WO2024206911A2

WO2024206911A2 - Clinical-grade organoids

Info

Publication number: WO2024206911A2
Application number: PCT/US2024/022355
Authority: WO
Inventors: Masaki Kimura; Takanori TAKEBE
Original assignee: Cincinnati Childrens Hospital Medical Center
Current assignee: Cincinnati Childrens Hospital Medical Center
Priority date: 2023-03-30
Filing date: 2024-03-29
Publication date: 2024-10-03
Anticipated expiration: 2025-09-30
Also published as: EP4689067A2; WO2024206911A3; KR20250165651A; CN121013904A

Abstract

Disclosed herein are improved methods of expanding cell populations and methods of producing organoids from pluripotent stem cells, as well as compositions and uses thereof, compositions including an amino acid supplemented liquid component which can be used for performing the methods, and kits including components for performing the methods and/or using the products of the methods. These methods may be performed without the need for xenogeneic and undefined basement membrane matrices for cell culture, which enables good manufacturing practice (GMP) compliant approaches for producing organoids for various uses such as clinical screening and therapeutics, including methods for treating a liver-related disease or disorder and screening candidate compounds or compositions for treating the same.

Description

CLINICAL-GRADE ORGANOIDS

STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with government support under UH3 DK119982-02 and DP2 DK128799-01 awarded by the National Institutes of Health. The government has certain rights to the invention.

CROSS REFERENCE TO RELATED APPLICATION

[0002] The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/493,269, entitled “CLINICAL-GRADE ORGANOIDS”, filed on March 30, 2023, which is incorporated by reference in its entirety.

FIELD

[0003] Aspects of the present disclosure relate generally to organoid compositions and methods of making the same. These methods may be performed without the use of xenogeneic basement membrane matrices during organoid differentiation and culture. Also disclosed herein are methods of cryopreserving organoids for long term storage and later use.

BACKGROUND

[0004] The culture of pluripotent stem cells and associated downstream cell types, including organoid compositions, commonly involve the use of a basement membrane matrix (also referred to as extracellular matrix), which provides a biological niche including extracellular proteins and growth factors that support cellular growth. A popular basement membrane matrix that is used for biological research is the extracellular matrix produced by murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, this basement membrane matrix is incompatible for human clinical purposes as it is composed of xenogeneic mouse components, is undefined (i.e. variability between batches), and may harbor pathogens.

[0005] Accordingly, there is a need for the ability to produce organoid compositions without the use of basement membrane matrices and other compounds involved in organoid production that are not derived from xenogeneic sources.

SUMMARY [0006] Embodiments of the disclosure include methods for expanding posterior foregut cells and/or posterior foregut endoderm cells, the methods including: a) dissociating a foregut endoderm cell monolayer to posterior foregut cells and/or posterior foregut endoderm cells; b) seeding the posterior foregut cells and/or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and/or posterior foregut endoderm cells with a TGF-b pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

[0007] In some embodiments, the foregut endoderm cell monolayer can be dissociated to the posterior foregut cells and/or posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be seeded onto the tissue container surface at a cell density of, or of about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities.

[0008] In some embodiments, wherein the tissue culture surface is coated with a basement membrane matrix or component thereof. In some embodiments, the basement membrane matrix or component thereof does not include non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or component thereof includes human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

[0009] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be cultured until three-dimensional (3D) spheroids can be formed spontaneously, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days.

[0010] In some embodiments, the TGF-b pathway inhibitor can be selected from A83-01, RepSox, EY365947, and SB431542, optionally A83-01. In some embodiments, the TGF-b pathway inhibitor can be provided at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the TGF-b pathway inhibitor is provided at a concentration of, or of about, 500 nM. In some embodiments, the FGF pathway activator can be selected from FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally FGF2. In some embodiments, the FGF pathway activator can be provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is provided at a concentration of, or of about, 5 ng/mL. In some embodiments, the Wnt pathway activator can be selected from Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119, optionally CHIR99021. In some embodiments, the Wnt pathway activator can be provided at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is provided at a concentration of, or of about, 3 pM. In some embodiments, the VEGF pathway activator can be selected from the group consisting of VEGF or GS4012, optionally VEGF. In some embodiments, the VEGF pathway activator can be provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is provided at a concentration of, or of about, 10 ng/mL.

[0011] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of step c) can be cultured in a media that further includes EGF, or cultured in a media that does not include EGF. In some embodiments, the EGF can be provided at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the EGF is provided at a concentration of, or of about, 20 ng/mL. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of step c) can be cultured in a media that further includes ascorbic acid, or cultured in a media that does not include ascorbic acid. In some embodiments, the ascorbic acid can be provided at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the ascorbic acid is provided at a concentration of, or of about, 50 pg/mL.

[0012] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of step c) can be cultured in a media that further includes a ROCK inhibitor, or cultured in a media that does not include the ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor can be provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally where the ROCK inhibitor is provided at a concentration of, or of about, 10 pM.

[0013] In some embodiments, the cells of step c) can be passaged one or more times, passaging In some embodiments, the cells of step c) can be passaged until the posterior foregut cells and/or posterior foregut endoderm cells do not form spheroids spontaneously. In some embodiments, the cells of step c) can be passaged not more than 3 times.

[0014] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be cultured, and the posterior foregut cells and/or posterior foregut endoderm cells can differentiated to liver organoids. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be cultured until three-dimensional (3D) spheroids are formed spontaneously, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells, and the posterior foregut cells and/or posterior foregut endoderm cells can be collected from the spheroids, optionally further comprising dissociating the spheroids into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be collected from the foregut endoderm cell monolayer by dissociating the foregut endoderm cell monolayer into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step.

[0015] Further embodiments of the disclosure include methods of differentiating posterior foregut cells and/or posterior foregut endoderm cells to liver organoids, the methods including: i) contacting posterior foregut cells and/or posterior foregut endoderm cells, optionally in the form of spheroids, optionally in the form of individual cells or cell clusters dissociated from spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells, and/or optionally cells aggregated in a microwell or other apparatus as described herein, with a retinoic acid pathway activator; and ii) contacting the cells of step i) with a medium for a period of time thereby differentiating the posterior foregut cells and/or posterior foregut endoderm cells to liver organoids, optionally wherein the medium is hepatocyte culture medium.

[0016] In some embodiments, the medium can be supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid. In some embodiments, the cMET tyrosine kinase receptor agonist can be selected from hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine can be selected from IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC). In some embodiments, the corticosteroid can be selected from a group consisting of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and mometasone. In some embodiments, the medium can be supplemented with HGF, OSM, and dexamethasone. In some embodiments, the medium can be supplemented with dexamethasone.

[0017] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can include the posterior foregut cells and/or posterior foregut endoderm cells produced by the aforementioned methods. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be in the form of spheroids or individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells derived from dissociating the spheroids, , optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

[0018] In some embodiments, the retinoic acid pathway activator can be selected from retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally retinoic acid. In some embodiments, the retinoic acid pathway activator can be provided at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator can be provided at a concentration of, or of about, 2.0 pM. In some embodiments, the HGF can be provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the HGF is provided at a concentration of, or of about 10 ng/mL. In some embodiments, the OSM is provided at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the OSM is provided at a concentration of, or of about 20 ng/mL. In some embodiments, the dexamethasone can be provided at a concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone is provided at a concentration of, or of about 100 nM. In some embodiments, the cells of step i) and/or step ii) are not contacted with EGF.

[0019] In some embodiments, the cells of step ii) can be cultured in a growth medium supplemented with non-essential amino acids, essential amino acids, and glycine. In some embodiments, the growth media after supplementation includes 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% non-essential amino acids by total volume, or a range defined by any two to the preceding values, optionally wherein the growth medium after supplementation is about 4- 10%, 6- 12%, 10-16%, 12-15%, 13-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% non- essential amino acids by total volume. In some embodiments, the growth medium after supplementation includes 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or a range defined by any two to the preceding values, optionally wherein the growth medium after supplementation is about 4-10%, 6-12%, 10-16%, 12-15%, 13-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% essential amino acids by total volume. In some embodiments, the supplemented glycine can be provided at a concentration of, or of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the supplemented glycine is provided at a concentration of, or of about, 18-22 mg/mL or 20 mg/mL.

[0020] In some embodiments, the cells of step ii) can be further contacted with a low/first concentration of bilirubin, wherein the liver organoids that are formed are mature liver organoids. In some embodiments, the low/first concentration of bilirubin can be a human fetal physiological concentration of bilirubin. In some embodiments, the low/first concentration of bilirubin can be, can be about, can be less than, or can be less than about: a) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or b) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.

[0021] In some embodiments, the mature liver organoids can exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, the mature liver organoids can express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoids can express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoids can express CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, the mature liver organoids can exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with the low/first dose of bilirubin.

[0022] In some embodiments, the cells of step ii) can be further contacted with a high/second concentration of bilirubin, wherein the liver organoids that are formed are hyperbilirubinemia liver organoids. In some embodiments, the high/second concentration of bilirubin can be, can be about, can be more than, or can be more than about: a) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L. In some embodiments, the hyperbilirubinemia liver organoids can express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

[0023] In some embodiments, the liver organoids can include a functional L- gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the liver organoids are able to synthesize ascorbate. In some embodiments, the functional GULO protein can be murine GULO (mGULO). In some embodiments, the gene that encodes for the functional GULO protein can be conditionally expressed, optionally using a tetracycline inducible system. In some embodiments, the liver organoids can be engineered with the gene that encodes for the functional GULO protein using CRISPR. In some embodiments, the gene or mRNA, or both, that encodes for the functional GULO protein can be introduced to the liver organoids by transfection. In some embodiments, the liver organoids including the functional GULO protein can express increased levels of NRF2 relative to liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids including the functional GULO protein can express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids that do not include the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate. In some embodiments, the liver organoids including the functional GULO protein exhibits reduced caspase-3 activity relative to liver organoids that do not include the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate. In some embodiments, the liver organoids including the functional GULO protein can express increased levels of ALB relative to liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids including the functional GULO protein can resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal liver markers can include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, the liver organoids including the functional GULO protein can exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids including the functional GULO protein can exhibit increased bilirubin conjugation activity relative to liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids including the functional GULO protein can exhibit increased viability in culture relative to liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids have been differentiated from pluripotent stem cells including a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate. In some embodiments, the liver organoids include an inactive UGT1A1 gene, wherein the liver organoids can be used as a model for Crigler-Najjar Syndrome.

[0024] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be aggregated in a microwell or other apparatus (e.g., Aggrewell) prior to step i), wherein aggregating the posterior foregut cells and/or posterior foregut endoderm cells can result in more uniformly sized liver organoids. In some embodiments, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof that is xenogeneic to humans, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of step i) and/or step ii) are not contacted with Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the cells of step i) and/or step ii), and/or the liver organoids formed therefrom, can be cultured in a static or non- static bioreactor, optionally a rotational bioreactor, optionally a 3D bioreactor, optionally a 3D rotational bioreactor. In some embodiments, after culturing in a static or non-static bioreactor, the liver organoids can be dissociated into single cells, and can be subsequently reconstructed and/or expanded via an additional culturing step in a static or non-static bioreactor, optionally a 3D bioreactor, optionally a 3D rotational bioreactor. In some embodiments, the methods further include cryopreserving the liver organoids. In some embodiments, cryopreserving the liver organoids includes slow-freezing or vitrification cryopreservation, optionally wherein the liver organoids can be cryopreserved with chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

[0025] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells have been derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells have been derived from a subject, optionally a subject having a liver-related disease or disorder. In some embodiments, the method can be used in a good manufacturing practice (GMP) compliant process. Embodiments of the disclosure further include the posterior foregut cells and/or posterior foregut endoderm cells or liver organoids produced by any of the aforementioned methods.

