs were compared ( Fig. 15A ). In animals injected subretinally with 100K (left panel) or 200K (middle panel) PRCs, 12 spatial frequencies (SPs) were performed and the OMR responses (tracking intensity) were recorded. The 1.2 response on the Y-axis is the threshold for the presence of OMR (shown by the green dotted line). Any value below 1.2 means that the eye cannot be tracked. At P35, P42, and P50, control eyes were blinded, and the eyes injected with 200k cells consistently showed larger and broader amplitude curves than the 100k group. The rightward shift in the SP threshold indicates better visual acuity in the 200k eyes.

RD10 mice were injected subretina with 100k or 200k PRCs and OMR was performed at P28, P35, P35, and P50 ( Fig. 15B ).

ICC analysis confirmed that ONL thickness was greater in 200k eyes than in 100k ( Fig. 15C ). Quantification of ONL thickness at transplanted sites relative to non-transplanted sites in animals that received 100K and 200K cells. The data are consistent with the hypothesis that higher doses result in greater efficacy.

OMRs were recorded for 300k rd10 mice injected with cryopreserved PRC (same as for rd10 mice above, subretinal injection at P14) ( Fig. 16A ). Cryopreserved PRC was functionally effective at P35 and P42. However, at P49, both vehicle and cryopreserved PRC eyes lacked OMRs. OMR analysis of RCS rats injected with 100,000 GB2R-PRC (CN2 batch), GB2R-PRC (P3 INT DS), or GB2R-PRC (cryopreserved PRC) cells per eye at P25 ( Fig. 16B ). Statistical analysis was performed using two-way ANOVA combined with Tukey's multiple comparison test (MCT), where test items were compared to uninjected eyes and eyes injected with GS2 vehicle. ERG analysis of RCS rats injected with 100,000 GB2R-PRC (CN2 batch), GB2R-PRC (P3 INT DS) or GB2R-PRC (cryopreserved PRC) cells per eye at P25 ( FIG. 16C ). Statistical analysis was performed using two-way ANOVA combined with Dukey's multiple comparison test (MCT), where the test article was compared to uninjected eyes and eyes injected with GS2 vehicle. Example 7 : Supplemental Immunosuppressive Drug Therapy ( IMT ) and Cellular Formulations

As previously mentioned, an exaggerated response was found in NIH-III mice following the surgical procedure. Supplemental immunosuppressive drug therapy (IMT) was tested to see if it could reduce the exaggerated response. Table 14 below shows the effects of GS2 buffer alone and different conditions including supplemental IMT. PRC was not used in the following experiments. Table 14 Grouping Total number of mice Test object Supplementary IMT Age at time of medication Autopsy (after injection) 1 10 (M + F) 5% DMSO low temperature buffer/ GS2+ (1:4) N/A 6-8 weeks 1 month 2 10 (M + F) 5% DMSO low temperature buffer/ GS2+ (1:4) 7 days Dex (IP, 2.5 mg/kg per day) 6-8 weeks 1 month 3 10 (M + F) 5% DMSO low temperature buffer/ GS2+ (1:4) 7 days Dex (IP, 2.5 mg/kg per day); CsA (in drinking water, 300 mg/L*) 6-8 weeks 1 month 4 8 (M + F) BSS N/A 6-8 weeks 1 month

Figures 17A and 17B show the ONL after GS2 was injected with or without IMT, such as dexamethasone (Dex) and/or cyclosporine (CsA). Based on optical coherence tomography (OCT), GS2 buffer without supplemental IMT showed the least damaged morphology overall, including compared to balanced salt solution (BSS).

18A to 18C show the ERG responses of each of the above-listed Groups 1 to 4. The effects of supplemental IMT on a-wave, twilight b-wave, and photopic b-wave levels were not significant among the groups.

Figure 19. Retinal morphology in each of Groups 1 to 4. GFAP and DAPI staining showed that each group had similar morphology and the addition of supplementary IMT did not change the retinal morphology.

In addition, C57BL/6 mice were used for testing to investigate the final concentrations of GS2 buffer alone and DMSO. Table 15 shows the experimental groups. Table 15 Grouping Total number of mice Test object Cryo Buffer : GS2+ Ratio ( for dilution ) Final DMSO % Age at time of medication Autopsy (after injection) 1 8 (M + F) GS2+ (Batch No. 9201) N/A 0 6-8 weeks 1 month 2 8 (M + F) GS2+ / 5% DMSO low temperature buffer 1:04 1% 6-8 weeks 1 month 3 8 (M + F) GS2+ / 5% DMSO low temperature buffer 1:14 0.33% 6-8 weeks 1 month 4 8 (M + F) BSS N/A 0 6-8 weeks 1 month

Table 16 shows the evaluation parameters and time intervals for Groups 1 to 4 studied in Table 15. Table 16 : Evaluation parameters and time intervals Parameters Approximate time interval ( days are days after injection ) Death/near death At least once a day Clinical observation At least once a day for the first three days after surgery and then three times a week for the remainder of the study Weight Once a week Eye examination before medication OU: Before giving medication Eye examination OU: 28 ± 5 days after administration Optical Coherence Tomography (OCT) OD: 1±1, 14±3, 28±5 days after administration Retinal electrogram (ERG) OU: 28 ± 5 days after administration Full autopsy At the end, 30 ± 5 days after dosing, including macroscopic lesions Tissue collection Eyes and tissues were fixed in 4% paraformaldehyde and stored at 4°C. Immunohistochemistry Eye

C57BL/6 mice were injected between weeks 6 and 8 and examined 4 weeks later. One eye was injected with GS2 buffer or control buffer. 2.5 mg/kg IMT, dexamethasone were administered daily for 7 days, and 300 mg/L cyclosporine was administered from D0 throughout the experiment.

Figures 20 and 21 show that the retinal morphology between Group 1 and Group 3 was similar to that of Control Group 4. Example 8 : Evaluation of PRCs derived from cell stock solutions

PCRs were prepared using the protocol described above and in Figures 12A to 12C and included freezing after P3, thawing, and completing the protocol until P4. Specifically, P4 PRCs using the direct/continuous process were compared to P4 PRCs that had been frozen at the P3 successive step and then thawed to complete differentiation.

RCS rat eyes that received P4 derived from P4(i) (P4(i) refers to P4 PRCs frozen and thawed between P3 and P4; “indirect”) showed increased focal retinal disruption at the injection site with enhanced PRC migration within the retina ( Fig. 22 ).

Table 17 shows the experimental design comparing P4(i) (“indirect”) and P4(d) (“direct”) PRC formulations. Table 17 Grouping Total number of mice Rd10 Test object Dosage Dosage volume (μl) IMT Age at time of medication Autopsy Group 1 1 6 P4(d) 126k 1ul dex +CsA P14 P50 2 6 P4(i) 118k 1ul dex +CsA P14 P50 Group 2 1 10 P4(d) 110k 1ul dex +CsA P14 P50 2 10 P4(i) 110k 1ul dex +CsA P14 P50

Immunosuppressive therapy consisted of daily intraperitoneal injections of dexamethasone (5 mg/kg) for 7 days after surgery. Oral cyclosporine A was administered in drinking water (300 mg/L) from P21 until the day of sacrifice.

Figure 23 shows the ratio of appropriate to inappropriate eye movements (OMR analysis) comparing PRC injected right eyes and left eye controls. The injected dose was 110k PRC as shown in Group 2 in Table 16. At ages P35, P42, and P49, P4(i) PRC showed reduced efficacy compared to P4(d) PRC.

Figure 24 shows that intraretinal migration was similar between P4(i) and P4(d) in Group 1. However, Figure 25 shows that migration into the inner plexiform layer (IPL) was enhanced in Group 2 after treatment with P4(i) PRC.

Table 18 shows Groups 1 and 2 to assess P4(i) PRC and investigate increased intraretinal migration/increased focal disruption in the rd10 mouse model (compared to rats). Table 18 Group 1 ​ Test object Direct P4 (P4d)/P4, via cell stock solution (P4i); Batch 2 CMC Dosage 62.5k/125k condition Finish Summary of results (n=4-6) Histology : Intraretinal migration/injection site disruption similar between P4d/P4i OMR : low dose, poor follow-up; high dose: similar between groups, P4d tends to be stronger than P4i Eye examination : P4i Some degree of increased incidence of vitreous cell/retinal detachment Group 2 Test object Direct P4 (P4d)/P4, via cell stock solution (P4i); Batch 3 CMC Dosage 125k condition Completed during life cycle Results Overview (n=10/10) Organizational Science : Analysis is ongoing OMR : P4d tracking is stronger than P4i. This indicates that the addition of cell reserve solution reduces the efficacy. Eye examination : The incidence of vitreous cell/retinal detachment is similar between P4d and P4i

Figures 26 and 27 show populations 1 and 2, respectively. For both populations tested, P4(i) (P4, via cell stock solution) showed reduced efficacy when injected. Example 9 : Evaluation of CellSTACK® Containers for Cell Cultures

Rd10 mice were tested with PRC prepared using PDL-coated T175 flasks compared to PRC prepared using CellSTACK® containers (Corning®) ( Figure 28 ). The control PRC preparation was batch 3 CMC. OMRs were analyzed at postnatal ages P35, P42, and P49 for each PRC preparation. Figure 28 represents the following: uninjected eyes, N=10; GS2 eyes, N=11; PRC-CTL (batch 3 CMC), N=5; CellSTACK, N=10; and PDL-precoated, N=6.

Figure 29 shows eyes injected with PRC prepared in CellSTACK® containers compared to PDL pre-coated flasks at D7 and D38 (days 7 and 38 after injection, respectively). Graft size increased in 6 of 10 eyes when using CellSTACK® PRC, and one eye had an abnormally large graft when comparing D7 to D38.

Subretinal injections and immunohistochemistry (IHC) were performed as described above. Figures 30 to 34 show IHC markers to assess engraftment after D38 injection. Markers stained included: human nuclear antigen (HuNu) (for assessment of engraftment) ( Figures 30A and 30B ); cone arrestin (CAR) (for assessment of cone morphology preservation) ( Figures 30A and 30B ); DAPI (for assessment of ONL thickness and preservation of rods/cones); GFAP (for assessment of Mullerian colloid degeneration, also a secondary marker of PRC) ( Figure 31 ); IBA1 (for assessment of microglia/macrophages) ( Figure 32 ); Ki67 (for assessment of proliferation) ( Figure 33 ); and Oct4 (for assessment of rich potential) ( Figure 34 ). Eyes were collected at P52. All three groups, including flasks pre-coated with PDL, PRCs prepared with CellSTACK®, and controls, showed engraftment. CellSTACK® PRCs induced significantly larger grafts compared to PDL pre-coated flask PRCs or control PRCs ( Figures 30A and 30B ).

The arrows in Figure 30B point to instances where the cone was well preserved. When treated with CellSTACK® PRC, instances where the cone was well preserved were more frequent. Instances where the ONL was 3-5+ layers thick in a wide area were more frequent in the CellSTACK® group.

Figure 31 shows IHC of GFAP in each test group. PRCs continued to express GFAP and showed Müller's jelly activation.

Figure 32 shows IBA staining in each test group. CellSTACK® showed slightly higher IBA infiltration than other groups. Arrows show microglial cell infiltration.

Figure 33 shows the Ki67 levels in each of the three groups. Higher Ki67 levels were found in the CellSTACK® group. However, Ki67+ cells were in small numbers, indicating limited proliferation over time.

Figure 34 shows that PRCs from all three groups were negative for the high potential marker Oct4. Example 10 : Delayed injection of PRCs

Delayed injection studies were performed in RCS rats to demonstrate that PRCs can be active after degeneration has occurred. Conclusions from this study: 1) As is typical with neuroprotective approaches, early intervention is best, however 2) late intervention can still be effective, even after disease has occurred.

Figure 35 shows a schematic diagram of the experimental design for delayed injection analysis. Rats were injected at P25, P45, and P60 after disease onset. OMR and ERG readings were obtained between P60 and P150, and eyes were subsequently collected for IHC.

FIG36A shows OMR readings obtained at P60 (note that in FIG36A , the injected eye was analyzed at P62 ), P90, P120, and P190. The OMR readings obtained on the earliest (P25) injected eye showed a stable threshold response over time. However, eyes injected at time points after P60 showed a reduced threshold response over time, but the threshold response was higher than the negative control group.

Figure 36B shows ERG readings obtained at P60, P90, P120, and P150. Delayed injections did not result in detectable ERG retention. P25 injections resulted in detectable ERG levels that decreased over time. Later injections resulted in sensory cell retention that was below ERG detection.

Figure 37 shows IHC of transplantation and retention. Quantification of transplantation and retention is shown in Figures 38A to 39B . Transplantation was calculated by the following formula: Transplantation = Subretinal transplant length in section / Retina length

ONL preservation was calculated by the following formula: ONL preservation = ONL length in slices / retinal length Example 11 : Overview of low survival rate study rd10 mouse model

This study was conducted to evaluate the efficacy of a PRC drug with a low survival rate (≤60%) (Group 1) compared to a PRC drug with a high survival rate (≥70%) (Group 2) in the rd10 mouse model.

In addition, some data were obtained to evaluate the safety of the PRC drug with low survival. Higher than expected losses were observed early in the first few days of the study, which were considered to be a result of perioperative analgesia in young mice and affected both groups uniformly (3 mice were lost in each group). Throughout the lifespan part of the study, except for the initial slow growth, especially after weaning from P21 to P28, body weight increased steadily in both treatment groups, and no serious welfare complications were reported. After examination, some treated eyes from both groups showed an increase in cells in the vitreous or retinal detachment, but there were no other complications for all 17 parameters evaluated. The frequency of complications was similar in both treatment groups. In the optomotor response (OMR) analysis, although vehicle-treated or untreated eyes showed a marked decrease and loss of tracking over time, tracking in cell-treated eyes in both groups resulted in statistically significant retention of tracking or insignificant but clear tracking function until P49, as reflected by an increase in tracking magnitude of 0.1-0.2 turns per degree and a decrease to baseline on either side of the peak. After necropsy, no major abnormalities were observed, and except for a slight increase in thymus weight in Group 1, the weights of various major organs were similar between groups and compared with untreated mice. After immunohistochemical evaluation, engraftment in both groups was confirmed by anti-human nuclear antigen (HNA) staining. Engraftment rates were similar between cell injection groups, with 2 of 4 eyes in Group 1 and 3 of 4 eyes in Group 2 showing HNA+ cell grafts. PRC injections were associated with a non-significant tendency toward cone elongated morphology (when stained for cone arrestin) or thickened ONL (when assessed with DAPI staining). In all eyes studied, GFAP staining intensity was prominent in the Müller neurojelly, consistent with the phenotype of this model and retinal degeneration.

The data obtained in this study support the hypothesis that PRC products with survival rates greater than 70% are similarly effective as PRC products with survival rates less than 60% (i.e., 50 to 60%). Other safety data collected also support the safety of the product at any level of survival. Statistical significance was not observed in many of the parameters evaluated due to higher than expected perioperative losses, but overall, the observed trends were consistent with the conclusions. Reference PRC and Test PRC

Reference : Preparation of GS2+/ Low Temperature Buffer Injection Mix. Low Temperature Buffer was added to GS2+ medium at a 1:4 ratio to produce the injection mix. In this study, the GS2+/Low Temperature Buffer Injection Mix was used as a diluent for the test article (ASP1819/PRC) as well as the vehicle control for injection.

Test Articles : Prepare ASP1819/PRC cells. Cryopreserved ASP1819/PRCs were stored in the vapor phase of a liquid nitrogen (LN2) tank at a mesophilic temperature ≤-135°C until the day of transplantation. ASP1819/PRC cells were thawed and diluted with GS2+ medium. The reconstituted cell suspension was stored at 2 to 8°C and assessed for viable cell count. After concentration in the GS2+/cryogenic buffer injection mixture to the target cell density (approximately 125,000 viable cells/µL) and viability (approximately 70% or ≤60%), the test articles were dispensed for injection within approximately 4 hours of concentration. Animal Systems

Species: Mouse

Strain: Pde6brd10 homozygous (C57BL6/J background)

Gender: M and F

Age range at time of medication: P14 to 15 days

Age at time of tissue collection: P51, P52

Weight range at time of administration: approximately 6 to 12 g

Source: AIRM (originally The Jackson Laboratory) rd10 mouse colony (#004297)

Number of animals studied: 14 (Group 1: 2M, 5F; Group 2: 1M, 6F) Cell preparation and treatment

Cells in frozen vials were thawed to generate test articles for Groups 1 and 2. Cells were counted manually using a hemacytometer and conch blue exclusion. To engineer specific cell viability for Groups 1 and 2, the initial cell suspension was subjected to Optiprep (an iodixanol-based density gradient) to separate dead/live cells. The appropriate amount of dead and live cells were then combined to generate test articles for injection. Cell preparation details are specified in the batch record. Study Design

Cohorts of 14 rd10 mice were randomly divided into two dose groups as described in Table 18. All mice received a subretinal injection of 1 µL PRC cells (≥70% or ≤60% survival) in the right eye (OD) via transscleral administration under anesthesia. The left eye (OS) was injected with vehicle solution or not injected. Animals were evaluated from the date of injection until final sacrifice as shown in Table 19. Comparative measurements were analyzed between the 4 groups of eyes (≥70% PRC, ≤60% PRC, vehicle, not injected). Mice were euthanized on day 37±3 after surgery, or at approximately P50 age. Table 18 : Experimental Study Design Grouping Number of mice Target cell number ( 1 µL volume ) Right eye ( OD ) Left Eye ( OS ) 1 7 (M or F) 125,000 Injection ≥ 70% viable cells (70% buffer) Vehicle buffer (3), or no injection (4) 2 7 (M or F) 125,000 Injection ≤ 60% viable cells (60% buffer) Vehicle buffer (4), or no injection (3) Table 19 : Evaluation parameters and time intervals Parameters Approximate time interval ( days are days after injection ) Death/near death At least once a day Clinical observation At least once a day for the first three days after surgery and then three times a week for the remainder of the study Weight Once a week Eye examination before medication OU: Before drug administration (day 0), in terms of gross abnormality Eye examination OU: 35±3 days after administration Opto-motor response (OMR) OU: 14±3, 21±3, 28±3 and 35±3 days after administration Optical Coherence Tomography (OCT) OU: 1±1, 7±3 and 31±3 days after administration Full autopsy At the end, 37 ± 3 days after dosing, including macroscopic lesions Tissue collection Eyes, optic nerves, brain, mandibular lymph nodes, heart, liver, kidneys, spleen, lungs, ovaries, testicles, thymus Organ weight Brain, heart, liver, kidneys, spleen, lungs, ovaries, testicles Immunohistochemistry Eye Immunosuppressive regimen

Dexamethasone (2.5 mg/kg per day) was administered intraperitoneally once daily on days 7 to 8 after surgery (approximately P14 to 21). After weaning at approximately P21, animals were maintained on oral cyclosporine A (CsA) (300 mg/L) in the drinking water for the remainder of the study until euthanasia .

Prior to surgery, the administration device (a microsyringe (Hamilton 600 series) equipped with a 34 ga flat-tip needle (Hamilton #207434)) was pre-filled with cell suspension or vehicle. A ketamine/xylazine mixture for anesthesia and Ethiqa for preoperative analgesia were administered to the test animals. When the test animals achieved deep anesthesia (indicated by slowed breathing and lack of response to toe pinch), they were placed on their side under a dissecting microscope with the eye undergoing the procedure positioned upward. The skin above and below the eye was pulled back to allow the eye to droop. Betadine eye drops (5%, sterile, ophthalmic grade) were administered to the eye for topical disinfection of the globe, and artificial tears were applied to flush excess betadine from the eye. Proparacaine eye drops were applied to render the eye and surrounding tissues numb, and phenylephrine (2.5%) and tropicamide eye drops were applied to dilate the pupil. Between eye drop/artificial tear applications, excess solution was removed using a fabric-tipped swab. For subretinal injections, a hole was cut in the conjunctiva to expose the sclera. Using a 30 ga beveled needle, a guide hole was created over the limbal region. Under visual guidance, a 34 ga needle microsyringe containing cell suspension or vehicle buffer was inserted into the hole and the injection material was pushed into the eye by manually depressing the plunger. After injection, the needle was slowly removed from the eye and the animal was positioned on the opposite side for the injection procedure in the second eye (or vehicle, if applicable). OCT imaging was performed immediately after the injection procedure to assess injection success. After imaging, ophthalmic erythromycin (0.5%) ointment was applied to the surgical site for antimicrobial and lubricating properties. Atipamezole (2 mg/kg) was administered intraperitoneally to aid in the recovery process. Normal saline was administered subcutaneously for moisture retention. Finally, to prevent hypothermia, the animals were moved to a heated cage until they were in sternal recumbency and then returned to their home cage. Optomotor Response ( OMR )

Visuomotor responses were measured using the Phenosys qOMR system. Head tracking responses to grating drifts of different spatial frequencies (0.05-0.6 turns/degree, 0.05 turns/degree increments) were measured twice on individual days for all animals at 4 time points (14±3, 21±3, 28±3, and 35±3 days after surgery), and the values were averaged to generate OMR response curves. Mice were placed unrestrained in the center of an area on an elevated platform, which was enclosed on 4 sides with monitors to simulate the presentation of a rotating drum with a vertical sine wave grating. The ratio of the time spent moving the head in the preferred direction relative to the time spent moving in the non-preferred direction was calculated, and the value of the ratio was considered the tracking strength. A mean value greater than 1.2 indicated the presence of tracking at that spatial frequency. Prior to the first time point collection (days 1 to 7 prior), animals were introduced to the device for acclimatization. Optical Coherence Tomography ( OCT )

Spectral domain optical coherence tomography (SD-OCT) was performed using a Leica Bioptigen Envisu system on days 1±3, 7±3, and 31±3 after surgery. Mice were anesthetized by IP injection or inhalation of anesthetics. Phenylephrine and tropicamide were applied to the eyes to dilate the pupils for better imaging of the retina. Lubricating eye drops (Genteal) were applied to maintain eye clarity for imaging. B-scans were obtained using an imaging system at the injection site to visualize the cell transplantation. At the end of the imaging period, mice were moved to a heated cage until sternal recumbency to prevent hypothermia and then returned to their home cages. Immunohistochemistry

For animals undergoing scheduled euthanasia at the terminal time point, double immunofluorescence staining was performed on slides prepared from all eyes injected with PRCs using anti-human nuclear antigen (HNA) staining to confirm PRC engraftment and anti-cone arrestin (CAR) to assess cone preservation and cone axon measurements. In addition, outer nuclear layer (ONL) thickness was analyzed using DAPI to assess overall photoreceptor cell preservation. Quantification of cone axon or ONL thickness was performed blinded to sample identity. For vehicle-injected eyes or non-injected eyes, CAR staining and ONL thickness analysis were performed, but HNA staining was omitted. Immunostaining for GFAP was performed to assess Müller's neurocolloid degeneration and confirm PRC identity. Cell Counts and Survival

The cells were counted and the viability was measured as indicated in the batch record. The final counts and viability are shown in Table 20 below. Table 20 : Cell Counts and Viability Group Number Target live cell count (cells/µL) Sample cell count (cells/µL) Target survival rate Sample survival rate 1 112,500- 137,300 125,000* ≥70% 68.4%# 2 112,500- 137,300 125,000* 53-57% 53.5% * Values adjusted to final count by addition of GS2 buffer, confirmatory counts were not performed to allow for cell conservation. #This survival rate was considered acceptable on the day of surgery due to the challenge of producing ≥70% viable preparations. Clinical Observations and Weight

No abnormal clinical signs of distress were observed during the course of the study. After an initial slow growth period during the first two weeks post-injection, the weight of all surviving mice increased steadily during the remainder of the study. No significant weight differences were observed between Group 1 and Group 2 at any time point. Eye Examination

A 17-point eye examination was performed ( Table 21 ). No abnormalities were observed on the ocular surface or in the anterior segment. Two cell-injected eyes in Group 1 showed cells in the vitreous, one of which showed mild (11-20 cells) and the other showed mild but slightly more cells (21-30). In one cell-injected eye in Group 2, mild cells (11 to 20 cells) were observed in the vitreous.

Retinal detachment was observed in the cell-treated eyes in both groups. Moderate detachment was observed in one eye in Group 1, which also showed a retinal tear at the transplant site. In Group 2, two eyes showed larger detachments, and the third eye showed smaller detachments.

Although the presence of these complications was limited to the cell-treated eyes, complications occurred with similar frequency in the 60% survival or 70% survival groups. In addition, retinal detachment is a known phenotype of rd10 mice (Pennesi et al., 2012, IOVS). Table 21 : Scoring of vitreous cells and retinal detachment obtained from ocular examination. Eyes showing abnormalities are indicated in red font. Data from complete ocular examination Grouping (OD) animal gender OS Vitreous cells (OS/OD) Retinal detachment (OS/OD) 1 (≥70%) 1 F Medium 0/0 0/0 6 F No injection 0/2 0/0 9 F No injection 0/0 0/0 10 F No injection 0/1 0/2 2 (≤60%) 2 M No injection 0/1 0/1 3 F Medium 0/0 0/0 5 F Medium 0/0 0/3 8 F No injection 0/0 0/3 Opto-motor response ( OMR )

Optomotor response analysis was performed at postnatal ages P28, P35, P42, and P49 ( Figures 40A to 40D ). For cell-injected eyes (eyes with 70% or 60% survival), there was a clear tracking response at all four time points evaluated, as observed by an inverted U-shaped curve centered at a spatial frequency of approximately 0.1 to 0.2 turns/degree. Meanwhile, in vehicle, tracking was weak (low amplitude) or absent. After statistical analysis, significant differences were observed only at P35 (*p=0.0376, 70% + buffer vs. buffer + vehicle; *p=0.0345, 70% + buffer vs. no injection) and P49 (*p=0.0446, 70% + buffer vs. buffer + vehicle). No statistical significance was observed in cell-treated eyes between experimental groups at any time point. Optical Coherence Tomography ( OCT )

OCT was performed on D0, 9, and 35 ( Figures 41A to 41F ). D0 OCT confirmed the formation of subretinal blister in all right eyes of Group 1, except for animal 11-F, which was the last to be found dead on D1. For Group 2, D0 OCT confirmed the formation of subretinal blister in all eyes of this group. OCT on D9 confirmed the persistence of subretinal material in all 4 right eyes of surviving mice in Group 1 and all 4 right eyes of surviving mice in Group 2. Group 2 mouse #8 showed larger retinal detachment on D9, which persisted until D35, when the graft appeared round in shape. OCT on D35 confirmed that the subretinal material in the two cell-treated groups had different sizes. Necropsy and Organ Weights

At the terminal time point, necropsy was performed on day 38. No abnormalities were observed. The weights of brain, heart, liver, kidney, spleen, lung, thymus, ovary, and testis were obtained. Except for a decrease in thymus weight between Group 1 and Group 2 (0.081 ± 0.012 g (Group 1) vs. 0.0447 ± 0.0085 g (Group 2)), there were no statistical differences in other organs between the experimental groups. The values were similar to those of untreated NIH-III mice (data not shown). Immunohistochemistry Human nuclear antigen ( HNA )

HNA immunostaining was performed to confirm engraftment of the administered PRCs. In the cell-treated eyes of Group 1, 2 of 4 eyes showed engrafted cells. One of those eyes showed many cells ( Fig. 42A , Group 1 ), while the other eyes contained smaller grafts. In the cell-treated eyes of Group 2, 3 of 4 eyes showed engrafted cells. Two grafts were large in size, while the graft in the third eye was small in size. In one of the eyes with a large graft in Group 2, the graft appeared as a sphere of cells in the subretinal space that appeared to be a larger retinal detachment in the subretinal space ( Fig. 42A , Group 2 ). Cone-arrestin ( CAR )

CAR immunostaining was performed to assess cone preservation. In both experimental groups, examples of elongated cone morphology were observed near the PRC transplant site, indicating that focal cone preservation can occur near the PRC graft when PRC is injected at high and low survival rates ( Figures 43A and 43B , relative to Figure 43C ). In eyes injected with vehicle or without injection, the gross morphology of cones was exclusively a flattened monolayer composed of stubby cells ( Figure 43D ). Although quantification of cone axon length did not reveal significant differences between the groups, one-way ANOVA was significant at p < 0.05, and cone axon length in cell-treated eyes tended to increase compared to control eyes, indicating that administration of PRC at any survival rate had a protective effect on cone morphology. Quantification of outer nuclear layer ( ONL )

ONL thickness was measured blindly at random sites near the graft by using DAPI to visualize the ONL. No statistical significance was observed for ONL thickness in the cell-treated groups compared to the uninjected or vehicle-injected control groups, but there was a trend toward an increase ( Figure 44 ).

Neurofibromin acidic protofibrotic protein （ GFAP ）

GFAP staining was performed to assess Müller's jelly in the retina ( Figure 45 ). The Müller's jelly in all eyes had prominent GFAP staining intensity, represented by vertical bands spanning the thickness of the retina, consistent with the rd10 mouse model at the age assessed (approximately P50). The persistent presence of GFAP immunoreactivity in the Müller's jelly following PRC treatment suggests that the host retina continues to experience some degree of disease. The implanted PRCs also expressed GFAP at high levels, consistent with previous observations following in vivo administration. Conclusions

The data obtained in this study support the hypothesis that PRC drugs with survival rates greater than 70% are similarly effective as PRC drugs with survival rates less than 60% (i.e., 50 to 60%). Other safety data collected also support the safety of the product at any survival level. Example 12 : Low Survival Study NIH-III Mouse Model

This study was conducted to evaluate the safety of a low survival (<60%) PRC drug product versus a high survival (>70%) PRC drug product in the immunodeficient NIH-III mouse model. No deaths occurred during the study period, except for one anesthesia-related death in Group 3 (<60%) during ERG recording. Weight gain was steady across groups throughout the life span portion of the study, and no serious well-being complications were reported. Following ophthalmological examination, some treated eyes in all three injection groups (Groups 1 to 3) showed vitreous hemorrhage, which was attributed to complications of the surgery. Additionally, within the injection groups (Groups 2 and 3), cells were present in the vitreous of some treated eyes, although the frequency was similar in Group 2 (high survival) and Group 3 (low survival). Apart from those observations, there were no other complications among the 17 parameters evaluated.

OCT imaging at day 28 identified the presence of a retinal injury pocket at the center of the injection in Groups 1 to 3, which may be related to the surgical procedure. Subretinal material of varying sizes at the injection site was also identified on OCT in Groups 1 to 3. The observation of this subretinal material in the vehicle-injected group (Group 1) suggests that host cells, likely immune cells, tend to infiltrate the injection site in this model following the procedure. Retinal electroretinogram (ERG) recordings obtained at approximately day 30 did not reveal significant differences between the study groups, suggesting that there were no gross effects on retinal function following cell or vehicle buffer administration. ERG results were consistent with ophthalmologic observations and OCT imaging, which similarly identified no gross effects. No adverse findings were observed after necropsy on day 36 after dosing, and organ weights were similar in all four groups. Hematology and clinical chemistry analyses did not reveal any major differences between the groups.

Upon histological examination of tissues collected on day 36, GFAP immunostaining with a unique PRC morphology (non-Mullerian jelly morphology) localized subretinally was observed in 1 sample from Group 2 and 2 samples from Group 3. However, nuclear anti-HNA staining was not observed in any sample, and therefore human cells could not be confirmed. Focal Mullerian jelly GFAP immunoreactivity and limited retinal laminar disruption at the injection site in Groups 1 to 3 were shown at similar levels in all three groups, consistent with surgically induced injection site damage. In addition, similar levels of elevated Iba1 immunoreactivity were detected at the injection site in Groups 1 to 3. Finally, staining for CD45, Bestrophin-1, and Ter119 was also performed to investigate the composition of the subretinal graft/cell mass, but none of these markers were clearly expressed in the subretinal graft.

In summary, the data obtained in this study support the safety of the drug in PRC with a survival rate of <60% (i.e., 50-60%) compared with PRC with a survival rate of >70%.

Species: Mouse

Strain: NIH-III nude mice

Gender: M and F

Age range at time of dosing: 6 to 8 weeks

Weight range at time of dosing: approximately 18 to 33 g

Source: Charles River Laboratories (strain code: 201, homozygous)

Number of animals studied: 36

Study Design

Thirty-six NIH-III nude mice were randomly assigned to four groups as described in Table 22. To facilitate the cell preparation and injection procedures, mice were divided into two injection groups on consecutive days, where all animals injected in the first group were injected with cells due to the complexity of cell preparation with survival as the goal. The right eye (OD) of mice in Groups 1 to 3 received a subretinal injection of 1.5 µL of vehicle or PRC via a transscleral administration route under anesthesia. The left eye (OS) was not injected. Group 4 mice were not injected. Animals were evaluated from the injection date until final sacrifice as shown in Table 23. Comparative measurements were analyzed between the four groups. Mice were euthanized approximately 5 weeks after injection. Table 22 : Experimental Study Design Grouping Total number of mice Test object Target survival rate Dose (cell count) Dose volume ( µL ) Injected cell density ( cells /µL ) Age at time of medication Autopsy (after injection) 1 10 (4M + 6F) Medium N/A N/A 1.5 N/A 6-8 weeks 5 wk 2 10 (4M + 6F) PRC (＞70% survival rate) 70-80% 125k 1.5 83,333 6-8 weeks 5 wk 3 10 (4M + 6F) PRC (<60% survival rate) 50-60% 125k 1.5 83,333 6-8 weeks 5 wk 4 6 (1M + 5F) N/A (untreated) N/A N/A N/A N/A N/A (age matched) 5 wk Table 23 : Evaluation parameters and time intervals Parameters Approximate time interval ( days are days after injection ) Death/near death At least once a day Clinical observation At least once a day for the first three days after surgery and then three times a week for the remainder of the study Weight Once a week Eye examination before medication OU: Before drug administration (day 0), in terms of gross abnormality Eye examination OU: 30 ± 3 days after administration Optical Coherence Tomography (OCT) OD: 1±1, 14±3, 30±3 days after administration Retinal electrogram (ERG) OU: 30 ± 3 days after administration Clinical Pathology At the end, 35 ± 3 days after administration Full autopsy At the end, 35 ± 3 days after dosing, including macroscopic lesions Tissue collection Eyes, optic nerves, brain, mandibular lymph nodes, heart, liver, kidney, spleen, lung, ovary, testis, thymus, or any organ with macroscopic lesions, are maintained for archival purposes. Tissues are fixed in 4% paraformaldehyde and stored at 4°C. For archival purposes. Tissues will be provided to the trial sponsor. Organ weight Brain, heart, liver, kidneys, spleen, lungs, ovaries, testicles, thymus Immunohistochemistry Eye Subretinal injection

Prior to surgery, the administration device (a microsyringe (Hamilton 600 series) equipped with a 34 ga flat-tip needle (Hamilton #207434)) was pre-filled with cell suspension or vehicle. The test animal was administered a ketamine/xylazine mixture. When the test animal achieved deep anesthesia (indicated by slowed breathing and lack of response to toe pinch), it was placed on its side under a dissecting microscope with the eye undergoing the procedure positioned upward. The skin above and below the eye was pulled back to allow the eye to droop. Betadine eye drops (5%, sterile, ophthalmic grade) were administered to the eye for topical disinfection of the globe, and artificial tears were applied to flush excess betadine from the eye. Proparacaine eye drops were applied to render the eye and surrounding tissues numb, and phenylephrine (2.5%) and tropicamide eye drops were applied to dilate the pupil. Between eye drop/artificial tear applications, excess solution was removed using a fabric-tipped swab. For subretinal injections, a hole was cut in the conjunctiva to expose the sclera. Using a 30 ga beveled needle, a guide hole was created over the limbal region. Under visual guidance, a 34 ga needle microsyringe containing a cell suspension or vehicle buffer was inserted into the hole and the injection material was pushed into the eye by manually depressing the plunger. After injection, the needle was slowly removed from the eye. OCT imaging was performed immediately after the injection procedure to assess injection success. After imaging, ophthalmic erythromycin (0.5%) ointment was applied to the surgical site for antimicrobial and lubricating properties. Atipamezole (2 mg/kg) was administered intraperitoneally to aid the recovery process. Normal saline was administered subcutaneously for moisturization. Finally, to prevent hypothermia, the animals were moved to a heated cage until sternal recumbency and then returned to their home cage.

Eye examination was performed by The Scientific Lead using a Phoenix Micron IV imaging microscope 30 ± 3 days after surgery. Eyes were dilated with 1% tropineamide and/or 2.5% phenylephrine (1 drop in each eye). The anterior segment (cornea and lens) and posterior segment (retina, vitreous) were examined under anesthesia, and pupillary reflexes, conjunctival secretion, conjunctival injection, conjunctival swelling, cornea, surface area of corneal involvement, pannus, aqueous flare, aqueous cells, iris involvement, lens, vitreous flare, vitreous cells, vitreous hemorrhage, retinal detachment, retinal hemorrhage, and choroidal/retinal inflammation were examined. All abnormalities were recorded by the examiner. Optical coherence tomography ( OCT )

Spectral domain optical coherence tomography (SD-OCT) was performed using a Leica Bioptigen Envisu system on days 1±3, 14±3, and 30±3 after surgery. Mice were anesthetized by IP injection or inhalation of anesthetics. Phenylephrine and tropicamide were applied to the eyes to dilate the pupils for better imaging of the retina. Lubricating eye drops (Genteal) were applied to maintain eye clarity for imaging. B-scans were obtained using an imaging system at the injection site to visualize the cell transplantation. At the end of the imaging period, mice were moved to a heated cage until sternal recumbency to prevent hypothermia and then returned to their cages. Retinal electroretinogram ( ERG )

ERG was performed 30 ± 3 days after surgery. Animals were kept in complete darkness overnight (at least 12 h) to allow for dark adaptation. During the entire recording period, animals were anesthetized with isoflurane on a recording platform or by intraperitoneal injection of ketamine/xylazine. Experimental procedures were performed under dim red lighting. Phenylephrine (2.5%) and tropicamide (1%) were added to dilate the pupil, while hydroxypropyl methylcellulose (0.3%) was added for lubrication. After the application of eye drops, the electrode contact recording lens (LKC) was placed on the animal's eye. Full-field illumination was provided by LED or Xenon flashlight via a Ganzfeld stimulator, and ERG responses were amplified, digitized, and recorded via the UTAS Bigshot ERG system (LKC). Depending on the stimulus intensity (3–20 stimulus presentations), responses were averaged to account for noise in the signal. Subcutaneous saline was administered for hydration (as needed). Finally, to prevent hypothermia, animals were moved to a heated cage until sternal recumbency and then returned to their home cage. If ketamine/xylazine anesthesia was used, atipamezole (2 mg/kg) was administered intraperitoneally to aid the recovery process. Immunohistochemistry

For animals that underwent scheduled euthanasia at the terminal time point, immunofluorescence staining was performed on slides prepared from all eyes injected with PRC by staining with anti-human nuclear antigen (HNA), which can confirm the human origin of the cell graft. Vehicle-injected eyes or non-injected eyes can be used as controls. In addition, all samples were evaluated using anti-GFAP and anti-Iba1 staining to evaluate Muller's neurocolloid degeneration and microglial/macrophage infiltration, respectively. The presence of hematopoietic-derived cells in some or all eyes was evaluated using anti-CD45 staining, the presence of erythrocytes in some or all eyes was evaluated using anti-TER119 staining, and the presence of retinal pigment epithelium in some or all eyes was evaluated using anti-bestrophin staining. Results Cell counts and survival

The cells were counted and viability was measured as indicated in the batch record. The final counts and viability are shown in Table 24 below. Table 24 Group Number Target live cell count (cells/µL) Sample cell count (cells/µL) 1.5 µL volume (viable cell count) Target survival rate Sample survival rate 2 (＞70%) 75,000-91.666 75,250 112,875 ≥70% 75.9% 3 (＜60%) 75,000-91.666 75,500 113,250 53-60% 58.0% 2 (＞70%) 75,000-91.666 75,375 113,063 ≥70% 73.7% 3 (＜60%) 75,000-91.666 76,125 114,188 53-60% 58.9% Eye examination

A 17-point eye examination was performed on day 28 ( Table 25 ). No abnormalities were observed on the ocular surface or in the anterior segment. Both eyes injected with cells in Group 2 showed moderate cells (approximately 100 or >100) in the vitreous. Meanwhile, in one cell-injected eye in Group 3, moderate cells (approximately 100 cells) were observed in the vitreous.