[0026] Further embodiments of the disclosure include in vitro compositions including: pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, and/or downstream liver cell types, and at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

[0027] In some embodiments, the composition includes posterior foregut cells and/or posterior foregut endoderm cells, and the posterior foregut cells and/or posterior foregut endoderm cells are dissociated posterior foregut cells and/or posterior foregut endoderm cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells can be at a cell density of greater than or equal to, exactly or about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities. In some embodiments, the tissue culture surface can be coated with a basement membrane matrix or component thereof. In some embodiments, the basement membrane matrix or component thereof does not include non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or component thereof includes human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

[0028] In some embodiments, at least a portion of the posterior foregut cells and/or posterior foregut endoderm cells can be spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, the TGF-b pathway inhibitor can be selected from A83-01, RepSox, LY365947, and SB431542; optionally wherein the TGF-b pathway inhibitor includes or is A83-01. In some embodiments, the TGF-b pathway inhibitor can be at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the TGF-b pathway inhibitor is at a concentration of, or of about, 500 nM. In some embodiments, the FGF pathway activator can be selected from FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally wherein the FGF pathway activator includes or is FGF2. In some embodiments, the FGF pathway activator can be at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mE, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is at a concentration of, or of about, 5 ng/mE. In some embodiments, the Wnt pathway activator can be selected from Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BME 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119, optionally wherein the Wnt pathway activator includes or is CHIR99021. In some embodiments, Wnt pathway activator can be at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is at a concentration of, or of about, 3 pM. In some embodiments, the VEGF pathway activator can be selected from VEGF or GS4012, optionally wherein the VEGF pathway activator includes or is VEGF. In some embodiments, the VEGF pathway activator can be at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is at a concentration of, or of about, 10 ng/mL. In some embodiments, the composition further includes exogenous EGF, or the composition does not include exogenous EGF. In some embodiments, the EGF can be at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the EGF is at a concentration of, or of about, 20 ng/mL. In some embodiments, the composition further includes exogenous and/or transgenically produced ascorbic acid, or the composition does not include exogenous and/or transgenically produced ascorbic acid. In some embodiments, the ascorbic acid can be at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the ascorbic acid is at a concentration of, or of about, 50 pg/mL. In some embodiments, the compositions can further include a ROCK inhibitor, or can be cultured in a media that does not include the ROCK inhibitor, optionally wherein the ROCK inhibitor includes or is Y-27632. In some embodiments, the ROCK inhibitor can be at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally where the ROCK inhibitor is at a concentration of, or of about, 10 pM.

[0029] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, ventral foregut endoderm, and/or downstream liver cells can be differentiated from stem cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, ventral foregut endoderm, and/or downstream liver cells can be differentiated from induced pluripotent stem cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, ventral foregut endoderm, and/or downstream liver cells have been passaged less than 4 times. In some embodiments, the cells include or consist essentially of posterior foregut cells and/or posterior foregut endoderm cells. In some embodiments, the TGF-b pathway inhibitor can be A83-01, the FGF pathway activator can be FGF2, the Wnt pathway activator can be CHIR99021, the VEGF pathway activator can be VEGF, and the ROCK inhibitor can be Y-27632.

[0030] Further embodiments of the disclosure include liver organoids produced by any of the aforementioned methods.

[0031] Further embodiments of the disclosure include in vitro compositions including: a) posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids, and b) a medium, wherein the medium optionally includes hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid, and wherein the composition optionally additionally includes c) a retinoic acid pathway activator. In some embodiments, the cMET tyrosine kinase receptor agonist can be selected from hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine can be selected from IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardio trophin-like cytokine (CLC). In some embodiments, the corticosteroid can be selected from dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and mometasone. In some embodiments, the medium can be supplemented with HGF, OSM, and dexamethasone. In some embodiments, the medium can be supplemented with dexamethasone. In some embodiments, the retinoic acid pathway activator can be selected from retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally wherein the retinoic acid pathway activator includes or is retinoic acid. In some embodiments, the retinoic acid pathway activator can be at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator is at a concentration of, or of about, 2.0 pM. In some embodiments, the HGF can be at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the HGF is at a concentration of, or of about 10 ng/mL. In some embodiments, the OSM can be at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the OSM is at a concentration of, or of about 20 ng/mL. In some embodiments, the dexamethasone can be at a concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone is at a concentration of, or of about 100 nM. In some embodiments, the composition does not include exogenous EGF.

[0032] In some embodiments, the composition further includes a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin can be at or near a human fetal physiological concentration of bilirubin. In some embodiments, the bilirubin can be, can be about, can be less than, or can be less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L. In some embodiments, the composition includes mature liver organoids, and the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, the mature liver organoids can express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid not contacted with a low dose of bilirubin. In some embodiments, the mature liver organoids can express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low dose of bilirubin. In some embodiments, the mature liver organoids can express CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, the mature liver organoids can exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with a low dose of bilirubin.

[0033] Further embodiments of the disclosure include in vitro compositions including mature liver organoids, wherein the cells of the mature liver organoid were contacted with a low dose of bilirubin, optionally wherein the low dose of bilirubin was exogenously provided, and the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, the mature liver organoids can express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin. In some embodiments, the mature liver organoids can express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin. In some embodiments, the mature liver organoids can express CYP2E1, CYP7A1, PR0X1, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, the mature liver organoids can exhibit increased CYP3A4 and CYP1A2 activity, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

[0034] In some embodiments, the compositions further include hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells were contacted with a high and/or second concentration of bilirubin. In some embodiments, the high/second concentration of bilirubin was, was about, was more than, or was more than about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L. In some embodiments, the hyperbilirubinemia liver organoids can express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids include a functional L- gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are able to synthesize ascorbate. In some embodiments, the functional GULO protein is murine GULO (mGULO). In some embodiments, the gene that encodes for the functional GULO protein can be conditionally expressed, optionally using a tetracycline inducible system. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids can be engineered to include a gene that encodes for the functional GULO protein using CRISPR. In some embodiments, the gene or mRNA, or both, that encodes for the functional GULO protein was introduced to the liver organoids by transfection. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can express increased levels of NRF2 relative to liver organoids and/or mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids and/or mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids include the functional GULO protein can exhibit reduced caspase-3 activity relative to liver organoids and/or mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can express increased levels of ALB relative to liver organoids and/or mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal liver markers can include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids an/dor mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can exhibit increased bilirubin conjugation activity relative to liver organoids and/or mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids including the functional GULO protein can exhibit increased viability in culture relative to liver organoids and/or mature liver organoids that do not include the functional GULO protein. In some embodiments, the liver organoids and/or mature liver organoids have been differentiated from pluripotent stem cells including a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.

[0035] Further embodiments of the disclosure include methods of administering the aforementioned liver organoids or compositions to a subject in need thereof, and methods for treating a liver-related disease or disorder in a subject in need thereof, including administering one or more of the aforementioned liver organoids or compositions to the subject.

[0036] In some embodiments, the liver organoid has been produced from cells derived from the subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells. In some embodiments, administering includes transplanting the liver organoid or composition into the subject. In some embodiments, the liver-related disease or disorder includes one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug- induced cholestasis, metabolic disease, autoimmune liver disease, Wilson’s disease, metabolic- associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, metabolic dysfunction-associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, hepatic cyst, acetaminophen acute liver injury, and/or alcohol-associated liver disease. In some embodiments, the liver dysfunction and/or failure includes hyperammonemia and/or hyperbilirubinemia; or the metabolic disease includes nonalcoholic fatty liver disease (NAFLD); or the nonalcoholic fatty liver disease (NAFLD) includes metabolic dysfunction-associated steatohepatitis (MASH); or the hepatitis includes hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and/or autoimmune hepatitis. In some embodiments, the subject can have reduced serum bilirubin and/or ammonia levels, and/or increased serum protein albumin following transplantation. In some embodiments, the subject can have improved symptoms of biliary stricture and/or liver regeneration following transplantation. In some embodiments, the subject can have an increased survival rate following transplantation. In some embodiments, the liver organoid engrafts onto the liver of the subject. In some embodiments, the liver organoid has been treated with amino acid (AA) supplementation. In some embodiments, the liver organoid has been treated with amino acid (AA) supplementation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to transplantation. In some embodiments, the liver-related disease or disorder includes acetaminophen acute liver injury. In some embodiments, the method can be used in a good manufacturing practice (GMP) compliant process.

[0037] Further embodiments of the disclosure include methods for screening, including contacting the aforementioned liver organoid with a candidate compound or composition, and assessing the effects of the candidate compound or composition on the liver organoid. In some embodiments, the liver organoid can be a model for a liver-related disease or disorder, and determining the effects of the candidate compound or composition on the liver organoid includes assessing the effects of the candidate compound or composition on the liver- related disease or disorder. In some embodiments, the liver organoid has been produced from cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject has a liver-related disease or disorder. In some embodiments, the method can be used in a good manufacturing practice (GMP) compliant process. [0038] Further embodiments of the disclosure include compositions including an amino acid supplemented liquid component according to Table 3. Additional embodiments include compositions including a cocktail of growth factors according to an embodiment of Table 1 or Table 2.

[0039] Further embodiments of the disclosure include compositions including an amino acid supplemented liquid component comprising exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% non-essential amino acid solution by volume (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL glycine. In some embodiments, the compositions include an amino acid supplemented liquid component comprising exactly or about 14% non-essential amino acid solution (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 6% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 80% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 20 g/L glycine. In some embodiments, the pH is between about pH 6-8, or pH 6.5-7.5, or exactly or about pH 7.0. In some embodiments, the compositions further include hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid. In some embodiments, the compositions further include hepatic lineage committed cells differentiated from definitive endoderm cells using retinoic acid. In some embodiments, the hepatic lineage committed cells are characterized as liver organoids. [0040] In some embodiments, the liver organoids can be characterized as secreting increased levels of albumin and urea relative to liver organoids included in HCM without amino acid supplementation. In some embodiments, the liver organoids can be characterized as expressing increased levels of hepatic maturation associated gene expression relative to liver organoids included in HCM without amino acid supplementation. In some embodiments, the liver organoids can be characterized as expressing reduced levels of Vimentin relative to liver organoids included in HCM without amino acid supplementation.

[0041] In some embodiments, the composition does not include non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans. In some embodiments, the composition does not include murine Engelbreth-Holm- Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and/or Geltrex®.

[0042] Further embodiments of the disclosure include, in vitro hyperbilirubinemia liver organoids including a naturally occurring and/or engineered mutation in the UDP glucuronosyltransferase family 1 member Al UGT1A1) gene. In some embodiments, the hyperbilirubinemia liver organoid was produced through at least two rounds of contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids to exogenous bilirubin. In some embodiments, the hyperbilirubinemia liver organoid was clonally derived and/or derived from iPSCs.

[0043] Further embodiments of the disclosure include cryopreserved compositions, including liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT), and/or cryopreserved compositions, including mature liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT), and/or cryopreserved compositions, including hyperbilirubinemia liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

[0044] Further embodiments of the disclosure include kits including means for conducting any of the aforementioned methods; and/or kits including the compositions or means for generating any of the aforementioned compositions; and/or kids for generating any of the aforementioned liver organoids. Further embodiments of the disclosure include uses of the methods, compositions, or kits, as a medicament, means for treatment and/or prevention of a disease, means of diagnosis, and/or medical research.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In addition to the features described herein, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict embodiments and are not intended to be limiting in scope.

[0046] FIG. 1A depicts an embodiment of a schematic for cryopreservation of liver organoids and optional subsequent steps.

[0047] FIG. IB depicts an embodiment of fluorescent microscope images of liver organoids that have been frozen using slow-freezing and vitrification cryopreservation approaches and thawing (compared with non-frozen organoids) to observe the abundance of live (labeled with Calcein AM) and dead cells (labeled with ethidium homodimer- 1).

[0048] FIG. 1C depicts an embodiment of quantification of albumin secretion by liver organoids that have been frozen using slow-freezing and vitrification cryopreservation approaches and thawing (compared with non-frozen organoids).

[0049] FIG. 2A depicts an embodiment of RT-qPCR quantification of expression of ALB, CYP3A4, PCK1 and G6PC in liver organoids cultured with amino acid (AA) supplementation medium added at various times of culture.

[0050] FIG. 2B depicts an embodiment of quantification of albumin secretion and urea production in liver organoids cultured with AA supplementation medium added at various times of culture.

[0051] FIG. 2C depicts an embodiment of a schematic for detecting activity, such as PCK1 activity, using a luciferase reporter approach with either live cells or cell lysate, in liver organoids grown with AA supplementation medium.

[0052] FIG. 2D depicts an embodiment of bright field microscopy images of organoids grown in AA supplementation medium and standard hepatocyte culture medium (HCM).

[0053] FIG. 2E depicts an embodiment of quantification of PCK1 expression as measured by a luciferase reporter in liver organoids grown in AA supplementation medium compared to those grown in standard hepatocyte culture medium in both live cell and cell lysate assays.

[0054] FIG. 3A depicts an embodiment of a schematic for two-dimensional (2D) hepatocyte differentiation from pluripotent stem cells and culture using AA supplementation medium added at various times of culture.

[0055] FIG. 3B depicts an embodiment of brightfield images of 2D hepatocyte cultures grown with AA supplementation medium added at various times of culture. [0056] FIG. 3C depicts an embodiment of quantification of lactate production in 2D hepatocytes grown in AA supplementation medium compared to those grown in standard hepatocyte culture medium.

[0057] FIG. 3D depicts an embodiment of quantification of albumin secretion in 2D hepatocytes grown in AA supplementation medium added at various times of culture compared to those grown in standard hepatocyte culture medium.

[0058] FIG. 3E depicts an embodiment of quantification of albumin secretion in 2D hepatocytes grown in AA supplementation medium for extended periods of time, where the rate of albumin secretion is normalized to the rate of hepatocytes grown in standard hepatocyte culture medium.

[0059] FIG. 3F depicts an embodiment of RT-qPCR quantification of gene expression of ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplementation medium added at various times of culture compared to those grown in standard hepatocyte culture medium.

[0060] FIG. 3G depicts an embodiment of fluorescence and brightfield microscopy images of 2D hepatocytes engineered to express mScarlet under a PCK1 reporter grown in AA supplementation medium with or without insulin starvation compared to those grown in standard hepatocyte culture medium with or without insulin starvation.

[0061 ] FIG. 3H depicts an embodiment of fluorescence microscopy images detecting expression of EpCAM (epithelial cells), Vimentin (mesenchymal cells) and DAPI or HNF4a (nuclei) in 2D hepatocytes grown in AA supplementation medium compared to those grown in standard hepatocyte culture medium.

[0062] FIG. 4A depicts an embodiment of a schematic for culturing liver organoids without the use of Matrigel® or other basement membrane matrix with xenogeneic components.

[0063] FIG. 4B depicts an embodiment of brightfield images showing spontaneous formation of 3D spheroids from a foregut cell monolayer, where the spheroids can be transferred to mature into liver organoids without the use of Matrigel® or other basement membrane matrix with xenogeneic components.

[0064] FIG. 4C depicts an embodiment of brightfield and fluorescence microscopy images showing that organoids grown in Matrigel®-free conditions exhibit normal organoid morphology. [0065] FIG. 4D depicts an embodiment of quantification of albumin secretion compared between organoids grown in Matrigel®-free conditions, organoids grown in Matrigel® conditions according to previous protocols, and 2D hepatocyte culture.

[0066] FIG. 4E depicts an embodiment of RT-qPCR quantification of expression of ALB, AFP, HNF4a, RBP4, and AAT between organoids grown in Matrigel®-free conditions, organoids grown in Matrigel® conditions according to previous protocols, and 2D hepatocyte culture.

[0067] FIG. 5A depicts an embodiment of a schematic for passaging and expansion of foregut cells after pluripotent stem cell differentiation for scaling organoid production.

[0068] FIG. 5B depicts an embodiment of brightfield microscopy images showing growth of foregut cells (days 1-7) and spontaneous formation of spheroids when plated on laminin or Matrigel®-coated plates.

[0069] FIG. 5C depicts an embodiment of brightfield microscopy images showing further growth of foregut cells (days 8-10) and full formation of spheroids, where cells plated on laminin-coated plates appear to result in more efficient spheroid formation compared to cells plated on Matrigel®-coated plates.

[0070] FIG. 5D depicts an embodiment of brightfield microscopy images showing growth of foregut cells (days 1-5) and spontaneous formation of spheroids when plated on laminin-coated plates at different seed densities.

[0071] FIG. 5E depicts an embodiment of brightfield microscopy images showing that when spheroids are collected from the initial spontaneous formation from foregut cells, additional spheroids arise from additional culture.

[0072] FIG. 5F depicts an embodiment of brightfield microscopy images and quantification of total potential cell number obtainable through multiple passages of foregut cells from an initial differentiation from pluripotent stem cells. Spheroids form during passages 1-3 and did not form from passage 4 foregut cells.

[0073] FIG. 5G depicts an embodiment of RT-qPCR quantification of expression of CDX2, FOXA2, AFP, VIM, SOX17, HNF4a, and ALB in foregut cells from passages 1-4.

[0074] FIG. 5H depicts an embodiment of a schematic for liver organoid formation starting from pluripotent stem cells and including foregut cell passaging for scaling.

[0075] FIG. 51 depicts an embodiment of a schematic for liver organoid formation starting from pluripotent stem cells and including foregut cell passaging for scaling and the use of an apparatus to aggregate foregut cells for improved uniformity in organoid size and shape. [0076] FIG. 6A depicts an embodiment of a schematic for 3D rotational culture of liver organoids grown under Matrigel®-free conditions.

[0077] FIG. 6B depicts an embodiment of brightfield images showing that liver organoids can be grown under Matrigel®-free conditions using 3D rotational culture.

[0078] FIG. 6C depicts an embodiment of RT-qPCR quantification of AFP, HNF4a, FOXA2, ALB, CDX2, and VIM in liver organoids grown in 3D rotational culture compared to passage 1 foregut cells.

[0079] FIG. 7A depicts an embodiment of a schematic for generation of HLOs and maturation with low dose bilirubin.

[0080] FIG. 7B depicts an embodiment of brightfield images of HLOs treated with low dose bilirubin (1 mg/L) compared to control, and luminal outline using ImageJ, where arrows indicate luminal projections that are similar to bile canaliculi found in human liver.

[0081] FIG. 7C depicts an embodiment of comparison of size and circularity of the lumen of the control and 1 mg/L bilirubin treated HLOs.

[0082] FIG. 7D depicts an embodiment of RT-qPCR of immature and maturation marker genes ALB, NANOG, SLC4A2, HO-1, AFP, and CDX2) for control organoids and organoids treated with 1 mg/L of bilirubin compared to a human liver sample.

[0083] FIG. 7E depicts an embodiment of CYP3A4 and CYP1A2 activity assays in response to rifampicin and omeprazole in control and 1 mg/L bilirubin treated HLOs (RLU: relative light units; CTG: CellTiter-Glo assay).

[0084] FIGs. 7F-H depict embodiments of immunofluorescence of mature liver enzymes and transport proteins in 1 mg/L bilirubin treated liver organoids. FIG. 7F depicts detection of CYP2E1 and MRP3. FIG. 7G depicts detection of CYP7A1 and MRP1. FIG. 7H depicts detection of PROXI and OATP2.

[0085] FIG. 8A depicts an embodiment of a brightfield image of HLOs with ascorbic acid depletion at day 15 compared to control.

[0086] FIG. 8B depicts an embodiment of an exemplary workflow for the generation of mGULO iPSCs.

[0087] FIG. 8C depicts an embodiment of a linear map of the synthetic mGULO- mCherry gene designed for the expression of mGULO under the doxycycline activated TetOn system in hiPSCs and vector map of the pAAVSl-Ndi-CRISPRi (Genl) plasmid used to clone the mGULO gene into the hiPSCs.

[0088] FIG. 8D depicts an embodiment of a schematic for the generation of HLOs using iPSCs modified to express the TetOn mGULO gene. [0089] FIG. 8E depicts an embodiment of brightfield and fluorescence images of mCherry expression in doxycycline (Dox) treated mGULO HLOs compared to control HLOs.

[0090] FIG. 8F depicts an embodiment of brightfield and fluorescence images of mCherry expression in ascorbate depleted mGULO HLOs with or without Dox treatment at day 18.

[0091] FIG. 8G depicts an embodiment of an ELISA for mGULO protein expression and cellular antioxidant concentration in mGULO HLOs treated with Dox (10 or 100 ng/mL) compared to control HLOs.

[0092] FIG. 8H depicts an embodiment of RT-qPCR of inflammatory and detoxification marker genes NRF2, 1L1B, IL6, and TNFa) in ascorbate depleted Dox treated mGULO HLOs compared to ascorbate depleted control or mGULO HLOs.

[0093] FIG. 81 depicts an embodiment of a caspase 3 activity assay for ascorbate depleted Dox treated mGULO HLOs compared to ascorbate depleted control or mGULO HLOs.

[0094] FIG. 8J depicts an embodiment of a heatmap from RNA-seq showing that Dox treated mGULO HLOs express periportal markers compared to control HLOs.

[0095] FIG. 8K depicts an embodiment of gene upregulation categorized by function, showing that periportal pathways are overrepresented in Dox treated mGULO HLOs.

[0096] FIG. 8L depicts an embodiment of brightfield images of Dox treated mGULO and control HLOs with or without 1 mg/L bilirubin treatment, and luminal outline using ImageJ, where arrows indicate luminal projections that are similar to bile canaliculi found in human liver.

[0097] FIG. 8M depicts an embodiment of comparison of size and circularity of the lumen of Dox treated mGULO HLOs or control HLOs with or without 1 mg/L bilirubin treatment.

[0098] FIG. 8N depicts an embodiment of quantification of albumin expression in Dox treated mGULO HLOs or control HLOs with or without 1 mg/L bilirubin treatment.

[0099] FIG. 80 depicts an embodiment of brightfield images of mGULO HLOs treated with bilirubin and Dox at varying concentrations (0, 10, 100, or 1000 ng/mL Dox).

[0100] FIG. 8P depicts an embodiment of CYP3A4 and CYP1A2 activity assays in response to rifampicin and omeprazole in control or Dox treated mGULO HLOs with 1 mg/L bilirubin.

[0101] FIG. 8Q depicts an embodiment of a UnaG assay showing loss of fluorescence indicating conjugation of bilirubin even in the presence of dark yellow bilirubin. [0102] FIG. 8R depicts an embodiment of a UnaG assay for mGULO organoids treated with Dox compared to control.

[0103] FIG. 8S depicts an embodiment of quantification of the total percentage of viable organoids and organoids carrying conjugated bilirubin in Dox treated mGULO organoids compared to control.

[0104] FIG. 9A depicts an embodiment for a schematic for generation of HLOs and treatment with bilirubin at various concentrations.

[0105] FIG. 9B depicts an embodiment of brightfield images of HLOs treated with bilirubin (0-10 mg/L) after 1 and 4 days.

[0106] FIG. 9C depicts an embodiment of RT-qPCR of UGT1A1 and NRF2 genes for organoids treated with varying concentrations of bilirubin compared to untreated organoids.

[0107] FIG. 9D depicts an embodiment of a profile of a patient with Crigler-Najjar Syndrome (CNS) from whom CNS iPSCs were generated. DNA sequencing of the patient revealed a nonsense mutation c.858C>A (p.Cys280X) in the UGT1A1 gene.

[0108] FIG. 9E depicts an embodiment of fluorescence images showing that the CNS iPSCs derived from the patient with Crigler-Najjar Syndrome express canonical pluripotency markers Sox2 and Oct4.

[0109] FIG. 9F depicts an embodiment of brightfield images showing that the CNS iPSCs can be differentiated to definitive endoderm (DE) and liver organoids (hLO) according to standard protocols.

[0110] FIG. 9G depicts an embodiment of fluorescence images showing that liver organoids produced from the CNS iPSCs express the proliferation marker Ki67, liver- specific marker AFP, and epithelial marker ECAD.

[0111] FIG. 9H depicts an embodiment of brightfield images of CNS HLOs treated with bilirubin (10 mg/L) and control (0 mg/L bilirubin) after 1 and 4 days, showing that these HLOs suffer from bilirubin toxicity.

[0112] FIG. 91 depicts an embodiment of brightfield images of CNS HLOs and CNS HLOs that have been transfected with UGT1A1 mRNA at 10 days after treatment with bilirubin (10 mg/L).