Vitreous hemorrhage was observed in 4 injected eyes in Groups 1 to 3. Two cases were found in Group 1, where the hemorrhage covered 10 to 20% of the fundus image. One case was found in Group 2, where the coverage of the fundus image was approximately 10%. One case was found in Group 3, where the coverage of the vitreous hemorrhage in the fundus image was approximately 5%.

No other retinal abnormalities were observed except for surgically induced lesions.

Although the presence of vitreous cells was limited to the cell-treated eyes, complications occurred with similar frequency in the 60% or 70% survival groups. In the case of vitreous hemorrhage, due to the observations in the vehicle-injected group (Group 1), it appears that this may be a risk of the subretinal injection procedure/surgery in the NIH-III strain, and that the administration of high- or low-survival PRC drugs does not pose additional risks.

Table 25 shows the values expressed as the number of mice observed to have complications relative to the total number of animals in that group. Animal groups showing abnormalities are indicated in bold. Table 25 : Scoring of vitreous cells and vitreous hemorrhage in ocular examination Grouping Vitreous cells Vitreous hemorrhage 1 (Vehicle) 0/10 2/10 2 (＞70%) 2/10 1/10 3 (<60%) 1/10 1/10 4 (untreated) 0/6 0/6 Optical Coherence Tomography ( OCT )

OCT was performed at D0, 14, and 30. D0 OCT confirmed the formation of subretinal edema in the right eyes of all mice in Groups 1 to 3. Some highly reflective material was detected in the vitreous of several eyes in all three groups, indicating partial misinjection into the vitreous or possible surgery-related bleeding.

In D14 OCT images of Groups 1 to 3, resorption of blister was observed in all injected mice. Some degree of focal surgical injury was observed in all vehicle- or cell-injected eyes, characterized by retinal thinning across a limited retinal surface area, particularly the outer nuclear layer (ONL). In Groups 2 to 3, some subretinal superfluorescent material (small/thin or large in size) was observed, consistent with the injection of PRC. Surprisingly, in Group 1, variable amounts of subretinal material were also observed, suggesting that injection of vehicle buffer caused subretinal accumulation of host cells, likely of immune origin. Additional evidence of inflammation was observed in approximately half of the eyes in Groups 1 to 3: scattered superfluorescence in the vitreous. In both eyes of Group 1, the retina lacked lamination at the injection site, and increased retinal thickness in one of those eyes indicated some degree of edema.

By D30, the findings observed at D14 were generally also observed at the final OCT time point, especially with respect to focal retinal thinning, subretinal material accumulation, and intravitreal superfluorescence. In the two eyes in Group 1 that developed retinal detachment from D14, those portions of the retina showed severe focal atrophy and thick fibrous morphology suggestive of scarring at D30.

In summary, OCT imaging revealed that a degree of surgical damage had occurred in the retina of the NIH-III model, which was associated with possible immune cell infiltration (despite the immunodeficiency of the model). The abnormalities observed in the vehicle buffer injected Group 1 eyes were severe or in some cases more severe than those produced in Groups 2 and 3, which supports the safety of the cells injected within the drug regimen at either cell viability (>70% or <60%). The data are shown in Figures 46A to 46J . Retinal electroretinogram ( ERG )

Full-field ERGs were performed in both eyes of all study animals at approximately D30 under both twilight and photopic conditions. Treated right eyes (Groups 1 to 3) were compared with uninjected fellow eyes and with the right eyes of all other groups (including the untreated right eye of Group 4) ( Figs. 47A to 47C ).

There were no statistically significant differences between the responses of the left and right eyes, or between the responses of the right eyes and the right eyes of the other groups, in the twilight recordings (a-wave or b-wave).

In the photopic recordings, the treated eyes of Groups 1 and 2 showed a statistically significant decrease (approximately 20-25%) relative to the uninjected fellow eyes. However, there were no statistically significant differences relative to all other right eyes of Groups 1 to 4.

Overall, ERG recordings did not reveal any systematic effects on the amplitude of ERG responses by any of the injected test substances.

At the terminal time point, necropsy was performed on day 36. No abnormalities were observed. Weights of brain, heart, liver, kidney, spleen, lung, thymus, ovary, and testis were obtained. There were no statistical differences in organ weights between the experimental groups (data not shown). Immunohistochemistry Human nuclear antigen ( HNA )

HNA immunostaining was performed to confirm engraftment of the administered PRC in Groups 2 and 3. In addition, samples from Group 1 were stained for comparison/control.

In Groups 1 to 3, no obvious positive nuclear staining for HNA was observed, as indicated by the lack of DAPI colocalization ( Figures 48A to 48F ). However, in one sample from Group 2 and two samples from Group 3, cellular-sized spontaneously fluorescent structures were observed within the subretinal mass, which is consistent with the morphology of subretinal PRC transplants measured in mice with retinal degeneration in previous studies. Despite the lack of nuclear HNA staining, we suspect that these transplants in Groups 2 and 3 are of human origin based on their morphology and GFAP expression pattern.

In addition, other spontaneously fluorescent cells and/or debris located within the subretinal mass or embedded in the lateral retina were observed in Groups 1 to 3. These bright spontaneously fluorescent structures, which usually appeared yellow/colored in bright field images ( Fig . 48B , bright field), were suspected to be damaged or dead photoreceptor cells due to their proximity to the outer core/inner segment/outer segment lamina. Given that this morphology was observed in all groups, this damage could be caused by the surgical injection procedure. Glial cell acidic protofibrotic protein ( GFAP )

GFAP staining was performed to assess Müller's jelly degeneration in the retina and provide a secondary marker for PRC. In Groups 1 to 3, Müller's jelly localized to the injection site was brightly labeled with GFAP as a vertical band across the thickness of the retina, suggesting that jelly degeneration may be associated with injury attributable to the injection procedure ( Figs. 49A to 49H ). The extent of Müller's jelly GFAP immunoreactivity was similar between the three injection groups, ranging from approximately 100 µm to several hundred microns along the transverse length of the retina. In untreated animals in Group 4, GFAP was restricted to the retinal nerve fiber layer, consistent with a healthy, untreated retina.

In addition, 3 subretinal DAPI+ graft-like structures in Groups 2 and 3 were also GFAP+ ( Figures 49C , 49E , and 49F ), with thinner curled finger-like omnidirectional staining morphology consistent with PRC after subretinal transplantation. GFAP expression in these grafts did not correlate with retinal Mullerian jelly GFAP immunoreactivity, indicating a different source from Mullerian jelly. In addition, the ONL overlying these grafts had lower GFAP expression, in contrast to examples of GFAP hypertrophy, in which GFAP was enriched in areas of damaged ONL ( Figure 49A ).

In one sample from Group 3, GFAP labeled structures with a pattern consistent with the optic nerve head (ONH), which is typical staining of this structure in the healthy retina.

Overall, the pattern of GFAP immunoreactivity at the injection site in the retina of Groups 1 to 3 was consistent with surgical injury induced by the injection procedure. In addition, in one sample from Group 2 and two samples from Group 3, the pattern of GFAP expression was consistent with suspected PRC expression after subretinal transplantation. Iba1 ( microglia / macrophages )

Anti-Iba1 staining was performed to assess the extent of microglial activation between groups. In the injected groups (Groups 1 to 3), Iba1+ cells were enriched at the injection site, while immunoreactivity was minimal in untreated eyes (Group 4) ( Figures 50A to 50D ). In general, the morphology of Iba1 staining in Groups 1 to 3 was consistent with activated microglia, which had a clump-like or apomorphic morphology, in contrast to dormant microglia, which had thin, branched processes. Overall, Iba1 expression and morphology were similar between Groups 1 to 3 and are suspected to be induced by the surgical procedure. CD45 , Ter119 , and Bestrophin -1

Since subretinal accumulation was observed in Group 1 by OCT and the presence of yellow or weakly stained material/cells was observed in Group 1 sections, immunostaining for CD45, Ter119, and bestrophin-1 was performed in a subset of samples from each group to determine whether the subretinal mass contained hematopoietic cells (including T cells), erythrocytes, or retinal pigment epithelium, respectively.

CD45 was largely negative in all groups. In several samples from Groups 1 to 3, bright spontaneous fluorescent clusters were observed in the subretinal grafts that did not co-label with DAPI. A few cells expressing membrane-bound CD45 signals appropriately localized for hematopoietic cells were occasionally observed in the retina or subretinal grafts.

Apart from occasional subretinal spontaneous fluorescence clusters observed in injected eyes, Ter119 expression was confined to single cells within the retina of Groups 1 to 4, consistent with blood cells localizing within retinal blood vessels.

In each sample, bestrophin-1 was negative in the subretinal graft or host retinal pigment epithelium (data not shown).

In conclusion, the staining for CD45, Ter119, and bestrophin-1 was largely negative or brightly autofluorescent within the subretinal grafts, indicating that the subretinal material in Groups 1 to 3 was largely non-hematopoietic in origin or composed of erythrocytes or retinal pigment epithelium.

The data obtained in this study support the safety of the drug for PRC with a survival rate of <60% (i.e., 50-60%) (compared to PRC with a survival rate of >70%).

without

[ Figure 1A ] Depicts cluster analysis of ESC/PRE/PRC. ESC: Undifferentiated research-grade J1-ESC and Kd-ESC. RPE: J1-RPE at P3+4 months. PRC: J1-PRC (P4), Kd-PRC (P4), GB2R-PRC-2019 (P4), GB2R-PRC-PR1 (P4), GB2R-PRC-CN2 (P4), GB2R-PRC-CN3 (P4).

[ Figure 1B ] depicts qPCR validation of neuronal genes including NANOG, NEUROD2, GAD1, DCX, MAP2, and NFIA.

[ Figure 1C ] depicts single cell analysis of PRCs-P4 (GB2R-PRC-2019 (P4), GB2R-PRC-PR1 (P4), GB2R-PRC-CN2 (P4), and GB2R-PRC-CN3 (P4)). The cells were classified into inhibitory neurons, excitatory neurons, mixed neurons, progenitor cells, and astrocytes.

[ Figure 1D ] depicts qPCR profiling comparing upregulation of markers FOXG1, MAP2, STMN2, DCX, and LINC00461 in GB2R-PRC, GB2R-ESC, RPE, and HMC cells. GB2R-FCP: GB2R-final cell products include GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer, and GB2R-PRC-2019.

[ FIG. 1E ] shows a heat map of the expression of eye movement area progenitor cell markers, rod/cone photoreceptor cell markers, and neuron markers in inhibitory neurons, excitatory neurons, selective neurons, progenitor cells, and astrocytes shown in FIG1C.

[ Figure 1F-1T ] shows the expression of cell markers in the PRC compositions listed in Table 10 on day 2 (D2), day 12 (D12), day 19 (D19), day 37 (D37/P0), day 55 (D55/P1), day 72 (D72/P2), day 90 (D90/P3) and day 107 (D107/P4). PRC markers FOXG1, MAP2, STMN2, and DCX ( Figure 1F ); PRC markers LINC00461, NEUROD2, GAD1, and NFIA ( Figure 1G ); inhibitory neuron markers DLX5, TUBB3, and SCGN ( Figure 1H ); inhibitory neuron markers ERBB4 and CALB2 ( Figure 1I ); excitatory neuron markers NEUROD2, NEUROD6, and SLA ( Figure 1J ); excitatory neuron markers NELL2 and SATB2 ( Figure 1K ); progenitor cell markers VIM, MKI67, CLU, and GLI3 ( Figure 1L ); astrocyte markers GFAP, MIR99AHG, and FBXL7 ( Figure 1M ); selective neuron markers MEIS2, PBX3, GRIA2, and CACNA1C ( Figure 1N ); eye movement zone progenitor cell markers PAX6, LHX2 and SIX3 ( Figure 1O ); eye movement zone progenitor cell markers NES and SOX2 ( Figure 1P ); rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL ( Figure 1Q ); neuron markers TUBB3, NFIA and NFIB ( Figure 1R ); neuron markers OTX2, ELAVL3 and ELAVL4 ( Figure 1S ); and neuron markers SLC1A2, SLC1A3, HCN1 and HES5 ( Figure 1T ).

[ Figure 2A ] Depicts a bright field image of a PRC-P4 cell.

[ Figure 2B ] Depicts a scorecard analysis of PRC differentiation focusing on self-renewal, ectodermal, mesoderm, and endoderm markers.

[ Figure 2C ] Depicts immunocytochemistry (ICC) staining of PRCs-P4, which shows the expression of PAX6/OTX2 in PRC-NPC cells (neural progenitor cells) and the expression of STMN2, CALB2, SCGN, and DCX in PRC-P4 cells.

[ Figure 2D ] Depicts the expression of markers NEUROD2, FOXG1, and HMGA1 in PRC-P4 cells, indicating successful differentiation.

[ Figure 2E ] Depicts flow cytometry of GB2R-CN3-P4 cells to measure purity based on FOXG1 and MAP2 expression. Percentage of cells expressing purity markers: Mean % (GB2R-PRC-CN3 FCP, N = 3). FOXG1: 95.6 (94.2-96.7); MAP2: 88.4 (84.7-91.4); and SSEA4: 0.44 (0.15-0.88).

[ Figure 2F ] Depicts differentially expressed genes between PRC-P3 and PRC-P4 as determined by RNA-seq analysis. These genes have a log10 average expression ratio higher than 3 or lower than -3.

[ Figure 3A ] Depicts the stimulation of PRCs with TBHP (oxidative stress) and the detection of factors secreted by stimulated PRCs using Luminex analysis. PRCs: GB2R-PRC-CN3, GB2R-PRC-CN2, precursor, and GB2R-PRC-2019.

[ Figure 3B ] Depicts the levels of secreted neuroprotective factors detected in the culture medium of PRC cells in the absence of oxidative stress. Stimulation of PRCs with TBHP (oxidative stress) and detection of factors secreted by stimulated PRCs using Luminex analysis. Quantification of CNTF, MIF, and S100B in the culture medium when not stimulated (in the absence of oxidative stress). PRCs: GB2R-PRC-CN3, GB2R-PRC-CN2, precursor, and GB2R-PRC-2019.

[ Fig. 3C ] shows confocal images demonstrating that subretinal transplantation of PRC inhibits microglial cell infiltration into the ONL in RCS rats (P60). Stained retinal sections were imaged on a Leica SP8 using confocal z-stack analysis.

[ Fig. 3D ] shows the stimulation of microglial cell line SIM-A9 by LPS and its inhibition by L-carnitine.

[ Figure 3E ] Depicts phagocytosis assay of GB2R-PRC-PR1 when incubated with pHrodo E. coli bioparticles.

[ Fig. 3F ] Depicts PRC internalization of rod outer segment (ROS) fragments in the retina of RCS (Royal College of Surgeons) rats. RHO+ fragments observed in GFP+ PRC transplants (left) and EM in the right shows ROS shedding in the PRC cytoplasm. GFAP+ PRC co-stained with HuNu (data not shown).

[ Figure 4A ] Schematic diagram depicting the drug administration and in vivo experimental schedule in RCS rats.

[ Figure 4B ] Depicts optomotor response (OMR) and electroretinogram (ERG) analysis of RCS rats injected with 100,000 GB2R-PRC-2019 PRC cells per eye at P25. Statistical analysis was performed using 2-way ANOVA combined with Tukey's multiple comparison test (MCT), where test articles were compared to uninjected eyes and eyes injected with GS2 vehicle. Cell survival rate: 78.4% (artificial).

[ Figure 4C ] shows representative images showing that PRC transplantation significantly attenuated outer nuclear layer (ONL) degeneration over a 3-month period after transplantation. Confocal images showed that PRC transplantation remained highly associated with the ONL. Stained retinal sections were imaged using a Leica MDi8 epi-fluorescence microscope.

[ Fig. 4D ] Quantification of outer nuclear layer (ONL) thickness is depicted, which shows that PRC transplantation is associated with significant preservation at P35, P60, and P120. Cell survival rate: P35: 57.1%, 54%; P60: 57.1% (cell counter); P120: >70% (manual). n=148 ROI/ 7 eyes.

[ Fig. 4E ] Plot depicting the area of GB2R-PRC-2019 (P4) implanted PRC relative to the area of photoreceptor ONL in the P120 RCS rat retinal degeneration model. Statistical analysis was performed using the Pearson correlation coefficient. Cell survival rate: >70% (artificial). n = 4 eyes (12 sections/eye).

[ Fig. 5A ] Depicts immunohistochemical analysis of neurofibromatosis protein (rabbit anti-GFAP; ABCAM) in P120 RCS rats. GFAP is expressed in Müller glia (MG) and optic nerve fiber astrocytes of the host rat retina (white arrows) and in PRCs implanted into the subretinal space. Increased GFAP expression in MG and astrocytes MG hypertrophy and expanded distribution of GFAP expression radially to MG cell bodies are characteristic of disease-related reactive neurofibrosis and can be found distal to the transplant site (yellow arrows, white arrows). Upregulation and increased distribution of GFAP, MG hypertrophy, and outer nuclear layer (ONL) degeneration were reduced or absent at the subretinal PRC transplant site. Cell survival rate: ＞70% (artificial).

[ Figure 5B ] Depicts TUNEL quantification (TUNEL labeling mix, Sigma catalog #11767291910, TUNEL enzyme Sigma catalog #11767305001) performed at P35, during which peak apoptotic activity is observed in the RCS retina. PRCs implanted into the subretinal space were identified based on Ku80+ staining (Abcam, ab80592) of adjacent sections (see below). Very few, if any, TUNEL+ nuclei were observed in the area of PRC implantation, indicating little apoptotic activity at the implantation site. However, non-implanted areas contained significant TUNEL+ nuclei in the ONL, indicating widespread photoreceptor cell death at P35. Quantification of TUNEL+ nuclei in the ONL showed a significant attenuation of apoptotic activity at the transplanted site relative to the non-transplanted area. Retinal sections through the PRC transplants were selected for TUNEL quantification and stained with Ku80 to determine the location of the implanted PRCs and DAPI to identify the ONL. A total of 3 to 4 regions of interest (ROIs) in the central and peripheral areas of each retina were imaged. TUNEL quantification was performed in a blinded manner using only the DAPI channel. ROIs were then identified as "transplanted" or "non-transplanted" based on the presence of subretinal Ku80+ PRCs. Cell survival rate: 3.5%/52.3% (cell counter).