[0113] FIG. 9J depicts an embodiment of a bilirubin assay measuring unconjugated (UCB) and conjugated (CB) bilirubin in the HLOs of FIG. 91.

[0114] FIG. 9K depicts an embodiment of a bilirubin assay measuring unconjugated (UCB) and conjugated (CB) bilirubin in mGULO HLOs treated with 10 mg/L bilirubin and Dox (0, 10, 100, or 1000 ng/mL). [0115] FIG. 10A depicts an embodiment of brightfield images of liver organoids treated with 10 mg/L bilirubin and glucocorticoid agonists hydrocortisone (HC; 1 or 5 pM) or dexamethasone (Dex; 1 or 5 pM).

[0116] FIG. 10B depicts an embodiment of a bilirubin assay measuring unconjugated and conjugated bilirubin in the liver organoids of FIG. 10A.

[0117] FIG. 10C depicts an embodiment of brightfield images of liver organoids treated with 10 mg/L bilirubin and glucocorticoid antagonists ketoconazole (KCZ; 1 or 5 pM) or mifepristone (Mif; 1 or 5 pM).

[0118] FIG. 10D depicts an embodiment of a bilirubin assay measuring unconjugated and conjugated bilirubin in the liver organoids of FIG. 10C.

[0119] FIG. 10E depicts an embodiment of RT-qPCR of UGT1A1 and NRF2 genes for organoids treated with 10 mg/L bilirubin compared to organoids treated with 10 mg/L bilirubin and hydrocortisone, dexamethasone, ketoconazole, or mifepristone.

[0120] FIG. 10F depicts an embodiment of a comparison of enriched pathways obtained from RNA sequencing between organoids treated with 10 mg/L bilirubin and 1 pM mifepristone compared to control and a GSEA plot comparing enriched ROS and xenobiotic metabolism.

[0121] FIG. 10G depicts an embodiment of a Venn diagram showing differentially expressed genes in ROS and xenobiotic metabolism.

[0122] FIG. 10H depicts an embodiment of ChlP-PCR and CHIP-qPCR for organoids treated with 10 mg/L bilirubin and either 1 pM mifepristone (Mife) or 1 pM dexamethasone (Dex).

[0123] FIG. 11A depicts an embodiment of a workflow for orthotopic transplantation of HLOs in rodents.

[0124] FIG. 11B depicts an embodiment of an albumin ELISA on blood serum collected from Gunn rats transplanted with mGULO HLOs or sham at different time points after transplantation.

[0125] FIG. 11C depicts an embodiment of a bilirubin assay on the Gunn rats of FIG. 11B after transplantation.

[0126] FIG. 11D depicts an embodiment of AST and ALT assays on the Gunn rats of FIG. 11B after transplantation.

[0127] FIG. 12A depicts an embodiment of an exemplary process for subjecting HLOs induced by conventional methods to expansion culture under conditions with or without Matrigel® in a 3D bioreactor culture. [0128] FIG. 12B depicts an embodiment of HLO growth after 15 days of 3D bioreactor culture, as compared to conventional static cultures.

[0129] FIG. 12C depicts an embodiment of obtaining large HLOs in the 3D bioreactor culture, even in the group without Matrigel® addition.

[0130] FIG. 13 depicts an embodiment of similar expression levels of E-cadherin, Vimentin, and Proxl were observed, indicating that HLOs obtained from 3D bioreactor culture have equivalent properties to those obtained from static culture.

[0131] FIG. 14A depicts an embodiment of dissociating HLOs induced by conventional methods into single cells, and performing passaging and reconstruction of HLOs using a 3D bioreactor.

[0132] FIG. 14B depicts an embodiment of inducing uniform HLOs after 6 days via reconstruction of HLOs in the 3D bioreactor, both with and without Matrigel®.

[0133] FIG. 15A depicts an embodiment of a schematic diagram of a model of acetaminophen acute liver injury rescue by HLO transplantation.

[0134] FIG. 15B depicts an embodiment of an image of an HLO used for transplantation.

[0135] FIG. 15C depicts an embodiment of Kaplan-Meier survival curves for acute liver injury rescue by HLO transplantation.

DETAILED DESCRIPTION

[0136] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Eigures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0137] The following description of various embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.

[0138] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0139] It should be understood that any use of subheadings herein are for organizational purposes, and should not be read to limit the application of those subheaded features to the various embodiments herein. Each and every feature described herein is applicable and usable in all the various embodiments discussed herein and that all features described herein can be used in any contemplated combination, regardless of the specific example embodiments that are described herein. It should further be noted that exemplary description of specific features are used, largely for informational purposes, and not in any way to limit the design, subfeature, and functionality of the specifically described feature.

Overview

[0140] The development of methods of producing human liver organoids from pluripotent stem cells has enabled the study of hepatic function that closely resembles the natural liver. Compared to methods that involve the combination of primary hepatic cells or differentiation from adult multipotent cells, the differentiation of pluripotent stem cells results in liver organoids with rich cell type diversity, including hepatocytes, stellate cells, Kupffer cells, and other epithelial and mesenchymal cell lineages that compose the normal liver.

[0141] However, as the maintenance of pluripotent stem cells and the formation of downstream intermediates is sensitive, previous methods of culture generally involve the use of a basement membrane matrix, which mimics the niche that promotes cells to proliferate. Common basement membrane matrices that are used in a laboratory setting are those isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. While these matrices offer great utility in cell culture, their presence prevents subsequent use in humans, as they contain xenogeneic animal components and may also potentially harbor pathogens. Accordingly, there is an ongoing process of developing cell culture methods that do not involve the use of xenogeneic basement membrane matrices.

[0142] Provided herein are methods for culturing organoids, such as liver organoids, without the use of Matrigel® or other xenogeneic basement membrane matrices. Other improvements for culturing liver organoids, such as supplementation of the growth medium and approaches to expanding cells for larger scale manufacturing beyond the limits of laboratory culture are also disclosed.

[0143] Also disclosed herein, human liver organoids (HLOs), such as those produced according to the methods disclosed herein, are used to model liver-related diseases and

- l- disorders. For example, the HLOs produced according to the methods disclosed herein can be used to model hyperbilirubinemia by treating them with varying concentrations of bilirubin.

[0144] {to be updated with any further independent claim embodiments once claims are agreed/finalized]

[0145] The incidence of liver diseases has increased at an accelerated pace. Neonatal hyperbilirubinemia (NH) is one condition that exacerbates the health of neonates. It affects 60% of all newborns and accounts for 114,000 annual deaths worldwide. Currently, the only treatments for NH involve 12 hours of phototherapy or exchange transfusion, but these can cause other complications. Therefore, efficient and scalable model systems for these liver diseases have now become a necessity to understand the molecular mechanism behind them and develop potential therapies.

[0146] There are currently two main model organisms for modeling NH: the Gunn rats and UGT1A1 knockout mice. However, these models lack key human proteins (OATP family) and epigenetic control involved in bilirubin metabolism. Recent studies have revealed that many aspects of UGT1A1 regulation do not translate well across the species. The genetic regulation of the UGT1A1 gene, which is the rate limiting enzyme in bilirubin metabolism, is significantly different in humans. Gunn rats with UGT deficiency are quite hard to maintain and require special accommodations. In addition, UGT1 KO mouse models display lethal hyperbilirubinemia and only live up to a couple of weeks. Many treatments have been tested with these models, but most have failed.

[0147] Breeding model organisms such as mice and rats takes months of work and planning, and the chance of getting the desired genotype is relatively low. Furthermore, model organisms show high variations in responses to biochemical perturbations over generations. These rodents also run the risk of losing the desired genotype when bred over long periods of time, and also require complex training and procedures to model diseases and evaluate the efficacy of treatments. Thus, there is a critical need to develop a more commensurable model to understand the dynamics of bilirubin metabolism.

[0148] HLOs, such as those produced according to the methods disclosed herein, are easy to work with and have very low variation across batches. Large batches of HLOs can be generated within a couple of weeks. Leveraging these qualities, several drugs were tested within a short span of time to identify a critical pathway that is involved in bilirubin metabolism. Therefore, liver organoids are a useful model for studying diseases and disorders associated with dysfunctional bilirubin metabolism, such as jaundice, Crigler-Najjar syndrome, Gilbert’s syndrome, Dubin- Johnson syndrome, or Rotor syndrome. [0149] These HLOs can be derived from patient derived induced pluripotent stem cells (iPSCs), where the patient can be healthy or having a diseased condition, and are identical in genetic content to the respective patient. They express most liver markers that are expressed in the pre-natal stages of development. Furthermore, they are clonal and therefore reacts similarly to external stimuli and biochemical perturbations. These HLOs are highly scalable and tractable, allowing screening approaches to test a vast array of drugs and small molecules.

[0150] Although abnormal metabolism of bilirubin results in disease, bilirubin is an important metabolite during early fetal development and acts as a metabolic hormone to play an antioxidant role in adults. HLO models prepared by previous methods, which did not involve the use of bilirubin, resemble immature tissue and express fetal and intestinal markers. Accordingly, as also disclosed herein, maturation of HLOs were induced by using low doses (mimicking normal fetal physiological levels) of bilirubin during the culture steps disclosed herein. Modulating the glucocorticoid receptor pathway in these HLOs, such as with treatment with mifepristone and ketoconazole, improved conjugation and metabolism of bilirubin.

[0151] Vitamin C is also necessary for proper development of the fetus and involved in the formation of the periportal zone of the liver. L-gulonolactone oxidase (GULO) is a naturally occurring enzyme that synthesizes vitamin C, but this enzyme is non-functional in human and some other animals such as Guinea pigs, necessitating exogenous vitamin C supplementation (typically through the diet). As shown in Guinea pig animal models, vitamin C deficiency causes significant metabolic disorders.

[0152] Compared to model organisms, genetic modifications are much easier in iPSC cell lines and they can be maintained easily over longer periods before differentiation into organoids. Taking advantage of this, iPSC-derived organoids (such as those produced according to the methods disclosed herein) expressing a functional L-gulonolactone oxidase (GULO), such as murine GULO (mGULO), were generated. When the iPSCs and organoids are human in origin, the expression of the functional L-gulonolactone allows for ascorbate synthesis, which is normally inactive in humans. These mGULO organoids exhibited increased efficiency in conjugating bilirubin and exhibited improved viability when treated with bilirubin. The production of ascorbate in mGULO organoids reduces oxidative stress in the organoids and drives expression of NRF2, which is a master regulator of cellular detoxification pathways and in turn promotes expression of UGT1A1, which catalyzes bilirubin conjugation. These mGULO organoids are otherwise genetically identical to the patients from which they are derived, and encompass the aspects of human bilirubin metabolism. Accordingly, these organoids can be used as model systems for elucidating the mechanistic development of liver diseases and disorders, such as NH, and developing therapeutic treatments thereto.

Definitions of Terms

[0153] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0154] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood when read in light of the instant disclosure by one of ordinary skill in the art to which the present disclosure belongs. For purposes of the present disclosure, the following terms are explained below.

[0155] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0156] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0157] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0158] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment. As used herein “another” may mean at least a second or more. [0159] The term “ones” means more than one.

[0160] As used herein, the term “plurality” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0161] As used herein, the term “set of’ means one or more. For example, a set of items includes one or more items.

[0162] As used herein, the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, step, operation, process, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, without limitation, “at least one of item A, item B, or item C” means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or item A and C. In some cases, “at least one of item A, item B, or item C” means, but is not limited to, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0163] As used herein, “substantially” means sufficient to work for the intended purpose. The term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent.

[0164] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0165] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in various embodiments.

[0166] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a nonhuman mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0167] As used herein, the terms “treatment,” “treating,” “treat,” and the like, with respect to a disease or condition, can refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. For example, a treatment can include executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0168] “Treatment,” as used herein, thus can cover any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms. “Treatment” can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition. [0169] The term “therapeutically effective” or “therapeutically effective amount” as used throughout this application can refer to an amount effective to achieve a desired and/or beneficial effect, and/or anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease. An effective amount can be administered in one or more administrations. In the methods, a therapeutically effective amount is an amount appropriate to treat an indication. By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease progression, increase the quality of life, or to prolong life. Such achievement can be measured by any suitable method, such as measurement of tumor size or blood cell count, or any other suitable measurement.

[0170] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and/or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0171] The term “disease state” as used herein, can generally refer to a condition that affects the structure or function of an organism. Disease states can include, for example, stages of a disease progression.

[0172] As used herein, the term “assessing” can include any form of measurement, and includes determining if an element is present or not. The terms “determining,” “measuring,” “evaluating,” “assessing” and “assaying” can be used interchangeably and can include quantitative and/or qualitative determinations.

[0173] As used herein, the terms “modulated” or “modulation,” or “regulated” or “regulation” and “differentially regulated” can refer to both up regulation (z.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (z.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

[0174] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.

[0175] As used herein, the term “marker” or “biomarker” can refer to any measurable substance taken as a sample from a subject whose presence is indicative of some phenomenon. Non-limiting examples of such phenomenon can include a disease state, a condition, or exposure to a compound or environmental condition. In various embodiments described herein, biomarkers may be used for diagnostic purposes e.g., to diagnose a disease state, a health state, an asymptomatic state, a symptomatic state, etc.). The term “biomarker” may be used interchangeably with the term “marker”. The term “marker” or “biomarker” can include a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expression pattern can be utilized as part of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, or which, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.

[0176] As used herein, the term “cellular phenotype” can refer to any determinable, observable, and/or measurable characteristic associated with a cell population.

[0177] As used herein, a “model” can include one or more in vitro or in vivo disease models; a model can also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof. A model can be used in a process and/or applied to an assay, in accordance with various embodiments as disclosed herein.

[0178] As used herein, a “process” can include one or more steps involving one or more features of one or more model as disclosed herein.

[0179] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to a biological, enzymatic, or therapeutic function

[0180] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0181] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and/or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and/or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0182] As used herein, “in vivo” is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0183] As used herein, “ex vivo” is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.

[0184] As used herein, “in vitro” is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube. [0185] The terms “nucleic acid” or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, or phosphoramidate. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g. plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and/or expression of the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof. [0186] A nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “upstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the 5 ’-end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “grouped” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.

[0187] The nucleic acids described herein comprise nucleobases. Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5- methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5, 6-dihydro uracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0188] The terms “peptide”, “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N-terminus of a subsequent sequence.

[0189] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity can be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0190] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between. Yield can be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0191] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and/or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, saltforming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0192] Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals. Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cryopreservation medium, which include other components such as nutrients (e.g. albumin, serum, bovine serum, fetal calf serum [FCS]) to enhance post-thawing survivability of the cells. In these cry opreservation media, at least one cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.

[0193] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, P-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w/w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0194] The term “pharmaceutically acceptable salts” has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are nontoxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0195] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0196] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery and/or incorporation of a compound to cells, tissues and/or bodily organs.

[0197] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.

[0198] The term “basement membrane matrix” or “extracellular matrix” as used herein has its plain and ordinary meaning in light of the specification and refers to any biological or synthetic compound, substance, or composition that enhances cell attachment and/or growth. Any extracellular matrix, as well as any mimetic or derivative thereof, known in the art can be used for the methods disclosed herein. Some examples of extracellular matrices, or mimetics or derivative thereof, include but are not limited to cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly- omithine, collagen, collagen IV, gelatin, fibronectin, vitronectin, laminin, laminin-511 elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan, fibrin, basement membrane, Matrigel®, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. A common basement membrane matrix that is used in laboratories are those isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, these basement membrane matrices are derived from non-human animals and therefore contain xenogeneic components that prevent its use towards humans. They are also not defined, which can lead to variability in manufacturing, as well as potentially harbor pathogens. Accordingly, in some embodiments, the methods for culturing cells may involve the use of synthetic and/or defined alternatives to these xenogeneic basement membrane matrices. The use of non-xenogeneic basement membrane matrices or mimetics or derivatives thereof enables manufacturing of biological products better suited for human use.

[0199] The terms “passage” and “passaging” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to the conventional approaches performed in biological cell culture methods to maintain a viable population of cells for prolonged periods of time. As cells are generally proliferative in cell culture, they undergo multiple cycles of mitosis until occupying the available space, which is typically a surface of a cell culture container (e.g., a plate, dish, or flask) submerged under culture medium. For example, the cells may grow out as a monolayer on a cell culture container surface. If the growing cells occupy the entire available space of surface, they cannot proliferate further and may exhibit senescent behavior. In order to continue growth of the cells, which may be performed to maintain the viability and proliferative nature of the cells and/or to expand the number of cells for downstream purposes, the cells may be passaged by taking a fraction of the cells and seeding this fraction onto a fresh surface (e.g., of a cell culture container) in culture medium. This fraction of the cells will continue to proliferate and multiply until they occupy the available space of the new surface, upon which this passaging can be repeated successively.

[0200] The microscopic architecture of the liver is made up of polygonal structures called “hepatic lobules”. Classically, these lobules take on a hexagonal structure, although other geometric shapes are observed depending on tissue specification. Each lobule unit comprises plates or layers of hepatocytes surrounding an internal central vein and encapsulated by bundles of vessels called portal triads, which are made up of a portal vein, hepatic artery, and bile duct. Hepatic activity occurs as blood flows from the portal triads at the periphery, across the hepatocytes, and into the central vein to return to the circulatory system. Due to the asymmetric organization of these lobules, the layers of hepatocytes are divided into three zones. Cells in the “periportal zone” (zone 1) are closest to the portal triad and receive the most oxygenated blood, the pericentral zone (zone 3) are closest to the central vein and therefore receive the least amount of oxygenated blood, and the transition zone (zone 2) is in between zone 1 and 3. Due to this separation, each zone of hepatocytes exhibit differing activities. For example, zone 1 hepatocytes are involved in oxidative liver functions such as gluconeogenesis and oxidative metabolism of fatty acids, whereas zone 3 hepatocytes are involved in glycolysis, lipogenesis, and cytochrome P450-mediated detoxification. In some embodiments, the liver organoids disclosed herein exhibit a periportal-like identity resembling the tissue found in the periportal zone of liver lobules, including the functional and cellular marker characteristics of the periportal zone.

[0201] The term “bilirubin” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the naturally occurring metabolite created by normal catabolic degradation of heme. Bilirubin arises from the catalysis of biliverdin by biliverdin reductase. In the liver, bilirubin is conjugated with glucuronic acid by a family of enzymes called UDT-glucuronosyltransferases (UGTs). This conjugation renders bilirubin water soluble, enabling it to be carried in bile to the small intestine and colon, whereby it is further metabolized to waste products. Dysfunctional bilirubin metabolism, particularly due to abnormal function of UGTs preventing conjugation of bilirubin, leads to accumulation of bilirubin and is associated with various diseases characterized by hyperbilirubinemia. Notably, however, while excessive bilirubin is detrimental, bilirubin also has antioxidant capabilities and therefore may have beneficial effects in reducing oxidative damage in cells. [0202] The term “hyperbilirubinemia” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the condition of elevated levels of bilirubin, which is a natural product of heme catabolism. Bilirubin is filtered from the blood by the liver and is converted to water soluble intermediates, which are then released to the intestinal tract in bile, metabolized by microbiota, and excreted as waste. In neonates, bilirubin levels, which were originally cleared by the mother through the placenta, might not be adequately cleared by the immature liver. Excessive levels of bilirubin may potentially cause severe neurological damage (kemicterus). In adults, hyperbilirubinemia may also result from diseases affecting the liver, such as hepatitis and cirrhosis. Neonatal hyperbilirubinemia is treated by phototherapy, or with blood transfusion in extreme cases, whereas treatments in adults are directed to the underlying cause.

[0203] The term “L-gulonolactone oxidase” and “GULO” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the enzyme that catalyzes L-gulonolactone to produce L-xylo-hex-3-gulonolactone and hydrogen peroxide. The L-xylo-hex-3-gulonolactone then spontaneously converts to ascorbate (vitamin C). Accordingly, this enzyme is involved in the biosynthesis of vitamin C, which is an essential nutrient that is involved in many biological functions such as use as a cofactor for several important enzymes and as an antioxidant. Notably, humans, as well as other haplorrhine primates, certain species of bats, and Guinea pigs have evolved to harbor a non-functional GULO gene. Therefore, these organisms are unable to synthesize ascorbate and require vitamin C intake from diet or supplementation, where a deficiency of vitamin C can lead to scurvy. As applied to the disclosure herein, a “functional GULO protein” is a GULO protein that has L- gulonolactone catalytic activity to result in the production of ascorbate. Conversely, an “inactive” GULO protein or “non-functional” GULO protein is one that does not have the catalytic activity to produce ascorbate. Humans and cells that are derived from humans comprise a non-functional GULO protein and do not have the ability to synthesize ascorbate. However, as disclosed herein, human cells may be engineered to express a functional GULO protein to enable ascorbate synthesis ability. These functional GULO proteins may be expressed in human cells (or other cells that are unable to normally synthesize ascorbate) through conventional methods of cloning, such as genetically engineering cells to have genetic sequences that encode for a functional GULO protein.

[0204] The term “% w/w” or “% wt/wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v/v” or “% vol/vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0205] The term “exogenous” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to external factors that originate outside of a biological specimen (e.g., a cell, population of cells, organoid, etc.), as opposed to being naturally occurring and/or produced by the biological specimen itself. As used herein, exogenous components, reagents, and/or conditions, are components, reagents, and/or conditions that are added to compositions described herein, although this does not necessarily preclude the possibility of the same components, reagents, and/or conditions also being present through a function endogenous to a biological specimen.

[0206] The terms “liver organoid” and “hepatocyte organoid” are used interchangeably herein, and refer to populations of cells differentiated in vitro to form selforganizing structures, which generally are three-dimensional (3D), and include one or more functional cell types. Liver organoids differ from naturally occurring liver tissue in a number of ways. For example, as compared with naturally occurring liver tissue, liver organoids can have a structure having a single lumen and generally a spherical shape, and can include a basement membrane which is unnatural. The single lumen of a liver organoid contains 3D tissues but generally does not make any hepatic lobular structure nor cord-like structure, as with naturally occurring liver tissue. Liver organoids also generally do not contain hematopoietic tissue and acquired immune cell subsets, such as T cell lineages. Further, as compared with naturally occurring liver tissue, liver organoids can have different efflux mechanisms, as a liver organoid can have a three-dimensional structure with a luminal structure but no ejection mechanism. In addition, liver organoids generally cannot receive dietary inputs, as they lack a gut and connected vascular channel.

[0207] Liver organoids can be derived from pluripotent stem cells (PSCs), including at least embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Liver organoids may also be formed from liver-derived stem cells. In general, liver organoids can self-organize through cell sorting and spatially restricted lineage commitment in a manner similar to that which occurs in vivo, but as directed in vitro by thoughtful introduction of exogenous and/or endogenous differentiating factors and/or conditions as described herein, optionally through one or more directed steps, optionally involving introduction of one or more components. [0208] The term “mature liver organoid” as used herein refers to liver organoids which have continued to develop from a liver organoid to include, in various embodiments, luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape. Mature liver organoids may exhibit lumens with smaller sizes and reduced circularity when compared to lumens of liver organoids. In some embodiments, mature liver organoids may be generated through addition of exogenous bilirubin and/or amino acid supplementation as described herein. In some embodiments, a mature liver organoid may be characterized as expressing reduced levels of AFP, CDX2, and/or NANOG relative to liver organoids, and/or as expressing increased levels of ALB, SLC4A2 and/or HO-1 relative to liver organoids. In some embodiments, a mature liver organoid may be characterized as expressing CYP2E1, CYP7A1, PROXI, MRP3, MRP3, and/or OATP2. In some embodiments, a mature liver organoid may exhibit increased CYP3A4 and/or CYP1 A2 protein levels and/or enzymatic activity relative to liver organoids.

[0209] The term “hyperbilirubinemia liver organoid” as used herein refers to liver organoids which have been exposed to high concentrations of bilirubin, generally provided exogenously through one or more dosings, to mimic a hyperbilirubinemia state. In some embodiments, hyperbilirubinemia liver organoids comprise genetic abnormalities that alter bilirubin metabolism, such as resulting in increased levels of bilirubin anabolism and/or reduced levels of bilirubin catabolism. Hyperbilirubinemia liver organoids can be characterized as expressing elevated levels of UGT1A1 and/or NRF2, relative to liver organoids not exposed to high concentrations of bilirubin.