[ Fig. 5C ] Depicts that extracellular vesicles (EVs) isolated from PRCs of RCS rats maintain OMR responses until P90. EVs were isolated from PRC-conditioned medium and injected subretinaally into RCS rats at a dose of 9.42*10 ⁸ EVs per eye. Other RCS rats received subretinal injections of PRCs (100,000 cells/eye) or vehicle. Injections were performed at age P25. OMR analysis was performed at P60, 90, 120 and compared with non-injected (NI) animals of the same age.

[ Fig. 5D ] Depicts that a single subretinal injection of PRC-EV preserves ONL thickness until P90. Quantification of retinal ONL thickness in PRC-EV-injected RCS rats relative to uninjected eyes. Retinal cryosections were stained with DAPI to visualize the ONL. Stained retinal sections were imaged using a Leica DMi8 epifluorescence microscope. ONL thickness in central and peripheral regions was determined by line drawing measurements through the ONL as described in Fig. 4C . P90: ONL preservation correlates with OMR preservation. ONL thickness in Long-evans rats (normal vision): approximately 55 µm (Weber et al., 1996). The P-suffix indicates postnatal age.

[ Figure 5E ] Depicts a single PRC-EV injection. ONL thickness was maintained until P90.

[ Fig. 5F ] Depicts the ONL after non-PRC-EV injection. The small patches of ONL that remained had an average of 1 to 3 rows of nuclei.

[ Figure 5G ] depicts the ONL after PRC-EV injection. Compared to the uninjected ONL, the longer ONL fragments that remained had an average of 3 to 6 rows of nuclei.

[ Figure 5H ] depicts the ONL at P120 after PRC-EV injection. At P120, a single PRC-EV injection failed to maintain ONL thickness.

[ Figure 5I ] Depicts the optomotor response after PRC-EV injection. Visual function was assessed by recording the optomotor response of PRC-EV injected rats at P60, P90, and P120.

[ Figure 5J ] Analysis of ONL in PRC-EV-treated rats showed that a single injection of PRC-EVs conferred ONL morphology that was maintained until P90, but not until P120.

[ Figure 5K ] Depicts the ONL after subretinal transplantation of PRC cells. Subretinal transplantation of PRC cells maintained ONL thickness until P120.

[ Figure 6A ] Toluidine blue staining of PRC implanted and non-implanted areas. ONL preservation was clearly observed, while the non-implanted area showed severe ONL degeneration. ONL was visualized by thick black bands (long arrows). Note: Lack of ONL preservation on the non-injected side (short arrows).

[ Figure 6B ] Transmission electron microscopy (TEM) analysis of the implanted and non-implanted ONL sites. The ONL in the implanted area was significantly preserved compared to the ONL site where PRC was not transplanted.

[ Fig. 6C ] shows TEM ultrastructural analysis. TEM shows that the overall morphology of photoreceptor cells and finer structures such as connecting ciliary organs of the inner segment of the retinal rod are maintained in the presence of subretinal PRCs. Finally, PRC transplantation minimizes the debris area in the subretinal space. In the absence of subretinal PRCs, the outer segment of the retinal rod fragments fill most of the subretinal space because the outer segment is not cleared after shedding. GB2R-PRC-2019 was injected at P25. The black arrows point to the maintained connecting ciliary organs of the inner and outer segments. Cell survival rate: 57.1% (cell counter).

[ Figure 7A ] Schematic diagram depicting administration of PRC using the rd10 mouse model. On postnatal day 14 (P14), 100k cells were transplanted into the subretinal space of the right eye, and GS2 vehicle was injected into the left eye as a control. OMR was performed at P21, P28, P35, P42, and P49. Cell survival rate: 68.5% (artificial).

[ FIG. 7B ] depicts a graph showing that at P21 and P28, both eyes showed the presence of tracking ability. At P35, P42, and P49, the control eye was blind, while the cell-injected eye still showed tracking ability.

[ FIG. 7C ] Depicted is a graph showing morphometric analysis of photoreceptor outer nuclear layer (ONL) preservation in P28rd10 mice. ONL preservation quantified by ONL area ( ^mm2 ) at PRC subretinal transplant sites was identified compared to adjacent central and peripheral retina without transplantation. Cell survival rate: 73.3% (artificial).

[ FIG. 7D ] Depicted is a graph showing analysis of cone length in P28 rd10 mice. The length of cone arrestin-labeled cone photoreceptor cells at the PRC subretinal transplant site was significantly increased compared to adjacent areas of the central and peripheral retina without subretinal transplantation. Cell survival rate: 73.3% (artificial).

[ FIG. 7E ] Depicts confocal imaging showing CtBP2/RIBEYE-labeled rod synapses at the transplant site and in central and peripheral retinal locations without transplantation.

[ Fig. 7F ] Depicted is a graphical representation of quantification of rod synapses in P28 rd10 mice. The number of ribeye-positive/cone-arrestin-negative rod synapses at the PRC-transplanted sites was significantly increased compared to non-transplanted central and peripheral retina, indicating that rod photoreceptor cells in the rd10 mouse retina exhibit PRC-associated preservation. Cell survival rate: 73.3% (artificial).

[ Figure 8A ] shows OMR at P35, and the survival rate of multiple batches of PRC was less than 70%. The survival rates of 66.05%, 68.5% and 68.8% PRC showed similar maintenance effects. PRC-injected eyes showed better OMR efficacy than control eyes.

[ FIG. 8B ] shows that PRC cells were anatomically preserved. Multiple batches of PRCs with viability less than 70% were observed to be anatomically preserved. Histological analysis confirmed the OMR data. At P35, injected PRCs with 66.05% viability preserved cone anatomy as assessed by ONL thickness, axon length, and morphology. At P50, injected PRCs with 68.5% viability preserved ONL thickness and density of ribeye+ synapses. At P70, injected PRCs with 68.8% viability also preserved ONL thickness and density of ribeye+ synapses.

[ Figure 9A ] shows ONL preservation in RD10 mice. On postnatal day 14 (P14), 100k cells were transplanted into the subretinal space of the right eye, and GS2 vehicle was injected into the left eye as a control. At P50, eyes were collected for IHC staining. DAPI staining showed only one row of photoreceptor cells in the non-transplanted retina, while multiple rows of photoreceptor cells were observed in the transplanted retina. Quantification of data from 5 animals, 3 sections per animal, showed that the ONL thickness and area in the transplanted retina were significantly higher than the ONL thickness and area of the non-transplanted retina (6 μm vs. 18 μm; 5000 ^μm2 vs. 10000 ^μm2 ). Cell survival rate: 68.5% (artificial).

[ Figure 9B ] Depicted (left) ICC staining images of cone arrestin (Millipore AB15282 1:500), which is used to detect cone morphology. In non-transplanted retina, arrestin is only expressed in the cell body, outer segment and axon degeneration of cones. In transplanted retina, cones are preserved by maintaining normal morphology, and arrestin is expressed in the outer segment, cell body, axon and peduncle. Quantitative data show that the axons of cones in transplanted retina are significantly longer than those in non-transplanted retina (8 μm vs. 4 μm). (Right) Ribeye protein (also known as ct-BP2, BD transduction assay 612044, 1:500) is a marker for presynaptic structures located at the axon terminals of photoreceptor cells. It shows the synaptic connection between photoreceptor cells and horizontal cells. Quantitative data showed that the expression of ribeye protein spots in transplanted retina was higher than that in non-transplanted retina (40 vs. 20/100 μm retina). Cell survival rate: 68.5% (artificial).

[ FIG. 9C ] Schematic diagram depicting administration of PRC cells to hemizygous rats. Overview of dosing and in vivo experimental schedule in P23H rats.

[ Fig. 9D ] Depicts the preservation of OMR in P23H hemizygous rats injected with 100,000 GB2R-PRC-P4-FDA PRC cells per eye at P25, revealing statistically significant preservation of OMR response compared to uninjected controls at P60, P90, and P120 and compared to vehicle-injected controls at P90. Statistical analysis was performed using two-way ANOVA coupled with Tukey's multiple comparison test (MCT). Cell survival rate: 71.7% (manual).

[ FIG. 10A ] Schematic diagram depicting the method for quantification of HuNu+ cells. RCS rats aged P23 to P25 were given subretinal injections of 100,000 GB2R PRCs or GS2 vehicle. Animals were sacrificed at P120 to obtain retinal tissue cryosectioning and histological analysis. Every 10th retinal section through the graft was selected for quantification, in which HuNu+ PRCs were quantified. Cell numbers were interpolated between sections and the total was calculated for all grafts. Total PRC grafts were calculated starting from n = 4.

[ Figure 10B ] depicts quantification of PRC engraftment in 4 transplanted animals at P120 or 3 months post-transplantation. Quantification of total HuNu+ PRCs transplanted subretinally showed an average of 80,000 cells engrafted per eye, which translates to an overall 80% engraftment rate compared to an initial injection of 100,000 cells per injection.

[ FIG. 10C ] Depicts differences in PRC engraftment in retinal degeneration models of varying severity at disease onset and after treatment. The specific batches and doses of PRC used in this analysis are shown in the boxes at the top of the figure. Bar graph of relative subretinal engraftment: RCS rat and P23H hemizygous rat retinal degeneration models demonstrated similar engraftment of GB2R-PRCs, with maximum engraftments found to cover 54 and 41% of the subretinal space, respectively. rd10 mouse and P23H homozygous rat retinal degeneration models showed significantly lower engraftment: 19% (rd10 mouse) and 2% (P23H homozygous rat) of the subretinal space showing maximum length of PRC engraftment. Relative retinal length graft coverage plots: Relative ratio calculations provide relative coverage of different sizes of mouse and rat eyes, while the maximum length plots show absolute graft lengths. rd10 mice and P23H rats show reduced grafts both in relative and absolute terms compared to RCS and P23H hemizygous rats.

[ FIG. 11A ] Depicts a graph showing that proliferation of subretinal PRCs is downregulated by P120. Quantification of subretinal transplanted HuNu (Millipore, MAB1281) and Ki67 (Abcam ab15580) double positive cells was performed in retinal cross sections from P35, P60 (not shown), and P120 animals. A total of three animals were used for each time point. Confocal images show Ki67 and HuNu double positive cells in the subretinal space at P35. However, no proliferative (Ki67+) PRCs were observed by P120. Quantification revealed that Ki67 and HuNu double positive PRCs were significantly downregulated by P120 or 3 months after transplantation (bar graph in lower left figure). Cell viability: P35: 43.5% and 52.3%; P60: 43.5% and 52.3% (cell counter); and P120: >70% (manual).

[ Figure 11B ] shows confocal images of implanted PRCs. Lack of Oct4 (Abcam ab27985) expression in implanted HuNu+ PRCs showed a lack of rich potential at all time points studied after transplantation (P35, P60, and P120). A total of three animals were used for each time point. Cultured GMP1 iPSCs were used as a positive control for Oct4 expression, where subcellular compartment localization was stained using WGA-647 (Thermo Scientific W32466) and DAPI (small images in the lower middle panel).

[ Figure 12A ] Schematic diagram depicting the PRC cell manufacturing protocol. P4-PRC(DS) exists at approximately 200M cells, and P4-PRC(DS) can currently be expanded to 500-600M cells. Prioritized protocol changes: i) replacement of culture dish (to T-flask), and ii) replacement of 2D to 3D lift method, from mechanical to enzymatic dissociation (expected to be implemented in GLP Tox studies and first-in-human studies). RIM: rescue induction medium; NDM: neural differentiation medium; ICC: immunocytochemistry; IFA: immunofluorescence assay.

[ Figure 12B ] Schematic diagram of the PRC cell manufacturing protocol, including the cryopreservation step between P3 and P4. P4-PRC(DS) exists at approximately 200M cells and can currently be expanded to 500-600M cells based on P4-PRC(DS). During method development, method changes implemented included: i) replacement of culture dishes (to T-flasks); ii) replacement of 2D to 3D promotion methods, from mechanical dissociation to enzymatic dissociation (expected to be implemented in GLP Tox studies and first-in-human studies), and iii) construction of P3-spheroid stock intermediates. RIM: rescue induction medium; NDM: neural differentiation medium; ICC: immunocytochemistry; IFA: immunofluorescence analysis.

[ Figure 12C ] Schematic diagram of the PRC manufacturing method.

[ Figure 13 ] Schematic diagram depicting the production, recovery, and injection of PRC cells. CZ: Crystal Zenith; CRF: controlled rate freezer.

[ FIG. 14A ] Depicts the post-thaw cell viability of photorescive rescue cells (PRCs) prepared in formulations containing 2.5% rHA, DPBS with Ca and Mg, 0.6% glucose, and different concentrations of DMSO (5% DMSO or 10% DMSO) and PRC cells formulated in a commercial CryoStor® CS10 cell freezing formulation containing 10% DMSO for comparison.

[ FIG. 14B ] Depicts the survival rate of P4 PRC cells after cryopreservation under different conditions. The graph shows the mean ± SD.

[ FIG. 14C ] Plots the percentage of survival of P4 PRC cells after cryopreservation under different conditions selected from those in FIG . 14B . Graphs represent mean ± SD.

[ FIG. 15A ] shows a comparison of OMR responses in animals injected subretinally with 100,000 or 200,000 GB2R PRCs. Twelve spatial frequencies (SPs) were performed and OMR responses (tracking intensity) were recorded in animals injected subretinally with 100K (left panel) or 200K (middle panel) PRCs. The 1.2 response on the Y-axis is the threshold for the presence of OMR (shown with a green dotted line). Any value below 1.2 means that the eye cannot be tracked. At P35, P42, and P50, control eyes were blind, and eyes injected with 200k PRC cells consistently showed larger and broader amplitude curves than the 100k group. The rightward shift in the SP threshold indicates better visual acuity in the 200k eyes. Survival rate: 80.6%/76.64% (manual, 2 vials).

[ FIG. 15B ] Schematic timeline depicting injection and analysis of rd10 mice.

[ FIG. 15C ] ICC analysis was used to show that the ONL thickness of the 200k eyes was greater than that of the 100k, as confirmed by ICC analysis. Quantification of ONL thickness at the transplanted site relative to the non-transplanted site in animals that received 100K and 200K cells. The data are consistent with the hypothesis that higher doses result in greater efficacy. On the P70: OMR curve, the ONL thickness of the 200k group relative to the 100k group consistently exhibited a greater or wider amplitude, and the ONL of the 200k group relative to the 100k group remained larger at P70. The data are consistent with the hypothesis that greater retinal coverage (higher dose) results in greater efficacy.

[ Fig. 16A ] shows OMR recordings of rd10 mice injected with cryopreserved 300k PRC formulation (Cryo-PRC) (same as previously described rd10 mice injected subretinally at P14). Cryo-PRC was functionally effective at P35 and P42. However, at P49, both vehicle and Cryo-PRC eyes lacked OMR. Cryopreserved formulations containing PRC or vehicle. Cell survival rate: 69.56% (artificial). N=8.

[ FIG. 16B ] shows OMR analysis of RCS rats injected with 100,000 cells per eye of GB2R-PRC (CN2 batch), GB2R-PRC (P3 INT DS), or GB2R-PRC (Cryo: cryopreserved formulation) at P25. Statistical analysis was performed using two-way ANOVA combined with Tukey's multiple comparison test (MCT), where the test article was compared to uninjected eyes and eyes injected with GS2 vehicle.

[ FIG. 16C ] shows ERG analysis of RCS rats injected with 100,000 cells per eye of GB2R-PRC (CN2 batch), GB2R-PRC (P3 INT DS), or GB2R-PRC (Cryo: cryopreserved formulation) at P25. Statistical analysis was performed using two-way ANOVA combined with Tukey's multiple comparison test (MCT), where the test article was compared to uninjected eyes and eyes injected with GS2 vehicle.

[ Figures 17A and 17B ] show OCT of eyes treated with GS2 buffer and supplemented with IMT. Figure 17A includes conditions: GS2+/hypothermic buffer (1:4) without IMT (left column) and GS2+/hypothermic buffer (1:4) with Dex (right column). Figure 17B shows GS2+/hypothermic buffer (1:4) with Dex/CsA (left column) and BSS without IMT (right column).

[ Figures 18A-18C ] show the ERG responses of Group 1 (GS2+/hypothermic buffer (1:4) without IMT); Group 2 (GS2+/hypothermic buffer (1:4) with Dex); Group 3 (GS2+/hypothermic buffer (1:4) with Dex/CsA); and Group 4 (BSS without IMT). Figure 18A shows the a-wave response. Figure 18B shows the twilight b-wave response. Figure 18C shows the photopic b-wave response.

[ Figure 19 ] shows the retinal morphology of Group 1 (GS2+/cold buffer (1:4) without IMT); Group 2 (GS2+/cold buffer (1:4) with Dex); Group 3 (GS2+/cold buffer (1:4) with Dex/CsA); and Group 4 (BSS without IMT).

[ Figure 20 ] shows the OCT images of Group 1 (GS2+/hypothermic buffer (1:4) without IMT); Group 2 (GS2+/hypothermic buffer (1:4) with Dex); Group 3 (GS2+/hypothermic buffer (1:4) with Dex/CsA); and Group 4 (BSS without IMT).

[ Figure 21 ] shows the ONL thickness of Group 1 (GS2+/hypothermia buffer (1:4) without IMT); Group 2 (GS2+/hypothermia buffer (1:4) with Dex); Group 3 (GS2+/hypothermia buffer (1:4) with Dex/CsA); and Group 4 (BSS without IMT).

[ Figure 22 ] shows the intraretinal migration after injection of PRCs that were frozen at the middle step of P3 and further differentiated to P4 after thawing.

[ Figure 23 ] shows the OMR readings after injection at time points P35, P42, and P49, comparing P4(d) with P4(i).

[ Figure 24 ] shows immunohistochemistry of the inner plexiform layer (IPL) demonstrating intraretinal migration between P4(d) and P4(i) injections.

[ Figure 25 ] shows the immunohistochemistry of the IPL, which shows that the migration into the IPL was enhanced after P4(i) PRC injection.

[ Figure 26 ] shows the OMR readings for Group 1 given high doses of P4(d) or P4(i).

[ Figure 27 ] shows the OMR readings for Group 1 given high doses of P4(d) or P4(i).

[ Figure 28 ] shows the OMR readings of eyes injected with PRCs prepared using either CellSTACK® or PDL pre-coated flasks compared to CMC controls (prepared using the culture dishes shown in Figures 12A-12B ).

[ Figure 29 ] shows OCT images of the retina at D7 and D38 (days after injection) comparing PRCs prepared using CellSTACK® and PDL pre-coated flasks.

[ Figures 30A-30B ] Immunohistochemical staining of CAR/HuNu ( Figures 30A and 30B ) is shown to compare PRC prepared using CellSTACK® or PDL pre-coated flasks (vs. CMC control). Both Figures 30A and 30B show that PRCs prepared using CellSTACK® have larger grafts and better cone retention.

[ Figure 31 ] shows immunohistochemical staining for GFAP and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks (vs. CMC control).

[ Figure 32 ] shows immunohistochemical staining for IBA1 and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks (vs. CMC control).