[0210] The term “tissue culture surface” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a substrate surface on which cells may aggregate and/or adhere to facilitate cell growth, differentiation, and/or function.

[0211] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In certain embodiments, a construct and/or vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins. Stem Cells

[0212] The term “totipotent stem cells” (also known as omnipotent stem cells) as used herein has its plain and ordinary meaning as understood in light of the specification and are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.

[0213] The term “embryonic stem cells (ESCs),” also commonly abbreviated as ES cells, as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that are pluripotent and derived from the inner cell mass of the blastocyst, an early-stage embryo.

[0214] The term “pluripotent stem cells (PSCs)” as used herein has its plain and ordinary meaning as understood in light of the specification and encompasses any cells that can differentiate into nearly all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system). PSCs can be the descendants of inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0215] The term “induced pluripotent stem cells (iPSCs),” also commonly abbreviated as iPS cells, as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a type of pluripotent stem cells artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing a “forced” expression of certain genes. hiPSC refers to human iPSCs. In some methods known in the art, iPSCs may be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcriptional regulators Oct-3/4 (POU5F1) and Sox2, although other genes may enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. As used herein, iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc. In other methods, a lentiviral system is used to transform somatic cells with 0CT4, SOX2, NANOG, and ETN28. Genes whose expression are induced in iPSCs include but are not limited to Oct-3/4 (P0U5FFy, certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Sox 15) certain members of the Klf family (e.g., Kl/l. Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, E1N28, Tert, Fbxl5, Eras, ECAT15-1, ECAT15-2, Tell, P-Catenin, ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Fthll7, Sall4, Rexl, UTF1, Stella, Stat3, Grb2, Prdml4, Nr5al , Nr5a2, or E-cadherin, or any combination thereof.

[0216] The term “precursor cell” as used herein has its plain and ordinary meaning as understood in light of the specification and encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types. In some embodiments, a precursor cell is pluripotent or has the capacity to becoming pluripotent. In some embodiments, the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell. In some embodiments, a precursor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein/peptide treatment. Precursor cells include embryonic stem cells (ESC), embryonic carcinoma cells (Ecs), and epiblast stem cells (EpiSC).

[0217] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which are in turn derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Methods for deriving embryonic stem cells from blastocytes are well known in the art. Human embryonic stem cells H9 (H9-hESCs) are used in the exemplary embodiments described in the present application, but it would be understood by one of skill in the art that the methods and systems described herein are applicable to any stem cells.

[0218] Additional stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to those provided by or described in the database hosted by the National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); WISC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW- SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Technion at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES- 1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (HI); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14). Exemplary human pluripotent cell lines include but are not limited to TkDA3-4, 1231 A3, 317- D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0219] In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. As used herein, the term “directed differentiation” describes a process through which a less specialized cell becomes a particular specialized target cell type. The particularity of the specialized target cell type can be determined by any applicable methods that can be used to define or alter the destiny of the initial cell. Exemplary methods include but are not limited to genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0220] In some embodiments, an adenovirus can be used to transport the requisite four genes, resulting in iPSCs substantially identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated. In some embodiments, non-viral based technologies are employed to generate iPSCs. In some embodiments, reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiment, generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions. [0221] The term “feeder cell” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying on the cell surface. Feeder cells are generally adherent cells and may be growth arrested. For example, feeder cells are growth-arrested by irradiation (e.g. gamma rays), mitomycin-C treatment, electric pulses, or mild chemical fixation (e.g. with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily have to be growth arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, the feeder cells are allogeneic or xenogeneic to the supported target stem cell, which may have implications in downstream applications. In some embodiments, the feeder cells are mouse cells. In some embodiments, the feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76/7 cells, human fibroblasts, human foreskin fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used in lieu of feeder cell co-culture or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the proliferation of the target stem cells.

Differentiation of PSCs

[0222] Known methods for making downstream cell types, such as definitive endoderm, foregut endoderm, posterior foregut endoderm, and/or hepatic lineages from pluripotent cells (e.g., iPSCs or ESCs) are applicable to some embodiments of the methods described herein. In some embodiments, pluripotent cells are derived from a morula. In some embodiments, pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells can be derived from the embryonic inner cell mass or from the embryonic gonadal ridges. Embryonic stem cells can originate from a variety of animal species including, but not limited to, various mammalian species including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm or other downstream cell types such as posterior foregut, posterior foregut endoderm, and/or hepatic lineages. In some embodiments, iPSCs are used to produce definitive endoderm or other downstream cell types such as posterior foregut, posterior foregut endoderm, and/or hepatic lineages. In some embodiments, human iPSCs (hiPSCs) are used to produce definitive endoderm or other downstream cell types such as posterior foregut, posterior foregut endoderm, and/or hepatic lineages.

[0223] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic germ layer cells, organ tissue progenitor cells, and then into tissue such as liver tissue or any other biological tissue. In some embodiments, the directed differentiation is done in a stepwise manner to obtain each of the differentiated cell types where molecules (e.g. growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses. In some embodiments, the directed differentiation is done in a nonstepwise manner where molecules (e.g. growth factors, ligands, agonists, antagonists) are added at the same time. In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in the PSCs or any downstream cells.

[0224] In some embodiments, the embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 6 hours to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0225] In some embodiments, the embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10 ng/mL, 20 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL, 120 ng/mL, 150 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL, 1200 ng/mL, 1500 ng/mL, 2000 ng/mL, 5000 ng/mL, 7000 ng/mL, 10000 ng/mL, or 15000 ng/mL, or any concentration that is within a range defined by any two of the aforementioned concentrations, for example, 10 ng/mL to 15000 ng/mL, 100 ng/mL to 5000 ng/mL, 500 ng/mL to 2000 ng/mL, 10 ng/mL to 2000 ng/mL, or 1000 ng/mL to 15000 ng/mL. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is varied during the course of the treatment. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can differ in concentrations.

[0226] In some embodiments, the ESCs or iPSCs are cultured in growth media that supports the growth of stem cells. In some embodiments, the ESCs or iPSCs are cultured in stem cell growth media. In some embodiments, the stem cell growth media is RPMI 1640, DMEM, DMEM/F12, or Advanced DMEM/F12. In some embodiments, the stem cell growth media comprises fetal bovine serum (FBS). In some embodiments, the stem cell growth media comprises FBS at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%. In some embodiments, the stem cell growth media does not contain xenogeneic components. In some embodiments, the growth media comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0227] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are cryopreserved. In some embodiments, the somatic cells are cryopreserved. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate.

[0228] In some embodiments, stem cells are treated with one or more growth factors to differentiate to definitive endoderm cells. Such growth factors can include growth factors from the TGF-beta superfamily. In some embodiments, the one or more growth factors comprise the Nodal/ Activin and/or the BMP subgroups of the TGF-beta superfamily of growth factors. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, Wnt3a or combinations of any of these growth factors. In some embodiments, the stem cells are contacted with Activin A. In some embodiments, the stem cells are contacted with Activin A and BMP4. [0229] In some embodiments, definitive endoderm (DE) can further undergo anterior endoderm pattering, foregut specification and morphogenesis, dependent on FGF, Wnt, BMP, or retinoic acid, or any combination thereof. In some embodiments, human PSCs are efficiently directed to differentiate in vitro into liver epithelium and mesenchyme. It will be understood that molecules such as growth factors can be added to any stage of the development to promote a particular type of hepatic tissue formation. In some embodiments, siRNA and/or shRNA targeting cellular constituents associated with the FGF, Wnt, BMP, or retinoic acid signaling pathways are used to inhibit or activate these pathways.

Culture and Expansion of Definitive Endoderm, Foregut Cells, and Downstream Cell Types

[0230] Methods of making liver organoids have been explored previously in, for example, Ouchi et al. “Modeling Steatohepatitis in Humans with Pluripotent Stem Cell- Derived Organoids” Cell Metabolism (2019) 30(2):374-384; Shinozawa et al. “High-Fidelity Drug-Induced Fiver Injury Screen Using Human Pluripotent Stem Cell-Derived Organoids” Gastroenterology (2021) 160(3):831-846; PCT Publications WO 2018/085615, WO 2018/191673, WO 2018/226267, WO 2019/126626, WO 2020/023245, WO 2020/069285, and WO 2021/262676, each of which is hereby expressly incorporated by references in its entirety. Disclosure of liver organoid compositions and methods of making thereof are applicable to the human liver organoids (HEOs) described herein.

[0231] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or foregut endoderm are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, and/or posterior foregut endoderm cells are cultured and expanded as described herein. In some embodiments foregut endoderm cells are cultured and expanded as described herein.

[0232] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with a TGF-b pathway inhibitor. In some embodiments, the TGF-b pathway inhibitor comprises one or more of A83-01, RepSox, EY365947, and SB431542. In some embodiments, the cells are not treated with a TGF-b pathway inhibitor. The TGF-b pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0233] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with an FGF pathway activator. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15/FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with an FGF pathway activator. The FGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0234] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with a Wnt pathway activator. In some embodiments, the Wnt pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl 1, Wntl6, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt pathway activator comprises CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, or TWS119, or any combination thereof. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the cells are not treated with a Wnt pathway activator. The Wnt pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0235] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with a VEGF pathway activator. In some embodiments, the VEGF pathway activator comprises one or more of VEGF or GS4012. In some embodiments, the cells are not treated with a VEGF pathway activator. The VEGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. [0236] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with an EGF pathway activator. In some embodiments, the EGF pathway activator comprises EGF. In some embodiments, the cells are not treated with an EGF pathway activator. The EGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0237] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with ascorbic acid. In some embodiments, the cells are not treated with ascorbic acid. Ascorbic acid as provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0238] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with a BMP pathway activator or BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises a BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP pathway inhibitor comprises Noggin, RepSox, LY364947, LDN-193189, SB431542, or any combination thereof. In some embodiments, the cells are not treated with a BMP pathway activator or BMP pathway inhibitor. The BMP pathway activator or BMP pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0239] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are contacted with a retinoic acid pathway activator. In some embodiments, the retinoic acid pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof. In some embodiments, the cells are not treated with a retinoic acid pathway activator. The retinoic acid pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0240] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are converted into liver cell types via a “one step process”. In some embodiments, pluripotent stem cells are converted into liver cell types via a “one step” process. For example, one or more molecules that can differentiate pluripotent stem cells into DE culture (e.g., Activin A) are combined with additional molecules that can promote directed differentiation of DE culture (e.g., FGF4, CHIR99021, RA) to directly treat pluripotent stem cells.

[0241] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and/or downstream liver cell types are expanded in cell culture. In some embodiments, pluripotent stem cells (e.g., ESCs and/or iPSCs), definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in cell culture. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in a basement membrane matrix. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in Matrigel®. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in a basement membrane matrix that does not comprise non-human animal components. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in a non-xenogeneic basement membrane matrix. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are not expanded in Matrigel®. In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel. In some embodiments, the pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632).

[0242] In some embodiments, the pluripotent stem cells (e.g. ESCs and/or iPSCs) are differentiated into definitive endoderm cells. In some embodiments, the pluripotent stem cells are differentiated into definitive endoderm cells by contacting the pluripotent stem cells with Activin A, BMP4, or both. In some embodiments, the pluripotent stem cells are contacted with a concentration of Activin A that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of Activin A within a range defined by any two of the aforementioned concentrations, for example, 10 to 200 ng/mL, 10 to 100 ng/mL, 100 to 200 ng/mL, or 50 to 150 ng/mL. In some embodiments, the pluripotent stem cells are contacted with Activin A at a concentration of 100 ng/mL or about 100 ng/mL. In some embodiments, the pluripotent stem cells are contacted with a concentration of BMP4 that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of BMP4 within a range defined by any two of the aforementioned concentrations, for example, 1 to 200 ng/mL, 1 to 100 ng/mL, 25 to 200 ng/mL, 1 to 80 ng/mL, or 25 to 100 ng/mL. In some embodiments, the pluripotent stem cells are contacted with BMP4 at a concentration of 50 ng/mL or about 50 ng/mL.