[ Figure 33 ] shows immunohistochemical staining for Ki67 and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks (vs. CMC control).

[ Figure 34 ] shows immunohistochemical staining for OCT4 and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks (vs. CMC controls).

[ Figure 35 ] Schematic diagram showing the experimental design of delayed PRC injection into RCS rats. Injections were performed at P25, P45, or P60 after disease onset. OMR and ERG readings were obtained from P60 to P150.

[ Figures 36A and 36B ] show the OMR ( Figure 36A ) and ERG ( Figure 36B ) readings at P60, P90, P120, and P150 after each injection time point.

[ Figure 37 ] shows the immunohistochemical staining of STEM21 in RCS rats injected with PRC at P45.

[ Figures 38A and 38B ] show quantification of subretinal PRC transplantation for injection time points of P25, P45, and P60. Figure 38A shows the maximum transplant length and Figure 38B shows the maximum transplant length/retinal length ratio.

[ Figures 39A and 39B ] show quantification of ONL preservation for injection time points P25, P45, and P60. Figure 39A shows the maximum preserved ONL length and Figure 39B shows the maximum ONL length/retinal length ratio. Gb2R-GMP-MCB PRC-P4 RCS injected at P25 (P150); Gb2R-GMP-MCB PRC-P4 RCS injected at P45 (P150); Gb2R-GMP-MCB PRC-P4 RCS injected at P60 (P150).

[ Fig. 40A-40D ] PRC-injected eyes maintained optomotor responses. OMR response analysis was performed at postnatal ages P28 ( Fig. 40A ), P35 ( Fig. 40B ), P42 ( Fig. 40C ), and P49 ( Fig. 40D ). Statistical significance relative to control eyes (vehicle or uninjected) was observed in Group 1 OD, while only a tendency relative to control eyes was observed in Group 2 OD. No statistical differences were observed between cell-injected groups. *p<0.05, **p<0.01.

[ Figures 41A-41F ] show retinal OCT images: D0 Group 1 (OD) ≥70% ( Figure 41A ); D0 Group 2 (OD) ≤60% ( Figure 41B ); D9 Group 1 (OD) ≥70% ( Figure 41C ); D9 Group 2 (OD) ≤60% ( Figure 41D ); D35 Group 1 (OD) ≥70% ( Figure 41E ); and D35 Group 2 (OD) ≤60% ( Figure 41F ).

[ Figures 42A and 42B ] Figure 42A shows that positive HNA staining was observed in the cell-injected eyes of Group 1 and Group 2. Red = HNA, Blue = DAPI. Scale bar is 50 µm. Figure 42B shows that Group 1 (OD) > 70% and Group 2 (OD) < 60%.

[ Figs. 43A-43F ] Examples of subretinal PRC transplantation associated with elongated cone morphology found in treated eyes of Groups 1 and 2. Example images of cone morphology in eyes injected with ≥70% viable PRC (Fig. 43A ), ≤60% viable PRC (Fig. 43B ), or vehicle (Fig. 43C ). Green = CAR, Red = HNA. Scale bar, 50 µm. Fig. 43D shows quantification of cone length. One-way ANOVA (p < 0.05). Error bars are SEM. See Fig. 43E (Group 1) and Fig. 43F (Group 2) for CAR staining in all eyes.

[ Figure 44 ] shows that subretinal PRC transplantation is associated with ONL thickening at the transplantation site. One-way analysis of variance (p=0.21). Error bars are SEM.

[ Figure 45 ] Substantial Müller's jelly GFAP immunoreactivity was present in all study eyes. Sample GFAP staining in study group eyes indicates dense vertical bands characteristic of the GFAP morphology of diseased retina. Green = GFAP, Red = HNA. Scale bar 50 µm.

[ Figures 46A-46J ] show OCT images at D28, revealing surgery-related damage at the injection site. Compared with the untreated Group 4 eyes ( Figure 46J ), OCT images showed surgical damage and accumulation of subretinal material in the injected eyes of Group 1 ( Figures 46A , 46B , and 46C ), Group 2 ( Figures 46D , 46E , and 46F ), and Group 3 ( Figures 46G , 46H , and 46I ). The presence of subretinal material in Group 1 eyes suggests that the subretinal mass observed by OCT in the cell-injected eyes (Groups 2 and 3) may contain considerable host cells. In both eyes of individual female mice from Group 1, individual medium-sized areas of retinal atrophy were observed at the center of the injection (dashed red lines).

[ Figures 47A-47C ] show ERG recordings (D29-32), which revealed no statistically significant differences between the study groups. Figure 47A shows a-wave amplitude measurements obtained by two-way ANOVA; Figure 47B shows b-wave amplitude measurements obtained by two-way ANOVA; and Figure 47C shows photopic b-wave amplitude measurements obtained by one-way ANOVA.

[ Figures 48A-48F ] show HNA immunostaining at the injection center and the retinal layered structure. Nuclear HNA staining (HNA channel alone, HNA and DAPI overlap, bright field) was not detected in Group 1 ( Figures 48A and 48B ), Group 2 ( Figures 48C and 48D ), and Group 3 ( Figures 48E and 48F ). However, subretinal graft-like structures (*) containing DAPI ⁺ cells and autofluorescent debris were found in Groups 2 and 3 ( Figures 48C , 48E , and 48F ). In Groups 1 to 3, additional autofluorescent debris (#) was observed under the retina or embedded in the outer retina ( Figures 48B and 48D ). Retinal laminae in all groups showed focal disruption at the center of injection (overlay of HNA and DAPI in Figures 48A-48F ), consistent with surgical injury. In many cases, structures that spontaneously fluoresce most brightly appear yellow/stained on bright field images (example in Figure 48B ). Red = HNA, blue = DAPI. Scale bar 100 µm.

[ Figures 49A-49H ] Showing enriched Müller jelly GFAP immunoreactivity at the injection site in injected eyes, consistent with injection-related injury. (AH) GFAP immunostaining was used to assess Müller jelly reactivity in Groups 1 ( Figures 49A and 49B ), 2 ( Figures 49C and 49D ), 3 ( Figures 49E and 49F ), and 4 ( Figures 49G and 49H ) (GFAP channel alone; GFAP and DAPI overlay). Figures 49C , 49E , and 49F GFAP also labels subretinal cell clusters that are tangled and thin, in contrast to the Müller jelly hypertrophy in Figure 49A . Green = GFAP, Blue = DAPI. Scale bar 100 µm.

[ Figures 50A-50D ] show increased Iba1 immunoreactivity at the injection site in eyes from Group 1 (Figure 50A ) , Group 2 ( Figure 50B ), and Group 3 ( Figure 50C ) compared to untreated eyes from Group 4 (Figure 50D), consistent with inflammation following injection-related injury. #Spontaneous fluorescent debris. Green = Iba1, Blue = DAPI. Scale bar 100 µm.

Claims (304)