[0243] Provided herein are methods for expanding posterior foregut cells. Exemplary methods are represented in FIG. 5H, without being limited by specific characteristics, such as timing and growth conditions exemplified in the figure. In some embodiments, the methods comprise a) dissociating a posterior foregut cell monolayer to posterior foregut cells and/or posterior foregut endoderm cells; b) seeding the posterior foregut cells and/or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and/or posterior foregut endoderm cells with a TGF-b pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are further cultured with ascorbic acid. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are not cultured with ascorbic acid. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are further cultured with EGF. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are not cultured with EGF. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are further cultured with a ROCK inhibitor. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are not cultured with a ROCK inhibitor. Some embodiments, the posterior foregut cell monolayer is dissociated to the posterior foregut cells and/or posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation. In some embodiments, the enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution generally known in the art, for example, Accutase, Accumax, trypsin, trypsin/EDTA, collagenase, dispase, TrypEE Express, or TrypLE Select. In some embodiments, mechanical dissociation may involve disrupting the cells using a pipette, microchannel, or other apparatus with an appropriately sized bore to mechanically shear groups of cells without disrupting the individual cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density that is, is about, is at least, is at least about, is not more than, or is not more than about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵ 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵ 9xl0⁵, IxlO⁶ 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities, for example, 1X10⁵-5X10⁶, IxlO⁵- 5xl0⁵, 5X10⁵-5X10⁶, or 3xl0⁵-7xl0⁵ cells/cm² of surface area of the tissue culture surface. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density that is or is about 5xl0⁵ cells/cm² of surface area of the tissue culture surface. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or component thereof. In some embodiments, basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is non-xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel or other substances that are not xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin-511. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids are formed spontaneously. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured for a number of days that is, is about, is at least, is at least about, is not more than, or is not more than about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or any number of days within a range defined by any two of the preceding number of days, for example, 4-6 days, 2-35 days, 2-15 days, 20-35 days, or 10-20 days. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or for at least any number of days within a range defined by any two of the preceding number of days, for example, 4-6 days, 4-35 days, 4-15 days, 20-35 days, or 10-20 days.

[0244] In some embodiments of the methods provided herein, the TGF-b pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the TGF-b pathway inhibitor is A83-01. In some embodiments, the TGF-b pathway inhibitor is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 100-1000 nM, 100-500 nM, 500-1000 nM, or 300-700 nM. In some embodiments, the TGF-b pathway inhibitor is provided at a concentration of, or of about, 500 nM.

[0245] In some embodiments of the methods provided herein, the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the FGF pathway activator is FGF2. In some embodiments, the FGF pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-10 ng/mL, 1-5 ng/mL, 5-10 ng/mL, or 3-7 ng/mL. In some embodiments, the FGF pathway activator is provided at a concentration of, or of about, 5 ng/mL.

[0246] In some embodiments of the methods provided herein, the Wnt pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the Wnt pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-8 pM, 1-3 pM, 3-8 pM, or 2- 4 pM. In some embodiments, the Wnt pathway activator is provided at a concentration of, or of about, 3 pM.

[0247] In some embodiments of the methods provided herein, the VEGF pathway activator is selected from the group consisting of VEGF or GS4012. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the VEGF pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 ng/mL, 1-10 ng/mL, 10-20 ng/mL, or 5-15 ng/mL. In some embodiments, the VEGF pathway activator is provided at a concentration of, or of about, 10 ng/mL.

[0248] In some embodiments of the methods provided herein, the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises EGF. In some embodiments, the EGF is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-30 ng/mL, 10-20 ng/mL, 20-30 ng/mL, or 15-25 ng/mL. In some embodiments, the EGF is provided at a concentration of, or of about, 20 ng/mL. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that does not comprise EGF.

[0249] In some embodiments of the methods provided herein, the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises ascorbic acid. In some embodiments, the ascorbic acid is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-100 pg/mL, 10-50 pg/mL, 50-100 pg/mL, or 30-70 pg/mL. In some embodiments, the ascorbic acid is provided at a concentration of, or of about, 50 pg/mL. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that does not comprise ascorbic acid.

[0250] In some embodiments of the methods provided herein, the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises a ROCK inhibitor. In some embodiments, the ROCK inhibitor is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 pM, 1-10 pM, 10-20 pM, or 5-15 pM. In some embodiments, the ROCK Inhibitor is provided at a concentration of, or of about, 10 pg/mL. In some embodiments, the ROCK inhibitor is Y- 27632 In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that does not comprise the ROCK inhibitor.

[0251] In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells of the methods provided herein may be cultured for multiple passages. In some embodiments, steps a) - c) of the methods provided herein are repeated with the cells of step c). In some embodiments, the methods further comprise passaging the cells of step c) one or more times. In some embodiments, the cells of step c) are passaged until the posterior foregut cells and/or posterior foregut endoderm cells do not form spheroids spontaneously. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are passaged and cultured for a number of days that is, is about, is at least, is at least about, is not more than, or is not more than about, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or any number of days within a range defined by any two of the preceding number of days, for example, 2-10 days, 2-4 days, 2-6 days, 4-10 days, 6-10 days, 4-6 days, or 3-7 days, before the posterior foregut cells and/or posterior foregut endoderm cells are passaged again. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are passaged and cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or for at least any number of days within a range defined by any two of the preceding number of days, for example, 2-10 days, 2-4 days, 2-6 days, 4-10 days, 6-10 days, 4-6 days, or 3-7 days, before the posterior foregut cells and/or posterior foregut endoderm cells are passaged again. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are passaged and cultured for 4, 5, or 6 days, before the posterior foregut cells and/or posterior foregut endoderm cells are passaged again. In some embodiments, not more than 1, 2, or 3 passages of the cells are performed. In some embodiments, the total yield of posterior foregut cells and/or posterior foregut endoderm cells (which may be in the form of spheroids) following the methods provided herein involving multiple passages is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, or 1000 times, or any times fold within a range defined by any two of the aforementioned times, for example, 50-1000 times fold, 50-200 time fold, 200-1000 time fold, or 100-300 times fold, the total yield of posterior foregut cells and/or posterior foregut endoderm cells obtained in a cell culture without passaging.

[0252] In some embodiments, the methods further comprise collecting the posterior foregut cells and/or posterior foregut endoderm cells and differentiating the posterior foregut cells and/or posterior foregut endoderm cells to liver organoids. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three- dimension (3D) spheroids are formed spontaneously, optionally wherein the spheroids comprise a structure with a single lumen, and the posterior foregut cells and/or posterior foregut endoderm cells are collected from the spheroids. In some embodiments, the methods further comprise dissociating the spheroids into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are collected from the posterior foregut cell monolayer by dissociating the posterior foregut cell monolayer into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step.

[0253] Provided herein in some embodiments are methods for expanding posterior foregut cells and/or posterior foregut endoderm and/or foregut endoderm cells. In some embodiments, the methods comprise a) dissociating a posterior foregut cell monolayer to posterior foregut cells and/or posterior foregut endoderm cells; b) seeding the posterior foregut cells and/or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and/or posterior foregut endoderm cells with a TGF-b pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, the posterior foregut cell monolayer is dissociated to the posterior foregut cells and/or posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation. In some embodiments, the enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution generally known in the art, for example, Accutase, Accumax, trypsin, trypsin/EDTA, collagenase, dispase, TrypLE Express, or TrypLE Select. In some embodiments, mechanical dissociation may involve disrupting the cells using a pipette, microchannel, or other apparatus with an appropriately sized bore to mechanically shear groups of cells without disrupting the individual cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density that is, is about, is at least, is at least about, is not more than, or is not more than about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶ 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities, for example, 1X10⁵-5X10⁶, 1X10⁵-5X10⁵, 5X10⁵-5X10⁶, or 3xl0⁵-7xl0⁵ cells/cm² of surface area of the tissue culture surface. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density that is or is about 5xl0⁵ cells/cm² of surface area of the tissue culture surface. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or component thereof. In some embodiments, basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is non- xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel or other substances that are not xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin-511. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids are formed spontaneously. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured for a number of days that is, is about, is at least, is at least about, is not more than, or is not more than about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or any number of days within a range defined by any two of the preceding number of days, for example, 2-35 days, 2-15 days, 20-35 days, or 10-20 days. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells are cultured for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or for at least any number of days within a range defined by any two of the preceding number of days, for example, 4-35 days, 4-15 days, 20-35 days, or 10-20 days. In some embodiments, steps a) - c) of the methods provided herein are repeated with the cells of step c). In some embodiments, the methods further comprise passaging the cells of step c) one or more times. In some embodiments, the cells of step c) are passaged until the posterior foregut cells and/or posterior foregut endoderm cells do not form spheroids spontaneously. In some embodiments, not more than 1, 2, or 3 passages of the cells are performed. In some embodiments, the total yield of posterior foregut cells and/or posterior foregut endoderm cells (which may be in the form of spheroids) following the methods provided herein involving multiple passages is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, or 1000 times, or any times fold within a range defined by any two of the aforementioned times, for example, 50-1000 times fold, 50-200 time fold, 200-1000 time fold, or 100-300 times fold, the total yield of posterior foregut cells and/or posterior foregut endoderm cells obtained in a cell culture without passaging. In some embodiments, Table 1 provides exemplary concentration ranges for each of the growth factors used in the present methods. In embodiments of the methods, any concentration or range thereof for a growth factor under a certain “sub-embodiment” may be used in combination with the concentration or range thereof for the other growth factors under the same or different “sub-embodiment”. Therefore, the combinations are not restricted to combinations under the same “sub-embodiment”. For example, the presence of the TGF-b pathway inhibitor, FGF pathway activator, Wnt pathway activator, and VEGF pathway activator as defined under any “sub-embodiment” for each may be combined with the presence of EGF (any one of EGF “sub-embodiments” 1-3) or the absence of EGF (“sub-embodiment” 4). The same will apply to any combination of the growth factors listed, whether they are present or absent in the methods embodied herein. Exemplary, non-limiting formulations are listed in Table 2. For each of the ranges provided in Table 1 and Table 2, this is to be interpreted to also include any concentration within the defined range. For example, 100-1000 nM TGF-b pathway inhibitor is to be interpreted to include 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM of TGF-b pathway inhibitor, or any concentration within a range defined by any two of the aforementioned concentrations. Exemplary concentrations within the defined ranges are provided throughout the present disclosure. In some embodiments, the TGF-b pathway inhibitor is A83-01. In some embodiments, the FGF pathway activator is FGF2. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the ROCK inhibitor is Y -27632.

Table 1. Exemplary combinations of growth factors used for expanding posterior foregut cells

Table 2: Exemplary combinations

[0254] Also disclosed herein are the posterior foregut cells and/or posterior foregut endoderm cells produced by the methods provided herein.

Gene Editing

[0255] In some embodiments, the iPSCs, definitive endoderm cells, posterior foregut spheroids, or organoids are genetically modified or edited according to methods known in the art. For example, gene editing using CRISPR nucleases such as Cas9 are explored in PCT Publications WO 2013/176772, WO 2014/093595, WO 2014/093622, WO 2014/093655, WO 2014/093712, WO 2014/093661, WO 2014/204728, WO 2014/204729, WO 2015/071474, WO 2016/115326, WO 2016/141224, WO 2017/023803, and WO 2017/070633, each of which is hereby expressly incorporated by reference in its entirety.

Methods and Compositions for Making Liver Organoids

[0256] Provided herein are methods and compositions for differentiating posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm to liver organoids. In some embodiments, the methods comprise i) contacting posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells, optionally in the form of spheroids, optionally in the form of individual cells or cell clusters dissociated from spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or optionally cells aggregated in a microwell or other apparatus as described herein, with a retinoic acid pathway activator; and ii) contacting the cells of step i) with medium (e.g., hepatocyte culture medium (HCM)) comprising hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone), for a period of time thereby differentiating the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells to liver organoids. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells may be any of the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells disclosed herein. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells are in the form of spheroids or individual posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells derived from dissociating the spheroids. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells may be produced by a method that does not involve the use of a xenogeneic basement membrane matrix. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells may be those generally known in the art.