A photoreceptor rescue cell composition comprises a plurality of heterogeneous photoreceptor rescue cells, wherein the plurality of heterogeneous photoreceptor rescue cells cumulatively express at least two markers selected from the group consisting of: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA. The photosensitive rescue cell composition of claim 1 further comprises a culture medium suitable for maintaining the viability of the cells. The photosensitive rescue cell composition of claim 1 or 2, wherein the cells are generated by in vitro differentiation of high-potential cells. The photosensitive rescue cell composition of claim 3, wherein the high-potential cells are embryonic stem cells (ESCs) or induced high-potential stem cells (iPSCs). The photosensitive rescue cell composition of any of the preceding claims, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the composition are photosensitive rescue cells. The photosensitive rescue cell composition of any of the preceding claims, wherein the plurality of heterogeneous cells cumulatively express FOXG1 and MAP2. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 50% to about 100%, about 50% to about 90%, about 55% to about 85%, or about 55% to about 73% of the cells in the composition express FOXG1; and/or (ii) at least about 50%, 55%, 57%, 60%, 65%, 70%, 71%, 75%, 78%, 79%, 80%, 81%, 85%, 87%, 90%, 92%, 95%, 97% or 100% of the cells in the composition express FOXG1, and/or (iii) the cells in the composition express about 55 transcripts per million (TPM) to about 200 TPM, about 60 TPM to about 170 TPM, about 140 TPM to about 165 TPM, or about 149 TPM to about 170 TPM. and/or (iv) the cells in the composition express at least 55 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM or 200 TPM of the FOXG1 transcript. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 75% to about 100%, about 75% to about 98%, about 75% to about 95%, about 77% to about 93% of the cells in the composition express MAP2; and/or (ii) at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the composition express MAP2; and/or (iii) the cells in the composition express about 250 transcripts per million (TPM) to about 700 TPM, about 290 TPM to about 650 TPM, about 450 TPM to about 625 TPM, or about 490 TPM to about 615 TPM of MAP2 transcripts; and/or (iv) cells in the composition express at least 250 TPM, 300 TPM, 350 TPM, 400 TPM, 450 TPM, 475 TPM, 490 TPM, 510 TPM, 525 TPM, 550 TPM, 575 TPM, 600 TPM, 610 TPM, 625 TPM, 650 TPM, 675 TPM or 700 TPM of the MAP2 transcript. The photosensitive rescue cell composition of claim 6, wherein the plurality of heteroplasmic cells cumulatively express at least one other marker selected from the group consisting of STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA. The photosensitive rescue cell composition of any of the preceding claims, wherein the plurality of heterogeneous cells cumulatively express at least 3, 4, 5, 6 or 7 of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA. A photorescrutable cell composition as claimed in any of the preceding claims, wherein (i) about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, or about 75% to about 80% of the cells in the composition express STMN2; and/or (ii) at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 100% of the cells in the composition express STMN2; and/or (iii) the cells in the composition express about 150 transcripts per million (TPM) to about 600 TPM, about 190 TPM to about 560 TPM, about 400 TPM to about 600 TPM, or about 450 TPM to about 560 TPM of STMN2 transcripts; and/or (iv) the cells in the composition express at least 150 TPM, 185 TPM, 200 TPM, 250 TPM, 300 TPM, 350 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 525 TPM, 550 TPM, 575 TPM TPM or 600 TPM of STMN2 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 65% to about 95%, about 65% to about 85%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 75% to about 95%, about 75% to about 90%, or about 75% to about 89% of the cells in the composition express DCX; and/or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95% of the cells in the composition express DCX; and/or (iii) the cells in the composition express about 200 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 900 TPM, about 600 TPM to about 900 TPM, or about 750 TPM to about 900 TPM. TPM to about 850 TPM of the DCX transcript; and/or (iv) the cells in the composition express at least 200 TPM, 250 TPM, 350 TPM, 400 TPM, 450 TPM, 500 TPM, 550 TPM, 600 TPM, 650 TPM, 700 TPM, 750 TPM, 800 TPM, 850 TPM or 900 TPM of the DCX transcript. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 65% to about 98%, about 65% to about 95%, about 70% to about 98%, about 70% to about 95%, about 70% to about 90%, about 75% to about 98%, about 75% to about 90%, or about 80% to about 95% of the cells in the composition express LINC00461; and/or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or 98% of the cells in the composition express LINC00461; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 100 TPM, about 50 TPM to about 95 TPM, about 85 TPM to about 95 TPM, about 10 ... TPM, or about 87 TPM to about 93 TPM of the LINC00461 transcript; and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 87 TPM, 89 TPM, 90 TPM, 92 TPM, 95 TPM or 100 TPM of the LINC00461 transcript. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 1% to about 8%, about 1% to about 7%, about 1% to about 5%, or about 2% to about 4% of the cells in the composition express NEUROD2; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% or 25% of the cells in the composition express NEUROD2; and/or (iii) the cells in the composition express about 0 transcripts per million (TPM) to about 10 TPM, about 0.01 TPM to about 9 TPM, about 0.2 TPM to about 2 TPM, or about 0.4 TPM to about 1.2 TPM of the NEUROD2 transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.4 TPM, 0.6 TPM, 0.8 TPM, 1.0 TPM, 1.2 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of the NEUROD2 transcript. A photorescrutable cell composition as claimed in any of the preceding claims, wherein (i) about 35% to about 70%, about 35% to about 68%, about 35% to about 67%, about 35% to about 66%, about 35% to about 65%, about 40% to about 70%, about 40% to about 68%, about 40% to about 67%, about 40% to about 66%, about 40% to about 65%, about 42% to about 70%, about 42% to about 68%, about 42% to about 67%, about 42% to about 66%, or about 42% to about 65% of the cells in the composition express GAD1; and/or (ii) at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69% or 70% of the cells in the composition express GAD1; and/or (iii) the cells in the composition express about 10 transcripts per million (TPM) to about 50 TPM, about 12 TPM to about 45 TPM, about 12 TPM to about 40 TPM, or about 25 TPM to about 42 TPM of the GAD1 transcript; and/or (iv) the cells in the composition express at least 10 TPM, 12 TPM, 16 TPM, 18 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 47 TPM, or 50 TPM of the GAD1 transcript. A photorescive cell composition as claimed in any of the preceding claims, wherein (i) about 60% to about 95%, about 60% to about 90%, about 60% to about 89%, about 60% to about 88%, about 60% to about 87%, about 60% to about 86%, about 65% to about 95%, about 65% to about 90%, about 65% to about 89%, about 65% to about 88%, about 65% to about 87%, about 65% to about 86%, about 69% to about 90%, about 69% to about 89%, about 69% to about 88%, about 69% to about 87%, or about 69% to about 86% of the cells in the composition express NFIA; and/or (ii) at least about 50%, 55%, 60%, 65%, 67%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 92% or 95% of the cells in the composition express NFIA; and/or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 120 TPM, about 33 TPM to about 117 TPM, about 60 TPM to about 85 TPM, or about 65 TPM to about 80 TPM of a NFIA transcript; and/or (iv) the cells in the composition express at least 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM or 120 TPM of a NFIA transcript. The photorescible rescue cell composition of any of the preceding claims, wherein the plurality of heterogeneous cells cumulatively express each of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA. The photosensitive rescue cell composition of any of the preceding claims, wherein the heteroplasmic cells each individually express at least one of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 or NFIA. The photosensitive rescue cell composition of any one of claims 1 to 18, wherein the plurality of heterogeneous photosensitive rescue cells comprises one or more cell types selected from the group consisting of inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and alternative neurons. The photosensitive rescue cell composition of claim 19, wherein the plurality of heterogeneous photosensitive rescue cells include each of inhibitory neurons, excitatory neurons, progenitor cells, astrocytes and selective neurons. The photosensitive rescue cell composition of claim 19 or claim 20, wherein the plurality of heterogeneous photosensitive rescue cells comprise inhibitory neurons expressing one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4 and CALB2. The photosensitive rescue cell composition of any one of claims 19 to 21, wherein the plurality of heterogeneous photosensitive rescue cells comprise a plurality of inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4 and CALB2. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 52% to about 69%, about 52% to about 75%, about 54% to about 69%, about 54% to about 68%, or about 54% to about 66% of the cells in the composition express DLX5; and/or (ii) at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 72%, 74%, 76%, 78% or 80% of the cells in the composition express DLX5; and/or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 150 TPM, about 50 TPM to about 140 TPM, about 80 TPM to about 138 TPM, or about 130 TPM to about 140 TPM of the DLX5 transcript; and/or (iv) the cells in the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 135 TPM, 140 TPM, or 150 TPM of the DLX5 transcript. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 60% to about 95%, about 70% to about 95%, about 72% to about 95%, about 75% to about 95%, about 76% to about 94%, about 77% to about 93%, about 78% to about 93%, about 70% to about 90%, about 72% to about 89%, about 73% to about 88%, about 74% to about 87%, about 75% to about 87%, about 76% to about 86%, about 77% to about 86%, or about 79% to about 86% of the cells in the composition express TUBB3; and/or (ii) at least about 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, or 95% of the cells in the composition express TUBB3; and/or (iii) the cells in the composition express about 150 transcripts per million (TPM) to about 500 TPM, about 160 TPM to about 450 TPM, about 300 TPM to about 450 TPM, about 350 TPM to about 430 TPM, or about 375 TPM to about 430 TPM of TUBB3 transcripts; and/or (iv) the cells in the composition express at least 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 350 TPM, 360 TPM, 370 TPM, 380 TPM, 390 TPM, 400 TPM, 410 TPM, 420 TPM, 430 TPM, 440 TPM, 460 TPM, 480 TPM, 490 TPM, 500 TPM, 510 TPM, 520 TPM, 530 TPM, 540 TPM, 550 TPM, 560 TPM, 570 TPM, 580 TPM, 590 TPM, 600 TPM, 610 TPM, 620 TPM, 630 TPM, 640 TPM, 650 TPM, 660 TPM, 670 TPM, 680 TPM, 690 TPM, 700 TPM TUBB3 transcripts of 100 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 430 TPM, 450 TPM, 475 TPM or 500 TPM. A photorescrutable cell composition as claimed in any of the preceding claims, wherein (i) about 45% to about 70%, about 45% to about 65%, about 50% to about 70%, about 50% to about 65%, about 50% to about 64%, about 50% to about 63%, about 50% to about 62%, about 50% to about 61%, about 46% to about 52%, about 47% to about 51%, about 48% to about 51%, or about 48% to about 50% of the cells in the composition express SCGN; and/or (ii) at least about 45%, 47%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 65%, 67% or 70% of the cells in the composition express SCGN; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 200 TPM, about 70 TPM to about 180 TPM, about 75 TPM to about 175 TPM, or about 120 TPM to about 175 TPM of the SCGN transcript; and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 171 TPM, 173 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, or 200 TPM of the SCGN transcript. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 60% to about 85%, about 60% to about 80%, about 60% to about 79%, about 60% to about 78%, about 60% to about 77%, about 60% to about 76%, about 63% to about 75%, about 63% to about 70%, about 63% to about 79%, about 63% to about 78%, about 63% to about 77%, about 63% to about 75%, about 63% to about 73%, about 63% to about 72%, about 63% to about 71%, about 65% to about 72%, or about 66% to about 71% of the cells in the composition express ERBB4; and/or (ii) at least about 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 75%, 80%, or 85% of the cells in the composition express ERBB4; and/or (iii) the cells in the composition express about 15 transcripts per million (TPM) to about 120 TPM, about 17 TPM to about 100 TPM, about 18 TPM to about 95 TPM, about 70 TPM to about 95 TPM, or about 73 TPM to about 94 TPM of ERBB4 transcript; and/or (iv) the cells in the composition express at least 15 TPM, 30 TPM, 50 TPM, 70 TPM, 73 TPM, 75 TPM, 80 TPM, 82 TPM, 85 TPM, 88 TPM, 90 TPM, 91 TPM, ERBB4 transcripts of TPM, 93 TPM, 95 TPM, 100 TPM, 110 TPM or 120 TPM. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 35% to about 75%, about 40% to about 70%, about 40% to about 65%, about 41% to about 75%, about 41% to about 70%, about 41% to about 65%, about 41% to about 64%, about 41% to about 63%, about 41% to about 62%, about 35% to about 55%, about 40% to about 52%, or about 41% to about 52% of the cells in the composition express CALB2; and/or (ii) at least about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 45%, 47%, 50%, 51%, 52%, 55%, 60%, 65%, 70% or 75% of the cells in the composition express CALB2; and/or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 220 TPM, about 50 TPM to about 200 TPM, about 70 TPM to about 200 TPM, or about 75 TPM to about 199 TPM of CALB2; and/or (iv) the cells in the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 195 TPM, 196 TPM, 220 TPM, 210 TPM, or 220 TPM of a CALB2 transcript. The photosensitive rescue cell composition of any one of claims 19 to 27, wherein the plurality of heterogeneous photosensitive rescue cells comprise excitatory neurons, and the excitatory neurons express one or more markers selected from the group consisting of NEUROD2, NEUROD6, SLA, NELL2 and SATB2. The photosensitive rescue cell composition of any one of claims 19 to 28, wherein the plurality of heterogeneous photosensitive rescue cells comprise a plurality of excitatory neurons, and the excitatory neurons cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2 and SATB2. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 24%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 24%, about 0.8% to about 30%, about 0.8% to about 25%, about 0.8% to about 24%, about 1% to about 5%, about 1% to about 4.5%, about 2% to about 5%, about 2% to about 4.5%, or about 2% to about 4% of the cells in the composition express NEUROD6; and/or (ii) at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 2%, 4%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, 23%, 24%, 26%, 28%, or 30% of the cells in the composition express NEUROD6; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 210 TPM, about 1 TPM to about 205 TPM, about 1 TPM to about 25 TPM, or about 10 TPM to about 20 TPM of NEUROD6 transcript; and/or (iv) the cells in the composition express at least 1 TPM, 5 TPM, 10 TPM, 12 TPM, 15 TPM, 19 TPM, 50 TPM, 100 TPM, 150 TPM, 175 TPM, 200 TPM, 203 TPM or 210 TPM NEUROD6 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express SLA; and/or (ii) at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 15%, 16%, 18% or 20% of the cells in the composition express SLA; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 60 TPM, about 0.1 TPM to about 50 TPM, about 1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 3 TPM to about 6 TPM of the SLA transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 10 TPM, 20 TPM, 30 TPM, 40 TPM, 50 TPM, or 60 TPM of the SLA transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 30%, or about 20% to about 28% of the cells in the composition express NELL2; and/or (ii) at least about 10%, 12%, 15%, 17%, 20%, 21%, 24%, 25%, 27%, 30%, 32%, 35%, 40% or 45% of the cells in the composition express NELL2; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 150 TPM, about 4 TPM to about 130 TPM, about 10 TPM to about 15 ... TPM, about 4 TPM to about 35 TPM, about 20 TPM to about 30 TPM, or about 25 TPM to about 28 TPM of NELL2; and/or (iv) the cells in the composition express at least 1 TPM, 5 TPM, 15 TPM, 20 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 120 TPM, or 150 TPM of a NELL2 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 12%, about 1% to about 11%, about 2% to about 20%, about 2% to about 15%, about 2% to about 12%, about 2% to about 11%, about 3% to about 20%, about 3% to about 15%, about 3% to about 12%, about 3% to about 11%, about 2% to about 6%, about 2% to about 5%, or about 3% to about 4% of the cells in the composition express SATB2; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 17% or 20% of the cells in the composition express SATB2; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 30 TPM, about 0.5 TPM to about 20 TPM, about 1 TPM to about 5 TPM, or about 2 TPM to about 3 TPM of a SATB2 transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 25 TPM, or 30 TPM of a SATB2 transcript. The photosensitive rescue cell composition of any one of claims 19 to 33, wherein the plurality of heterogeneous photosensitive rescue cells comprise progenitor cells expressing one or more markers selected from the group consisting of VIM, MKI67, CLU and GLI3. The photosensitive rescue cell composition of any one of claims 19 to 34, wherein the plurality of heterogeneous photosensitive rescue cells comprises a plurality of progenitor cells that cumulatively express each of the markers VIM, MKI67, CLU and GLI3. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 40% to about 75%, about 40% to about 70%, about 40% to about 69%, about 40% to about 60%, or about 42% to about 47% of the cells in the composition express VIM; and/or (ii) at least about 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 69%, 72%, 75% or 80% of the cells in the composition express VIM; and/or (iii) the cells in the composition express about 250 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 865 TPM, about 200 TPM to about 350 TPM, or about 250 TPM to about 340 TPM of the VIM transcript; and/or (iv) the cells in the composition express at least 250 TPM, 260 TPM, 270 TPM, 300 TPM, 320 TPM, 350 TPM, 370 TPM, 400 TPM, 500 TPM, 600 TPM, 700 TPM, 800 TPM, or 900 TPM of the VIM transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 5% to about 20%, about 5% to about 15%, about 5% to about 12%, about 6% to about 15%, about 6% to about 12%, about 7% to about 15%, about 7% to about 12%, or about 6% to about 8% of the cells in the composition express MKI67; and/or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the cells in the composition express MKI67; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 10 TPM to about 35 TPM, about 15 TPM to about 25 TPM, or about 18 TPM to about 22 and/or (iv) the cells in the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 33 TPM, 35 TPM, 37 TPM or 40 TPM of the MKI67 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 10% to about 60%, about 15% to about 55%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 40%, about 20% to about 35%, or about 25% to about 32% of the cells in the composition express CLU; and/or (ii) at least about 10%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 55% or 60% of the cells in the composition express CLU; and/or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 400 TPM, about 40 TPM to about 150 TPM, about 60 TPM to about 150 TPM or about 60 TPM to about 105 TPM of CLU transcript; and/or (iv) the cells in the composition express at least 30 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 365 TPM, 375 TPM or 400 TPM of CLU transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 12% to about 50%, about 12% to about 35%, about 12% to about 29%, about 15% to about 29%, about 15% to about 29%, or about 15% to about 17% of the cells in the composition express GLI3; and/or (ii) at least about 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45% or 50% of the cells in the composition express GLI3; and/or (iii) the cells in the composition express from about 5 transcripts per million (TPM) to about 60 TPM, from about 10 TPM to about 45 TPM, from about 15 TPM to about 30 TPM, or from about 20 TPM to about 25 TPM of a GLI3 transcript; and/or (iv) the cells in the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM, or 60 TPM of a GLI3 transcript. The photoreceptor rescue cell composition of any one of claims 19 to 39, wherein the plurality of heterogeneous photoreceptor rescue cells comprise astrocytes expressing one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG and FBXL7. The photoreceptor rescue cell composition of any one of claims 19 to 40, wherein the plurality of heterogeneous photoreceptor rescue cells comprises a plurality of astrocytes, and the astrocytes cumulatively express each of the markers GFAP, LUCAT1, MIR99AHG and FBXL7. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 50%, about 1% to about 20%, about 1% to about 15%, about 1% to about 13%, about 1% to about 10%, about 1% to about 7%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express GFAP; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GFAP; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 150 TPM, about 0.1 TPM to about 125 TPM, about 1 TPM to about 20 TPM or about 3 TPM to about 15 TPM of GFAP transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.5 TPM, 1 TPM, 5 TPM, 7 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 30 TPM, 40 TPM, 50 TPM, 80 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 140 TPM or 150 TPM of GFAP. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 5% to about 20%, about 5% to about 17%, about 5% to about 15%, about 5% to about 13%, about 7% to about 20%, about 7% to about 17%, about 7% to about 15%, about 7% to about 13%, about 5% to about 12%, or about 7% to about 10% of the cells in the composition express LUCAT1; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17% or 20% of the cells in the composition express LUCAT1. A photorescive cell composition as claimed in any of the preceding claims, wherein (i) about 50% to about 100%, about 50% to about 90%, about 50% to about 88%, about 60% to about 100%, about 60% to about 90%, about 60% to about 88%, about 70% to about 90%, about 70% to about 88%, or about 75% to about 82% of the cells in the composition express MIR99AHG; and/or (ii) at least about 50%, 60%, 65%, 70%, 72%, 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 96%, 98% or 100% of the cells in the composition express MIR99AHG; and/or (iii) the cells in the composition express from about 5 transcripts per million (TPM) to about 40 TPM, from about 5 TPM to about 30 TPM, from about 6 TPM to about 25 TPM, or from about 10 TPM to about 15 TPM of the MIR99AHG transcript; and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM, or 40 TPM of the MIR99AHG transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 20% to about 70%, about 20% to about 60%, about 25% to about 70%, about 25% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 40%, about 32% to about 39%, or about 34% to about 39% of the cells in the composition express FBXL7; and/or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 60%, 65% or 70% of the cells in the composition express FBXL7; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 30 TPM, about 7 TPM to about 25 TPM, about 10 TPM to about 15 TPM, or about 11 TPM to about 14 TPM of the FBXL7 transcript; and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM, or 40 TPM of the FBXL7 transcript. A photosensitive rescue cell composition as in any one of claims 19 to 45, wherein the plurality of heterogeneous photosensitive rescue cells comprise selective neurons expressing one or more markers selected from the group consisting of: MEIS2, PBX3, GRIA2 and CACNA1C. A photosensitive rescue cell composition as in any one of claims 19 to 46, wherein the plurality of heterogeneous photosensitive rescue cells comprises a plurality of selective neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2 and CACNA1C. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 30% to about 80%, about 30% to about 90%, about 40% to about 90%, about 45% to about 85%, about 45% to about 80%, about 49% to about 79%, or about 50% to about 78% of the cells in the composition express MEIS2; and/or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 81% or 82% of the cells in the composition express MEIS2; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 200 TPM, about 10 TPM to about 180 TPM, about 15 TPM to about 200 TPM, about 10 TPM to about 200 TPM, about 15 ... TPM, about 50 TPM to about 180 TPM, or about 60 TPM to about 173 TPM of the MEIS2 transcript; and/or (iv) the cells in the composition express at least 5 TPM, 10 TPM, 15 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 135 TPM, 136 TPM, 138 TPM, 140 TPM, 145 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, or 200 TPM of the MEIS2 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 30% to about 90%, about 35% to about 85%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, or about 49% to about 75% of the cells in the composition express PBX3; and/or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 80%, 85% or 90% of the cells in the composition express PBX3; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 100 TPM, about 5 TPM to about 90 TPM, about 25 TPM to about 90 TPM, or about 29 TPM to about 88 TPM of the PBX3 transcript; and/or (iv) the cells in the composition express at least 5 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM or 100 TPM of the PBX3 transcript. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 20% to about 60%, about 20% to about 50%, about 20% to about 50%, about 20% to about 48%, about 22% to about 55%, about 22% to about 50%, about 22% to about 47%, or about 23% to about 47% of the cells in the composition express GRIA2; and/or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 46%, 47%, 50%, 52%, 54%, 56%, 58% or 60% of the cells in the composition express GRIA2; and/or (iii) the cells in the composition express about 2 transcripts per million (TPM) to about 40 TPM, about 2 TPM to about 30 TPM, about 4 TPM to about 35 TPM, or about 10 TPM to about 30 TPM of the GRIA2 transcript; and/or (iv) the cells in the composition express at least 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 17 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 26 TPM, 27 TPM, 28 TPM, 29 TPM, 30 TPM, 35 TPM, or 40 TPM of the GRIA2 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 20% to about 70%, about 30% to about 60%, about 35% to about 70%, about 35% to about 60%, about 33% to about 60%, about 35% to about 60%, or about 39% to about 60% of the cells in the composition express CACNA1C; and/or (ii) at least about 20%, 25%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65% or 70% of the cells in the composition express CACNA1C; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 15 TPM, about 1 TPM to about 10 TPM, about 1 TPM to about 7 TPM, or about 3 TPM to about 7 TPM of the CACNA1C transcript; and/or (iv) the cells in the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 10 TPM, 12 TPM, or 15 TPM of the CACNA1C transcript. A photosensitive rescue cell composition as claimed in any one of claims 1 to 51, wherein the plurality of heterogeneous photosensitive rescue cells comprises: (i) inhibitory neurons expressing one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4 and CALB2; (ii) excitatory neurons expressing one or more markers selected from the group consisting of NEUROD2, NEUROD6, SLA, NELL2 and SATB2; (iii) progenitor cells expressing one or more markers selected from the group consisting of VIM, MKI67, CLU and GLI3; (iv) astrocytes expressing one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG and FBXL7; and (v) selective neurons expressing one or more markers selected from the group consisting of: MEIS2, PBX3, GRIA2 and CACNA1C. A photosensitive rescue cell composition as claimed in any one of claims 1 to 52, wherein the plurality of heterogeneous photosensitive rescue cells comprises: (i) a plurality of inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4 and CALB2; (ii) a plurality of excitatory neurons that cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2 and SATB2; (iii) a plurality of progenitor cells that cumulatively express each of the markers VIM, MKI67, CLU and GLI3; (iv) a plurality of astrocytes that cumulatively express each of the markers GFAP, LUCAT1, MIR99AHG and FBXL7; and (v) a plurality of selective neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2, and CACNA1C. A photosensitive rescue cell composition as claimed in any one of claims 1 to 53, wherein the composition comprises: (i) about 25% to about 55%, about 25% to about 50%, about 30% to about 55%, about 30% to about 50%, about 35% to about 55%, about 35% to about 50%, or about 38% to about 49% inhibitory neurons; and/or (ii) about 0% to about 15%, about 0% to about 12%, about 0% to about 10%, about 0% to about 8%, or about 0.5% to about 9% excitatory neurons; and/or (iii) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 17% to about 45%, about 17% to about 40%, about 17% to about 35%, or about 20% to about 35% progenitor cells; and/or (iv) about 0% to about 6%, about 0% to about 5%, about 0% to about 4%, about 0% to about 3%, about 0% to about 2%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, or about 0.5% to about 1.5% stellate cells; and/or (v) about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, or about 17% to about 37% mixed neurons. A photoreceptor rescue cell composition as claimed in any one of claims 1 to 54, wherein the cells in the composition further express one or more eye movement zone progenitor cell markers, rod/cone photoreceptor cell markers and/or neuron markers. The photoreceptor rescue cell composition of claim 55, wherein the eye movement area progenitor cell markers are selected from the group consisting of: PAX6, LHX2, SIX3, NES and SOX2. The photoreceptor rescue cell composition of claim 55 or claim 56, wherein the plurality of heterogeneous cells cumulatively express at least 1, 2, 3 or 4 of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES or SOX2. A photorescible rescue cell composition as claimed in any one of claims 55 to 57, wherein the plurality of heterogeneous cells cumulatively express at least one of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2. The photorescive cell composition of any one of claims 55 to 58, wherein the plurality of heterogeneous cells cumulatively express SOX2. A photorescible rescue cell composition as claimed in any one of claims 55 to 59, wherein the composition is substantially free of cells expressing the eye movement area progenitor cell markers RAX, SIX6 and/or TBX3. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 25% to about 60%, about 25% to about 55%, about 25% to about 52%, about 25% to about 45%, about 30% to about 60%, about 30% to about 55%, about 30% to about 45%, or about 30% to about 42% of the cells in the composition express PAX6; and/or (ii) at least about 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% of the cells in the composition express PAX6; and/or (iii) the cells in the composition express about 20 transcripts per million (TPM) to about 125 TPM, about 30 TPM to about 110 TPM, about 35 TPM to about 100 TPM, about 70 TPM to about 80 TPM, or about 73 TPM to about 78 TPM. and/or (iv) the cells in the composition express at least 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 78 TPM, 80 TPM, 82 TPM, 85 TPM, 87 TPM, 90 TPM, 92 TPM, 95 TPM, 97 TPM, 100 TPM, 105 TPM, 110 TPM, 115 TPM, 120 TPM or 125 TPM of the PAX6 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 3% to about 35%, about 5% to about 35%, about 5% to about 35%, about 6% to about 35%, about 7% to about 35%, about 3% to about 30%, about 5% to about 30%, about 6% to about 30%, about 7% to about 30%, about 3% to about 25%, about 5% to about 25%, about 6% to about 25%, or about 7% to about 9% of the cells in the composition express LHX2; and/or (ii) at least about 3%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 32% or 35% of the cells in the composition express LHX2; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 36 TPM, about 5 TPM to about 10 TPM, or about 6 TPM to about 8 TPM of the LHX2 transcript; and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 36 TPM, 38 TPM, or 40 TPM of the LHX2 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12% or 2% to about 10% of the cells in the composition express SIX3; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% or 25% of the cells in the composition express SIX3; and/or (iii) the cells in the composition express about 1 transcript per million (TPM) to about 50 TPM, about 2 TPM to about 30 TPM, about 1 TPM to about 25 TPM, or about 5 TPM to about 20 TPM of the SIX3 transcript; and/or (iv) the cells in the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM 9 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, or 50 TPM of the SIX3 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 15% to about 40%, about 15% to about 35%, about 15% to about 34%, about 15% to about 33%, about 15% to about 32%, about 15% to about 31%, about 18% to about 40%, about 18% to about 35%, about 18% to about 34%, about 18% to about 33%, about 18% to about 32%, or about 18% to about 31% of the cells in the composition express NES; and/or (ii) at least about 15%, 17%, 20%, 25%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or 40% of the cells in the composition express NES; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 35 TPM, about 5 TPM to about 28 TPM, about 7 TPM to about 15 TPM, about 10 TPM to about 15 TPM, or about 11 TPM to about 13 TPM of the NES transcript; and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 22 TPM, 24 TPM, 25 TPM, 26 TPM, 27 TPM, 30 TPM, or 35 TPM of the NES transcript. A photorescrutable cell composition as claimed in any of the preceding claims, wherein (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 75% of the cells in the composition express SOX2; and/or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% or 90% of the cells in the composition express SOX2; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 250 TPM, about 90 TPM to about 200 TPM, about 125 TPM to about 190 TPM, or about 155 TPM to about 175 TPM of a CACNA1C transcript; and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, 200 TPM, 210 TPM, 220 TPM, 230 TPM, 240 TPM, or 250 TPM of a CACNA1C transcript. The photoreceptor rescue cell composition of any one of claims 55 to 65, wherein the rod/cone photoreceptor cell markers are selected from the group consisting of ASCL1, RORB, NR2E3 and NRL. The photoreceptor rescue cell composition of claim 66, wherein the plurality of heterogeneous cells cumulatively express each of the rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL. The photoreceptor rescue cell composition of any one of claims 55 to 67, wherein the composition is substantially free of cells expressing rod/cone photoreceptor cell markers CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1 and GNAT2. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 10% to about 60%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 22% to about 45%, about 22% to about 43%, about 25% to about 420%, or about 28% to about 30% of the cells in the composition express ASCL1; and/or (ii) at least about 10%, 15%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 41%, 42%, 45%, 50%, 55% or 60% of the cells in the composition express ASCL1; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 150 TPM, about 60 TPM to about 140 TPM, about 65 TPM to about 130 TPM or about 95 TPM to about 130 TPM of the ASCL1 transcript; and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 125 TPM, 130 TPM, 140 TPM or 150 TPM of the ASCL1 transcript. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 10% to about 25%, or about 11% to about 22% of the cells in the composition express RORB; and/or (ii) at least about 5%, 7%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 25%, 30%, 35%, 40%, 45% or 50% of the cells in the composition express RORB; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 20 TPM, about 0.1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 1 TPM to about 6 TPM of the RORB transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 18 TPM, or 20 TPM of the RORB transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or about 2% to about 5% of the cells in the composition express NR2E3; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% or 20% of the cells in the composition express NR2E3; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 3 TPM, or about 0.1 TPM to about 1 TPM of the NR2E3 transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 2 TPM, 5 TPM, 7 TPM, 9 TPM, or 10 TPM of the NR2E3 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, or about 2% to about 8% of the cells in the composition express NRL; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% or 20% of the cells in the composition express NRL; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 7 TPM or about 0.1 TPM to about 2 TPM of the NRL transcript; and/or (iv) the cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of the NRL transcript. The photorescible rescue cell composition of any one of claims 55 to 72, wherein the neuronal markers are selected from the group consisting of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5. The photorescible rescue cell composition of claim 73, wherein the plurality of heterogeneous cells cumulatively express each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 50% to about 100%, about 60% to about 100%, about 60% to about 95%, about 70% to about 100%, about 70% to about 95%, about 80% to about 100%, about 80% to about 95%, or about 80% to about 90% of the cells in the composition express NFIB; and/or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98% or 100% of the cells in the composition express NFIB; and/or (iii) the cells in the composition express about 150 transcripts per million (TPM) to about 650 TPM, about 180 TPM to about 610 TPM, about 400 TPM to about 500 TPM, or about 400 TPM to about 480 TPM of the NFIB transcript; and/or (iv) the cells in the composition express at least 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 550 TPM, 600 TPM, 625 TPM, or 650 TPM of the NFIB transcript. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, or about 2% to about 6% of the cells in the composition express OTX2; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% or 20% of the cells in the composition express OTX2; and/or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about 50 TPM, about 8 TPM to about 40 TPM, about 8 TPM to about 25 TPM, or about 12 TPM to about 15 TPM of the OTX2 transcript; and/or (iv) the cells in the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 26 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM or 50 TPM of the OTX2 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 78%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 78%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 78% of the cells in the composition express ELAVL3; and/or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% or 90% of the cells in the composition express ELAVL3; and/or (iii) the cells in the composition express from about 10 transcripts per million (TPM) to about 120 TPM, from about 15 TPM to about 100 TPM, from about 20 TPM to about 90 TPM, from about 60 TPM to about 90 TPM, or from about 70 TPM to about 80 TPM of the ELAVL3 transcript; and/or (iv) the cells in the composition express at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM or 120 TPM ELAVL3 transcript. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 78%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 78%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, or about 50% to about 65% of the cells in the composition express ELAVL4; and/or (ii) at least about 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 75%, 80%, 85% or 90% of the cells in the composition express ELAVL4; and/or (iii) the cells in the composition express about 10 transcripts per million (TPM) to about 120 TPM, about 15 TPM to about 100 TPM, about 20 TPM to about 90 TPM, about 50 TPM to about 90 TPM, about 70 TPM to about 90 TPM or about 79 TPM to about 85 TPM of the ELAVL4 transcript; and/or (iv) the cells in the composition express at least 10 TPM, 15 TPM, 20 TPM to about 100 TPM, about 20 TPM to about 90 TPM, about 50 TPM to about 90 TPM, about 70 TPM to about 90 TPM or about 79 TPM to about 85 TPM of the ELAVL4 transcript; ELAVL4 transcripts of TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 82 TPM, 84 TPM, 86 TPM, 90 TPM, 100 TPM, 110 TPM, or 120 TPM. A photosensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 73%, about 51% to about 73%, or about 52% to about 66% of the cells in the composition express SLC1A2; and/or (ii) at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 75%, 80%, 85%, or 90% of the cells in the composition express SLC1A2; and/or (iii) the cells in the composition express about 10 transcripts per million (TPM) to about 120 TPM, about 10 TPM to about 90 TPM, about 20 TPM to about 90 TPM, about 40 TPM to about 70 TPM, about 50 TPM to about 70 TPM, or about 58 TPM to about 63 TPM of the SLC1A2 transcript; and/or (iv) the cells in the composition express at least 10 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM or 120 TPM SLC1A2 transcripts. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 11% to about 19%, or about 10% to about 12% of the cells in the composition express SLC1A3; and/or (ii) at least about 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 25%, 30%, 35%, or 40% of the cells in the composition express SLC1A3; and/or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 200 transcripts per million (TPM); TPM, about 70 TPM to about 190 TPM, about 60 TPM to about 100 TPM, about 60 TPM to about 80 TPM, or about 72 TPM to about 79 TPM of the SLC1A3 transcript; and/or (iv) the cells in the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 79 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 175 TPM, 180 TPM, or 200 TPM of the SLC1A3 transcript. A photorescible cell composition as claimed in any of the preceding claims, wherein (i) about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 3% to about 4% of the cells in the composition express HCN1; and/or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the cells in the composition express HCN1; and/or (iii) the cells in the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 5 TPM, about 0.1 TPM to about 1 TPM, or about 0.2 TPM to about 0.6 TPM of HCN1 transcripts; and/or (iv) cells in the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1.0 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of the HCN1 transcript. A light-sensitive rescue cell composition as claimed in any of the preceding claims, wherein (i) about 5% to about 30%, about 5% to about 25%, about 1% to about 30%, about 1% to about 25%, about 5% to about 25%, about 10% to about 25%, or about 10% to about 15% of the cells in the composition express HES5; and/or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or 30% of the cells in the composition express HES5; and/or (iii) the cells in the composition express about 10 transcripts per million (TPM) to about 60 TPM, about 5 TPM to about 50 TPM, about 12 TPM to about 46 TPM, or about 15 TPM to about 39 TPM of the HES5 transcript; and/or (iv) the cells in the composition express 10 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM of the HES5 transcript. A photoreceptor rescue cell composition as claimed in any one of claims 1 to 82, wherein the plurality of heterogeneous cells cumulatively express: (i) one or more of the eye movement area progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2; (ii) one or more of the rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL; and (iii) one or more of the neuron markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5. A photoreceptor rescue cell composition as claimed in any one of claims 1 to 83, wherein the plurality of heterogeneous cells cumulatively express: (i) each of the eye region progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2; (ii) each of the rod/cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL; and (iii) each of the neuron markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5. The photoreceptor rescue cell composition of any of the preceding claims, wherein the composition is substantially free of at least one cell type selected from the group consisting of high-potential stem cells, retinal ganglion cells, photoreceptor cells, and axonal neurons. The photoreceptor rescue cell composition of any of the preceding claims, wherein the composition is substantially free of high-potential stem cells, retinal ganglion cells, photoreceptor cells, and axonal neurons. The photoreceptor rescue cell composition of any of the preceding claims, wherein the composition is substantially free of retinal progenitor cells expressing VSX2 and/or POU5F1. The photorescible rescue cell composition of any of the preceding claims, wherein the composition has less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% of cells expressing SSEA4 as determined by flow cytometry, or wherein the composition contains no cells expressing SSEA4 as determined by flow cytometry. A photoreceptor rescue cell composition as claimed in any of the preceding claims, wherein the cells in the composition have phagocytic activity, and optionally have the ability to phagocytose isolated photoreceptor cell outer segments, dye conjugates or both. The photosensitive rescue cell composition of any of the preceding claims, wherein the cells in the composition secrete one or more neuroprotective factors. The photosensitive rescue cell composition of claim 90, wherein the neuroprotective factors are selected from the group consisting of CNTF, MIF, S100B, GFAP, TAU, NCAM1 and TNC. The photorescible rescue cell composition of any of the preceding claims, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75% or at least 80% of the cells in the composition are alive. The photosensitive rescue cell composition of any of the preceding claims, wherein about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable. The photosensitive rescue cell composition of any of the preceding claims, wherein at least 50% of the cells in the composition are alive. The photosensitive rescue cell composition of any preceding claim, wherein at least 55% of the cells in the composition are alive. The photosensitive rescue cell composition of any preceding claim, wherein at least 60% of the cells in the composition are alive. A photorescue cell composition as claimed in any preceding claim, wherein at least 65% of the cells in the composition are alive. The photosensitive rescue cell composition of any preceding claim, wherein at least 68% of the cells in the composition are alive. A photorescible rescue cell composition as claimed in any one of claims 1 to 98, wherein the composition is produced according to a method comprising: 1) culturing enriched potential stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells obtained from step 1 in neural differentiation medium (NDM) and noggin; 3) expanding the cells obtained from step 2 in NDM in the absence of noggin, comprising a) culturing the cells in NDM without noggin under low-adherence or non-adherence conditions; and b) culturing the cells in NDM without noggin under adhesion conditions. The photosensitive rescue cell composition of claim 99, wherein the high-potential cells are embryonic stem cells (ESCs) or induced high-potential stem cells (iPSCs). The photosensitive rescue cell composition of claim 99 or 100, wherein step 3 is performed at least once, at least twice, at least three times, at least four times, at least five times, or at least six times. The photosensitive rescue cell composition of any one of claims 99 to 101, wherein the cells in the composition are collected after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. The photosensitive rescue cell composition of claim 102, wherein the cells in the composition are collected after the fifth repetition of step 3. The photosensitive rescue cell composition of claim 102 or 103, wherein the collected cells in the composition are stored at low temperature. A photosensitive rescue cell composition as claimed in any one of claims 99 to 104, wherein the composition is stored at low temperature between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3. A photosensitive rescue cell composition as claimed in any one of claims 99 to 104, wherein the composition is cryopreserved after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. The photosensitive rescue cell composition of claim 106, wherein step 3 is repeated five times and wherein the composition is stored at low temperature after the fifth repetition of step 3. The photosensitive rescue cell composition of any one of claims 99 to 107, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75% or at least 80% of the cells are alive after cryopreservation and thawing. The photosensitive rescue cell composition of any one of claims 99 to 108, wherein after cryopreservation and thawing, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% is viable. The photorescue cell composition of any one of claims 99 to 109, wherein at least about 50% of the cells in the composition are alive after cryopreservation and thawing. The photorescue cell composition of any one of claims 99 to 110, wherein at least about 55% of the cells in the composition are alive after cryopreservation and thawing. The photorescue cell composition of any one of claims 99 to 111, wherein at least about 60% of the cells in the composition are alive after cryopreservation and thawing. The photorescue cell composition of any one of claims 99 to 112, wherein at least about 65% of the cells in the composition are alive after cryopreservation and thawing. A photorescue cell composition as claimed in any one of claims 99 to 113, wherein at least 68% of the cells in the composition are alive after cryopreservation and thawing. A photosensitive rescue cell composition as claimed in any one of claims 99 to 114, wherein the composition comprises a spheroid. The photosensitive rescue cell composition of claim 115, wherein about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are spheroids. A photosensitive rescue cell composition as claimed in any one of claims 99 to 116, wherein during steps 1, 2 and/or 3, the cells in the composition are cultured in a cell culture container selected from the group consisting of: a culture dish, a culture bottle and a culture chamber. The photosensitive rescue cell composition of claim 117, wherein the cell culture container has a size of about 50 cm ² to about 800 cm ² , about 60 cm ² to about 800 cm ² , about 100 cm ² to about 800 cm ² , about 150 cm ² to about 800 cm ² , about 175 cm ² to about 800 cm ² , about 200 cm ² to about 800 cm ² , about 250 cm ² to about 800 cm ² , about 300 cm ² to about 800 cm ² , about 400 cm ² to about 800 cm ² , about 500 cm ² to about 800 cm ² , about 600 cm ² to about 800 cm ² , about 700 cm ² to about 800 cm ² , about 30 cm ² to about 100 cm ² , about 50 cm ² to about 100 cm ² , about 100 cm ² to about 300 cm ² , about 150 cm ² to about 250 cm ² , or about 150 cm ² to about 200 cm ² cell growth area. The photosensitive rescue cell composition of claim 117 or 118, wherein the cell culture container has a cell growth area of at least about 60 cm ² , about 175 cm ² , or about 636 cm ² . A photosensitive rescue cell composition as claimed in any one of claims 117 to 119, wherein the culture chamber is a stackable rectangular chamber. The photosensitive rescue cell composition of any one of claims 117 to 120, wherein step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5, or 1 to 2 culture chambers. The photosensitive rescue cell composition of claim 121, wherein step 3 has at least 1, 2, 5, 10 or 40 culture chambers. The photosensitive rescue cell composition of any one of claims 117 to 122, wherein the cell culture container is coated for low-attachment or non-attachment cell culture in step 3a and/or for adherence cell culture in step 3b. The photosensitive rescue cell composition of any one of claims 99 to 123, wherein the cells are enzymatically dissociated from the culture plate into a cell suspension. The photosensitive rescue cell composition of claim 124, wherein the enzymatic lysis uses an enzyme selected from the group consisting of thermolysin, liberase, accutase, and combinations thereof. The photosensitive rescue cell composition of claim 125, wherein the enzyme used to dissociate the cells is acurase. The photosensitive rescue cell composition of any one of claims 99 to 126, wherein the dissociation of the cells from the culture dish does not include manual scraping. A pharmaceutical preparation comprising the photosensitive rescue cell composition of any one of claims 1 to 127 and a pharmaceutically acceptable formulation. The pharmaceutical preparation of claim 128, wherein the pharmaceutically acceptable formulation is suitable for ocular delivery. A formulation comprising: a) a composition as in any one of claims 1 to 127, or a pharmaceutical preparation as in claim 128 or claim 129; and b) about 4 to 10% (v/v) of a cryoprotectant, about 2 to 3% (w/v) of albumin, about 0 to 1.5% (w/v) of glucose, and a buffer. The formulation of claim 130, wherein the low temperature protective agent is selected from DMSO, glycerol and ethylene glycol. The formulation of claim 130 or 131, wherein the cryoprotectant is DMSO. The formulation of any one of claims 130 to 132, wherein the formulation comprises about 4 to 6% (v/v) cryoprotectant. The formulation of any one of claims 130 to 133, wherein the formulation comprises about 0.08 to 0.10% (w/v) glucose. The formulation of any one of claims 130 to 133, wherein the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.09% (w/v) glucose, and a buffer. The formulation of any one of claims 130 to 134, wherein the formulation comprises about 0.6% (w/v) glucose. The formulation of any one of claims 130 to 134 or 136, wherein the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and buffer. The formulation of any one of claims 130 to 137, wherein the albumin is human albumin. The formulation of any one of claims 130 to 138, wherein the albumin is recombinant human albumin. The formulation of any one of claims 130 to 139, wherein the buffer is buffered saline. The formulation of any one of claims 130 to 140, wherein the buffer is phosphate buffered saline (PBS). The formulation of claim 140 or 141, wherein the buffered saline comprises Ca2+ and Mg2+. The formulation of claim 140 or 141, wherein the buffered saline solution does not contain Ca2+ and Mg2+. The formulation of any one of claims 130 to 143, wherein the formulation is stored at low temperature. A formulation comprising: a) a composition as claimed in any one of claims 1 to 127, or a pharmaceutical preparation as claimed in claim 128 or claim 129; and b) a solution comprising (1) a buffer solution which maintains the solution at physiological pH; and (2) at least 2 mM or at least 0.05% (w/v) glucose; and (3) an osmotically active agent which maintains the solution at physiological osmotic pressure. The formulation of claim 145, wherein the solution comprises at least 5 mM or at least 0.1% (w/v) glucose; or at least 7.5 mM or at least 0.14% (w/v) glucose; or at least 10 mM or at least 0.2% (w/v) glucose; or at least 15 mM or at least 0.25% (w/v) glucose; or at least 20 mM or at least 0.4% (w/v) glucose; or at least 25 mM or at least 0.5% (w/v) glucose. The formulation of claim 145 or 146, wherein the solution further comprises (4) a divalent cation source, wherein the divalent cation source comprises a calcium source and/or a magnesium source, and/or (5) an acetate buffer and/or a citrate buffer. A formulation comprising: a) a composition as claimed in any one of claims 1 to 127, or a pharmaceutical preparation as claimed in claim 128 or claim 129; and b) a solution comprising (1) a buffer which maintains the solution at physiological pH, wherein the buffer is not a bicarbonate buffer; and (2) glucose; and (3) an osmotic active agent which maintains the solution at physiological osmotic pressure; and (4) a source of divalent cations, optionally wherein the source of divalent cations comprises a calcium source and/or a magnesium source, and/or wherein the buffer comprises an acetate buffer and/or a citrate buffer. The formulation of claim 147 or 148, wherein the calcium source comprises a pharmaceutically acceptable calcium salt and/or the magnesium source comprises a pharmaceutically acceptable magnesium salt. The formulation of claim 149, wherein the pharmaceutically acceptable calcium salt and/or the pharmaceutically acceptable magnesium salt is selected from the group of calcium salts and/or magnesium salts formed with an acid, and the acid is selected from the group comprising acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid. The formulation of any one of claims 147 to 150, wherein the calcium source comprises calcium chloride, optionally wherein the calcium source comprises calcium chloride dihydrate. The formulation of any one of claims 147 to 151, wherein the magnesium source comprises magnesium chloride, optionally wherein the magnesium source comprises magnesium chloride hexahydrate. The formulation of any one of claims 147 to 152, wherein the citrate buffer is provided in the form of sodium citrate. The formulation of any one of claims 145 to 153, wherein the glucose is D-glucose (dextrose). A formulation as claimed in any one of claims 145 to 154, wherein the osmotic active agent is a salt, optionally wherein the osmotic active agent is a sodium salt, and further optionally wherein the osmotic active agent is sodium chloride. The formulation of any one of claims 145 to 155, wherein the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and glucose. The formulation of any one of claims 145 to 156, wherein the pH of the solution is 6.8-7.8, or 7.4-7.5, or about 7.5. The formulation of any one of claims 145 to 157, wherein the solution is isotonic or hypertonic. The formulation of any of claims 145 to 157, wherein the solution exhibits an osmotic pressure of about 270-345 mOsm/1 or about 315 mOsm/1. A formulation as claimed in any one of claims 145 to 159, wherein the concentration of the calcium source is (a) 0.25-0.75 mM, or 0.4-0.65 mM, or 0.5-0.6 mM, or about 0.6 mM; or (b) 0.5-0.9 mM, or 0.6-0.8 mM, or about 0.7 mM. The formulation of any one of claims 145 to 160, wherein the concentration of the magnesium source is 0.05-5 mM, or 0.1-0.3 mM, or about 0.3 mM. The formulation of any one of claims 145 to 161, wherein the concentration of glucose is 5-50 mM, or 10-25 mM, or 10-20 mM, or about 16 mM. The formulation of any one of claims 145 to 162, wherein the concentration of the osmotic active agent is about 100-200 mM, or about 125-175 mM, or about 150 mM. The formulation of any one of claims 145 to 163, wherein the concentration of the citrate or acetate is 0.1-5 mM, or 0.5-2 mM, or about 1 mM. The formulation of any one of claims 145 to 164, wherein the solution further comprises a potassium salt, optionally wherein the potassium salt is potassium chloride, further optionally wherein the KCl concentration is 0.2-5 mM, or 1-2.5 mM, or about 2 mM. The formulation of any of claims 145 to 165, wherein the solution comprises (a) about 0.7 mM CaCl ₂ (calcium chloride), about 0.3 mM MgCl ₂ (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, and about 145 mM NaCl, or (b) about 0.5-0.9 mM CaCl ₂ (calcium chloride), about 0.2-0.4 mM MgCl ₂ (magnesium chloride), about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, and about 116-174 mM NaCl, or (c) about 0.85% NaCl, about 0.01% CaCl ₂ dihydrate (calcium chloride dihydrate), about 0.006% MgCl ₂ hexahydrate (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate and about 0.29% dextrose, or (d) about 0.68-1.02% NaCl, about 0.008-0.012% CaCl ₂ dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl ₂ hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate and about 0.23-0.35% dextrose. A formulation as claimed in any one of claims 145 to 166, wherein the solution further comprises (a) about 2 mM KCl, and/or (b) a viscoelastic polymer, optionally wherein the polymer is hyaluronic acid or a salt or solvent thereof, and optionally wherein the polymer is sodium hyaluronate. The formulation of claim 167, wherein the polymer is present in the solution at a concentration effective to reduce exposure of cells to shear stress, optionally wherein the polymer is at a concentration of 0.005-5% w/v or about 0.05% w/v. The formulation of any of claims 145 to 168, wherein the solution comprises (a) about 0.7 mM CaCl ₂ (calcium chloride), about 0.3 mM MgCl ₂ (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl, and about 0.05% hyaluronic acid, or (b) about 0.5-0.8 mM CaCl ₂ (calcium chloride), about 0.2-0.4 mM MgCl ₂ (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl, and about 0.04-0.06% hyaluronic acid. A formulation as claimed in any one of claims 145 to 169, wherein the solution does not contain (a) a carbonate buffer, and/or (b) glutathione, or glutathione disulfide (GSSG), and/or (c) a zwitterionic organic buffer. The formulation of any of claims 145 to 170, wherein the solution (a) can be stored at 25°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least one week, at least two weeks, at least three weeks, or at least one month without measurable precipitation of solutes and/or without measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution, and/or (b) can be stored at 2-8°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least one week, at least two weeks, at least three weeks, or at least one month without measurable precipitation of solutes and/or without measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution. A formulation as claimed in any one of claims 145 to 171, wherein the solution is (a) suitable for administration to a subject, suitable for administration to an eye of a subject and/or suitable for implantation into an eye of a subject, and/or (b) substantially free of pyrogens, and/or (c) sterile, and/or (d) for lavage, cell recovery, cell storage, cell transport and/or administration to a subject. A method for producing a composition as claimed in any one of claims 1 to 127, comprising: 1) culturing high-potential stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells obtained from step 1 in neural differentiation medium (NDM) and noggin; 3) expanding the cells obtained from step 2 in NDM without noggin, comprising a) culturing the cells in NDM without noggin under low attachment or non-attachment conditions, and b) culturing the cells in NDM without noggin under non-attachment conditions, thereby differentiating the high-potential stem cells into photosensitive rescue cells. A method for producing a photosensitive rescue cell composition comprising a plurality of heterogeneous cells, the method comprising: 1) culturing high-potential stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells in neural differentiation medium (NDM) and noggin; 3) expanding the cells in NDM without noggin, comprising a) culturing the cells in NDM without noggin under low attachment or non-attachment conditions, and b) culturing the cells in NDM without noggin under attachment conditions, thereby differentiating the high-potential stem cells into a plurality of cells in the photosensitive rescue cell composition. The method of claim 173 or claim 174, wherein the high-potential stem cells are embryonic stem cells (ESCs) or induced high-potential stem cells (iPSCs). The method of any of claims 173 to 175, wherein step 3 is performed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. The method of any one of claims 173 to 176, wherein the cells in the composition are collected after a third repetition of step 3, after a fourth repetition of step 3, or after a fifth repetition of step 3. The method of claim 177, wherein the composition is collected after the fifth repetition of performing step 3. The method of claim 177 or 178, wherein the collected cells in the composition are cryogenically stored. A method as in any of claims 173 to 179, wherein the composition is stored at a low temperature between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3. A method as in any one of claims 173 to 180, wherein the composition is stored at low temperature after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. The method of claim 180, wherein step 3 is repeated five times and wherein after the fifth repetition of step 3, the composition is stored at a low temperature. The method of any one of claims 173 to 182, wherein after cryopreservation and thawing, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells in the composition are viable. The method of any one of claims 173 to 183, wherein after cryopreservation and thawing, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable. The method of any one of claims 173 to 184, wherein after cryopreservation and thawing, less than about 50% of the cells in the composition are viable. The method of any one of claims 173 to 185, wherein after cryopreservation and thawing, less than about 55% of the cells in the composition are viable. The method of any one of claims 173 to 186, wherein after cryopreservation and thawing, less than about 60% of the cells in the composition are viable. The method of any one of claims 173 to 187, wherein after cryopreservation and thawing, less than about 65% of the cells in the composition are viable. The method of any one of claims 173 to 188, wherein after cryopreservation and thawing, less than about 68% of the cells in the composition are viable. The method of any one of claims 173 to 189, wherein the composition comprises spheroids. The method of claim 190, wherein about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are spheroids. A method as in any one of claims 173 to 191, wherein during steps 1, 2 and/or 3, the cells in the composition are cultured in a cell culture container selected from the group consisting of: a culture dish, a culture bottle and a culture chamber. The method of claim 192, wherein the cell culture container has a size of about 50 cm ² to about 800 cm ² , about 60 cm ² to about 800 cm ² , about 100 cm ² to about 800 cm ² , about 150 cm ² to about 800 cm ² , about 175 cm ² to about 800 cm ² , about 200 cm ² to about 800 cm ² , about 250 cm ² to about 800 cm ² , about 300 cm ² to about 800 cm ² , about 400 cm ² to about 800 cm ² , about 500 cm ² to about 800 cm ² , about 600 cm ² to about 800 cm ² , about 700 cm ² to about 800 cm ² , about 30 cm ² to about 100 cm ² , about 50 cm ² to about 100 cm ² , about 100 cm ² to about 300 cm ² , about 150 cm ² to about 250 cm ² , or about 150 cm ² to about 200 cm ² of cell growth area. The method of claim 192 or 193, wherein the cell culture vessel has a cell growth area of at least about 60 cm ² , about 175 cm ² , or about 636 cm ² . The method of claim 192 or 193, wherein the culture chamber is a stackable rectangular chamber. The method of claim 195, wherein step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5, or 1 to 2 culture chambers. The method of claim 196, wherein step 3 comprises at least 1, 2, 5, 10 or 40 culture chambers. The method of any one of claims 192 to 197, wherein the cell culture container is coated for low-attachment or non-attachment cell culture in step 3a, and/or for adherence cell culture in step 3b. The method of any one of claims 173 to 198, wherein the cells are enzymatically dissociated from the culture plate into a cell suspension. The method of claim 199, wherein the enzyme used to dissociate the cells is thermolysin, liberase and/or akurin. The method of claim 200, wherein the enzyme used to dissociate the cells is acurase. The method of any one of claims 173 to 201, wherein the dissociation of the cells from the culture dish does not comprise manual scraping. A photosensitive rescue cell composition produced by the method of any one of claims 173 to 202. A method for treating an eye disease or disorder in a subject, comprising administering to the subject a photorescrutin cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. A method for increasing the secretion of neuroprotective factors by the eye of an individual suffering from an eye disease or disorder, comprising administering to the individual a photorescive cell composition as described in any one of claims 1 to 127 or 203, a pharmaceutical preparation as described in any one of claims 128 or 129, or a formulation as described in any one of claims 130 to 172. The method of claim 205, wherein the method increases the secretion of the neuroprotective factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the secretion of the neuroprotective factor before administration. A method for improving visual acuity in an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescrutin cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. The method of claim 207, wherein the method increases visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to visual acuity prior to administration. The method of claim 207 or 208, wherein visual acuity is measured based on optomotor response (OMR) and/or electroretinogram (ERG). The method of claim 209, wherein the spatial frequency threshold of the treated eye is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the spatial frequency threshold before administration as measured by OMR. A method as claimed in claim 209 or 210, wherein the glimmer b-wave amplitude of the treated eye is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the glimmer b-wave amplitude before administration based on ERG measurement. A method for preventing or slowing photoreceptor cell loss in a subject suffering from a retinal disease or disorder, comprising administering to the subject a photoreceptor rescue cell composition as claimed in any one of claims 1 to 127 or 203, a pharmaceutical preparation as claimed in any one of claims 128 or 129, or a formulation as claimed in any one of claims 130 to 172. The method of claim 212, wherein prevention of photoreceptor cell loss is measured based on expression of CNFT. The method of claim 213, wherein the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to CNFT expression in the eye before administration. A method of increasing phagocytic activity in the eye of a subject suffering from a retinal disease or disorder, comprising administering to the subject a photorescive cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. The method of claim 215, wherein the method increases the phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the phagocytic activity before administration. A method of inhibiting microglial cell activation in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescrutin cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. The method of claim 217, wherein inhibition of microglial cell activation is measured based on expression of CNFT and/or MIF. The method of claim 218, wherein the method increases CNFT performance by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to CNFT performance before administration. The method of claim 217 or claim 218, wherein the method increases the expression of MIF by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of MIF before administration. A method of reducing oxidative stress in the eye of a subject suffering from a retinal disease or disorder, comprising administering to the subject a photorescive cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. The method of claim 221, wherein the reduction of oxidative stress is measured based on the expression of CNFT. The method of claim 222, wherein the method increases CNFT performance by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to CNFT performance before administration. A method of increasing the expression of an anti-apoptotic factor in the eye of a subject suffering from a retinal disease or disorder, comprising administering to the subject a photorescive cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. The method of claim 224, wherein the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of the anti-apoptotic factor before administration. The method of claim 224 or 225, wherein the anti-apoptotic factor is S100B. A method for preventing degeneration of the outer nuclear layer (ONL) in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual a photorescive cell composition of any one of claims 1 to 127 or 203, a pharmaceutical preparation of any one of claims 128 or 129, or a formulation of any one of claims 130 to 172. The method of any one of claims 204 to 227, wherein the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, secondary map atrophy of macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. The method of claim 228, wherein the eye disease is macular degeneration or retinitis pigmentosa. The method of any one of claims 204 to 229, wherein the disease is a retinal degenerative disease. The method of any of claims 204 to 230, wherein the disease is associated with loss of photoreceptor cells. The method of claim 231, wherein the disease is associated with loss of photoreceptor cells in the extranuclear layer of the retina. The method of any one of claims 204 to 232, wherein the photoreceptor rescue cell composition, formulation or pharmaceutical preparation is administered into the subretinal space, the supracordial space, into the eye via a reservoir, or into other parts of the individual's body via systemic delivery. The method of any one of claims 204 to 233, wherein the cell preparation or formulation is administered by injection or implantation. The method of claim 234, wherein the injection is administered intraocularly. The method of claim 234 or 235, wherein the intraocular administration comprises injecting an aqueous solution, an isotonic solution and/or a saline solution into the subretinal space to form an anterior blister; and removing the aqueous solution, and then administering the photoreceptor rescue cell composition in the form of the aqueous solution into the same subretinal space. The method of any one of claims 204 to 237, wherein the cell preparation or formulation is administered within at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years of symptom onset. The method of any of claims 204 to 237, wherein (i) the individual has intermediate or near-advanced disease; (ii) the individual has a best corrected visual acuity (BCVA) in the range of 20/50 to 20/200; (iii) the individual has a BCVA worse than 20/200 but maintains light perception; or (iv) the individual is diagnosed with retinitis pigmentosa by genotyping. The method of any one of claims 204 to 238, further wherein one or more anti-inflammatory agents are administered to the individual. The method of claim 239, wherein the one or more anti-inflammatory agents are administered concurrently. The method of claim 239, wherein the one or more anti-inflammatory agents are administered separately. The method of claim 241, wherein the one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before the one or more anti-inflammatory agents. The method of any of claims 239, 241 and 242, wherein the one or more anti-inflammatory agents are administered before and after the cell preparation or formulation. The method of any one of claims 204 to 238, wherein the cell preparation or formulation is administered without administering one or more anti-inflammatory agents. The method of any of claim 243, wherein the one or more anti-inflammatory agents comprises dexamethasone and/or cyclosporine. A population of extracellular vesicles (EVs) secreted by the photosensitive rescue cell composition of any one of claims 1 to 127 and 203. The EV population of claim 246, wherein the EVs secreted by the photosensitive rescue cells express at least one protein selected from the group consisting of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA, DLX5, TUBB3, SCGN, ERBB4, CALB2, NEUROD2, NEUROD6, SLA, NELL2, SATB2, VIM, MKI67, CLU, GLI3, GFAP 、LUCAT1、MIR99AHG、FBXL7、MEIS2、PBX3、GRIA2、CACNA1C、PAX6、LHX2、SIX3、NES、SOX2、ASCL1、RORB、NR2E3、NRL、TUBB3、NFIA、NFIB、OTX2、ELAVL3、ELAVL4、SLC1A2、SLC1A3、HCN1、HES5、AGT、ACBLN2、CDH7、DNAH11、 EGR1, FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, nestin, TRbeta2, MASH1, RORbeta, MAP2, ELAVL3, NFIA, DCX, LHX2 、SLC1A2、ELAVL4、PAX6、EMX2、ASCL1、DLL1、NFIB、ENOX1、TUBB3、MAP2、DCLK1/2、DCX、KALRN、LINC00461、C1orf61、NCAM1、SETBP1、PAK3、AKAP6、RTN1、CRMP1、FOXG1、TRIM2、BACH2、rescuerin、opsin、rhodopsin、rod and cone cGMP phosphodiesterase。 A pharmaceutical composition comprising the EV group as claimed in claim 246 or claim 247 and a pharmaceutically acceptable carrier. A formulation comprising: a) an EV population as in claim 192 or 193, or a pharmaceutical composition as in claim 194; and b) about 4-10% (v/v) of a cryoprotectant, about 2-3% (w/v) of albumin, about 0-1.5% (w/v) of glucose, and a buffer. The formulation of claim 249, wherein the low temperature protectant is selected from DMSO, glycerol and ethylene glycol. The formulation of claim 249 or 250, wherein the cryoprotectant is DMSO. The formulation of any one of claims 248 to 251, wherein the formulation comprises about 4-6% (v/v) cryoprotectant. The formulation of any one of claims 248 to 252, wherein the formulation comprises about 0.08-0.10% (w/v) glucose. The formulation of any one of claims 248 to 253, wherein the formulation comprises about 5% (v/v) DMSO, about 2.5% (w/v) albumin, about 0.09% (w/v) glucose, and a buffer. The formulation of any one of claims 248 to 252, wherein the formulation comprises about 0.6% (w/v) glucose. The formulation of any one of claims 248 to 252 or 255, wherein the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and buffer. The formulation of any one of claims 248 to 256, wherein the albumin is human albumin. The formulation of any one of claims 248 to 257, wherein the albumin is recombinant human albumin. The formulation of any one of claims 248 to 258, wherein the buffer is buffered saline. The formulation of any one of claims 248 to 259, wherein the buffer is phosphate buffered saline (PBS). The formulation of claim 259 or 260, wherein the buffered saline comprises Ca2+ and Mg2+. The formulation of claim 259 or 260, wherein the buffered saline solution does not contain Ca2+ and Mg2+. The formulation of any one of claims 249 to 262, wherein the formulation is stored at low temperature. A method for treating an eye disease in an individual, comprising administering to the individual an EV population as described in claim 246 or 247, a pharmaceutical composition comprising an EV population as described in claim 248, or a formulation comprising an EV population as described in any one of claims 249 to 263. A method for increasing the secretion of neuroprotective factors by the eye of an individual, comprising administering to the individual an EV population as described in claim 246 or 247, a pharmaceutical composition comprising an EV population as described in claim 248, or a formulation comprising an EV population as described in any one of claims 249 to 263. The method of claim 265, wherein the method increases the secretion of the neuroprotective factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the secretion of the neuroprotective factor before administration. A method for improving visual acuity in an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as claimed in claim 246 or 247, a pharmaceutical composition comprising an EV population as claimed in claim 248, or a formulation comprising an EV population as claimed in any one of claims 249 to 263. The method of claim 267, wherein the method increases visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to visual acuity prior to administration. The method of claim 267 or 268, wherein visual acuity is measured based on optomotor response (OMR) and/or electroretinogram (ERG). The method of claim 269, wherein the spatial frequency threshold of the treated eye is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the spatial frequency threshold before administration as measured by OMR. A method as claimed in claim 269 or 270, wherein the glimmer b-wave amplitude of the treated eye is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the glimmer b-wave amplitude before administration as measured by ERG. A method for preventing or slowing photoreceptor cell loss in an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as claimed in claim 246 or 247, a pharmaceutical composition comprising an EV population as claimed in claim 248, or a formulation comprising an EV population as claimed in any one of claims 249 to 263. The method of claim 272, wherein the prevention or reduction of photoreceptor cell loss is measured based on the expression of CNFT. The method of claim 273, wherein the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to CNFT expression in the eye prior to administration. A method for increasing phagocytic activity in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as in claim 246 or 247, a pharmaceutical composition comprising an EV population as in claim 248, or a formulation comprising an EV population as in any one of claims 249 to 263. The method of claim 275, wherein the method increases the phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the phagocytic activity before administration. A method for inhibiting microglial cell activation in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as claimed in claim 246 or 247, a pharmaceutical composition comprising an EV population as claimed in claim 248, or a formulation comprising an EV population as claimed in any one of claims 249 to 263. The method of claim 277, wherein inhibition of microglial cell activation is measured based on expression of CNFT and/or MIF. The method of claim 278, wherein the method increases CNFT performance by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to CNFT performance before administration. The method of claim 277 or claim 278, wherein the method increases the expression of MIF by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of MIF before administration. A method for reducing oxidative stress in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as claimed in claim 246 or 247, a pharmaceutical composition comprising an EV population as claimed in claim 248, or a formulation comprising an EV population as claimed in any one of claims 249 to 263. The method of claim 281, wherein the reduction of oxidative stress is measured based on the expression of CNFT. The method of claim 282, wherein the method increases CNFT performance by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to CNFT performance before administration. A method for increasing the expression of anti-apoptotic factors in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as described in claim 246 or 247, a pharmaceutical composition comprising an EV population as described in claim 248, or a formulation comprising an EV population as described in any one of claims 249 to 263. The method of claim 284, wherein the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of the anti-apoptotic factor before administration. The method of claim 284 or 285, wherein the anti-apoptotic factor is S100B. A method for preventing degeneration of the outer nuclear layer (ONL) in the eye of an individual suffering from a retinal disease or disorder, comprising administering to the individual an EV population as claimed in claim 246 or 247, a pharmaceutical composition comprising an EV population as claimed in claim 248, or a formulation comprising an EV population as claimed in any one of claims 249 to 263. The method of any one of claims 264 to 287, wherein the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, secondary map atrophy of macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber's congenital amaurosis, or Stargardt's disease. The method of claim 288, wherein the eye disease is macular degeneration or retinitis pigmentosa. The method of any one of claims 264 to 289, wherein the disease is a retinal degenerative disease. The method of any of claims 264 to 290, wherein the disease is associated with loss of photoreceptor cells. The method of claim 291, wherein the disease is associated with loss of photoreceptor cells in the extranuclear layer of the retina. A method as in any one of claims 264 to 292, wherein the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population is administered to the subretinal space or supracortal space of the individual. A method as in any one of claims 264 to 293, wherein the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population is administered by injection or implantation. The method of claim 294, wherein the injection is administered intraocularly. The method of claim 294 or 295, wherein the intraocular administration comprises injecting an aqueous solution, an isotonic solution and/or a saline solution into the subretinal space to form an anterior hydatid; and removing the aqueous solution, and then administering the photoreceptor rescue cell composition in the form of the aqueous solution into the same subretinal space. A method as in any of claims 287 to 296, wherein the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population is administered within at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years of symptom onset. The method of any one of claims 287 to 297, further wherein one or more anti-inflammatory agents are administered to the subject. The method of claim 298, wherein the one or more anti-inflammatory agents are administered concurrently with the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population. The method of claim 298, wherein the one or more anti-inflammatory agents are administered separately from the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population. The method of claim 300, wherein 1) the one or more anti-inflammatory agents are administered before the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population, or 2) the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population is administered before the one or more anti-inflammatory agents. A method as in any of claims 298, 300 and 301, wherein the one or more anti-inflammatory agents are administered before and after administration of the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population. A method as in any of claims 287 to 297, wherein the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population is administered without administering one or more anti-inflammatory agents. The method of claim 303, wherein the one or more anti-inflammatory agents comprises dexamethasone and/or cyclosporine.