[0257] In some embodiments of the methods provided herein, the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580. In some embodiments, the retinoic acid pathway activator is retinoic acid. In some embodiments, the retinoic acid pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1.0-3.0 pM, 1.0-2.0 pM, 2.0-3.0 pM, or 1.5-2.5 pM. In some embodiments, the retinoic acid pathway activator is provided at a concentration of, or of about, 2.0 pM.

[0258] In some embodiments, the medium (e.g., hepatocyte culture medium) is supplemented with a cMET tyrosine kinase receptor agonist, an interleukin 6 (IL-6) family cytokine, and/or a corticosteroid. In some embodiments. The cMET tyrosine kinase receptor agonist is selected from the group consisting of HGF, PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine is selected from a group consisting of IL-6, OSM, leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC). In some embodiments, the corticosteroid is selected from a group consisting of dexamethasone, 67 eclomethasone, betamethasone, fluocortolone, halometasone, and mometasone. In some embodiments, the medium (e.g., hepatocyte culture medium) is supplemented with HGF, OSM, and dexamethasone. In some embodiments, the medium (e.g., hepatocyte culture medium) is supplemented with dexamethasone.

[0259] In some embodiments of the methods provided herein, the HGF is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 ng/mL, 1-10 ng/mL, 10-20 ng/mL, or 5-15 ng/mL. In some embodiments, the HGF is provided at a concentration of, or of about 10 ng/mL.

[0260] In some embodiments of the methods provided herein, the OSM is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-30 ng/mL, 10-20 ng/mL, 20-30 ng/mL, or 15-25 ng/mL. In some embodiments, the OSM is provided at a concentration of, or of about 20 ng/mL.

[0261] In some embodiments of the methods provided herein, the dexamethasone is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 50-200 nM, 50-100 nM, 100-200 nM, or 50-150 nM. In some embodiments, the dexamethasone is provided at a concentration of, or of about 100 nM.

[0262] In some embodiments of the methods provided herein, the cells of step i) and/or step ii) are contacted in a media that further comprises EGF. In some embodiments, the EGF is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-30 ng/mL, 10-20 ng/mL, 20-30 ng/mL, or 15- 25 ng/mL. In some embodiments, the EGE is provided at a concentration of, or of about, 20 ng/mL. In some embodiments, the cells of step i) and/or step ii) are contacted in a media that does not comprise EGE.

[0263] In some embodiments of the methods provided herein, the cells of step ii) are cultured in a growth medium supplemented with non-essential amino acids, essential amino acids, and glycine. In some embodiments, the growth media after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% non-essential amino acids by total volume, or a range defined by any two of the preceding values, for example 7-9%, 6-10%, 5-12%, 8-14%, 10-15%, 4-15%, 15-17%, 13-19%, 12-24%, or 10-25% and so forth. In some embodiments, the growth medium after supplementation is about 6-10%, 8-14%, 10-15%, 4-15%, 15-17%, 13-19%, 12-24%, or 10-25%, or about 4%, 6%, 8%, 10%, 12%, 14%, or 16% non-essential amino acids by total volume. In some embodiments, the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or a range defined by any two of the preceding values, for example, 7-9%, 6-10%, 5-12%, 8-14%, 10-15%, 4-15%, 15-17%, 13- 19%, 12-24%, or 10-25%. In some embodiments, the growth medium after supplementation is about 6-10%, 8-14%, 10-15%, 4-15%, 15-17%, 13-19%, 4%, 6%, 8%, 10%, 12%, 14%, or 16% essential amino acids by total volume. In some embodiments, the supplemented glycine is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 19-21 mg/mL, 18-22 mg/mL, 16-24 mg/mL, 10-30 mg/mL, or 5-34 mg/mL. In some embodiments, the supplemented glycine is provided at a concentration of, or of about, 18-22 or 20 mg/mL. In some embodiments, the growth medium is any standard growth medium generally used for cell culture, and their compositions thereof. For example, the growth medium is Eagle’s minimal essential medium (MEM), Eagle’s minimal essential medium with alpha modification (a- MEM), Basal Medium Eagle (BME), Dulbecco’s modified Eagle’s medium (DMEM) or hepatocyte culture medium (HCM). Merely for exemplary purposes, Table 3 depicts the standard non-essential amino acid and essential amino acid concentrations for MEM; and exemplary concentrations of these amino acids after supplementation in some embodiments provided herein. In some embodiments, the supplemented medium concentrations are ± 10%, ± 5%, or ± 1% of the values listed in Table 3.

Table 3. Exemplary amounts of amino acid supplementation

[0264] In some embodiments of the methods provided herein, the cells of step ii) are further contacted with a low/first concentration of bilirubin, wherein the liver organoids that are formed are mature liver organoids. In some embodiments, the low/first concentration of bilirubin is a human fetal physiological concentration of bilirubin. In some embodiments, the low/first concentration of bilirubin is, is about, is less than, or is less than about, 0.1 to 1 mg/L, 0.5 to 1 mg/L, or 1 mg/L. In some embodiments, the low/first concentration of bilirubin is, is about, is less than, or is less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L. In some embodiments, the low/first concentration of bilirubin is, is about, is less than, or is less than about, 0.1 to 3 mg/L, 0.5 to 3 mg/L, or 3 mg/L. In some embodiments, the low/first concentration of bilirubin is, is about, is less than, or is less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L. In some embodiments, the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape. In some embodiments, the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoids express CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with the low/first dose of bilirubin.

[0265] In some embodiments of the methods provided herein, the cells of step ii) are further contacted with a high/second concentration of bilirubin, wherein the liver organoids that are formed are hyperbilirubinemia liver organoids. In some embodiments, the liver organoids are, are about, are at least, or are at least about, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days old, or a range defined by any two of the aforementioned values, for example, 18-35, 18-30, 20-25, or 18-25 days when used in the methods disclosed herein. In some embodiments, the high/second concentration of bilirubin is, is about, is more than, or is more than about, 2-10 mg/L, 5-10 mg/L, 10 mg/L, or 20 mg/L. In some embodiments, the high/second concentration of bilirubin is, is about, is more than, or is more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L. In some embodiments, the liver organoids are contacted with the high/second concentration of bilirubin for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or a range defined by any two of the aforementioned values, for example, 1-10, 1-5, 3-8, 5-10, or 7-10 days to form the hyperbilirubinemia liver organoids. In some embodiments, the hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

[0266] In some embodiments of the methods provided herein, the liver organoids comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the liver organoids are able to synthesize ascorbate. In some embodiments, the functional GULO protein is mGULO. However, the functional GULO may alternatively be derived from any other animal species that comprises a functional GULO protein. In some embodiments, the gene that encodes for the functional GULO protein is conditionally expressed. In some embodiments, the gene is conditionally expressed using a tetracycline inducible system or any other system for conditional expression generally known in the art. In some embodiments, the liver organoid is engineered with the gene that encodes for the functional GULO protein using CRISPR or any other method of genetic engineering generally known in the art. In some embodiments, the gene or mRNA, or both, that encodes for the functional GULO protein is introduced to the mature liver organoid by transfection. In some embodiments, the liver organoids comprising the functional GULO protein express increased levels of NRF2 relative to liver organoids that do not comprise the functional GULO protein. In some embodiments, the liver organoids comprising the functional GULO protein expresses reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids that do not comprise the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate. In some embodiments, the liver organoids comprising the functional GULO protein exhibits reduced caspase-3 activity relative to liver organoids that do not comprise the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate. In some embodiments, the liver organoids comprising the functional GULO protein express increased levels of ALB relative to liver organoids that do not comprise the functional GULO protein. In some embodiments, the liver organoids comprising the functional GULO protein resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal markers comprise FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, the liver organoids comprising the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that do not comprise the functional GULO protein. In some embodiments, the liver organoids comprising the functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids that do not comprise the functional GULO protein. In some embodiments, the liver organoids comprising the functional GULO protein exhibit increased viability in culture relative to liver organoids that do not comprise the functional GULO protein. In some embodiments, the liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.

[0267] In some embodiments of the methods provided herein, the liver organoids comprise an inactive UGT1A1 gene, wherein the liver organoids are a model for Crigler-Najjar Syndrome.

[0268] In some embodiments of the methods provided herein, the methods further comprise aggregating the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells in a microwell or other apparatus (e.g., Aggrewell) prior to step i). An exemplary schematic for culturing liver organoids from posterior foregut cells and/or posterior foregut endoderm cells aggregated, for example, in a microwell, is embodied in FIG. 51. In some embodiments, each aggregate of posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells comprises about 250, about 500, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 9000, about 9500, or about 10000 posterior foregut cells and/or posterior foregut endoderm cells, or any number of posterior foregut cells and/or posterior foregut endoderm cells within a range defined by any two of the aforementioned number of cells In some embodiments, aggregating the posterior foregut cells and/or posterior foregut endoderm cells and/or foregut endoderm cells results in more uniformly sized liver organoids. Additional information about methods of aggregating posterior foregut cells and/or posterior foregut endoderm cells to produce more uniformly sized liver organoids, such as with a microwell or other apparatus, such as an Aggrewell can be found in PCT Publication WO 2021/030373, which is hereby expressly incorporated by reference in its entirety.

[0269] In some embodiments of the methods provided herein, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof. In some embodiments, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof that is xenogeneic to humans. In some embodiments, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the cells of step i) and/or step ii) are not contacted with Matrigel®, Cultrex®, or Geltrex®.

[0270] In some embodiments of the methods provided herein, the cells of step i) and/or step ii) are cultured in a non-static bioreactor. In some embodiments, the cells of step i) and/or step ii) are cultured in a rotational bioreactor.

[0271] In some embodiments of the methods provided herein, the methods further comprise cry opreserving the liver organoids. In some embodiments, cry opreserving the liver organoids comprises slow-freezing or vitrification cryopreservation. In some embodiments, the liver organoids are cryopreserved with chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT). In some embodiments, chroman 1 is provided at a concentration of or of about 50 nM. In some embodiments, emricasan is provided at a concentration of or of about 5 pM. In some embodiments, polyamine is provided at a concentration of or of about 1:1000. In some embodiments, trans-ISRIB is provided at a concentration of or of about 7 pM.

[0272] Disclosed herein are the liver organoids produced by the methods disclosed herein.

[0273] As applied to any of the cells disclosed herein, such as pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or liver organoids, the cells may be derived from a patient. In some embodiments, the patient has a liver disease. In some embodiments, the definitive endoderm, posterior foregut, posterior foregut endoderm, and/or liver organoids may be derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

Methods of Use

[0274] Also disclosed are methods comprising administering any of the liver organoids disclosed herein to a subject in need thereof. Also disclosed herein are methods for treating a liver-related disease or disorder in a subject in need thereof. In some embodiments, the methods comprise administering any of the liver organoids disclosed herein to the subject. In some embodiments, the liver organoid has been produced from cells derived from the subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells.

[0275] Also disclosed herein are methods for screening. In some embodiments, the methods comprise contacting any of the liver organoids disclosed herein with a candidate compound or composition, and assessing the effects of the candidate compound or composition on the liver organoid. In some embodiments, the liver organoid is a model for a liver-related disease or disorder, and assessing the effects of the candidate compound or composition on the liver organoid comprises assessing the effects of the candidate compound or composition on the liver-related disease or disorder. In some embodiments, the liver organoid has been produced from cells derived from a subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject has a liver-related disease or disorder.

Liver-Related Diseases and Disorders

[0276] The liver organoids of the disclosure can be used in treatment and/or studying or modeling liver-related diseases and disorders. In some embodiments, the methods include administering any of the liver organoids or liver cells disclosed herein. Also disclosed herein are the m liver organoids or liver cells disclosed herein for use in the manufacture of a medicament for the treatment of a liver-related disease or disorder. Also disclosed herein are the liver organoids or liver cells disclosed herein for use in the treatment of a liver-related disease or disorder in a subject in need thereof.

[0277] Liver-related diseases and disorders relevant to the disclosure can include conditions such as liver dysfunction and/or failure (e.g. hyperammonemia and/or hyperbilirubinemia, and the like), hepatitis (e.g. hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and/or autoimmune hepatitis, and the like), viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease (e.g. metabolic dysfunction-associated liver disease (MASLD), MetALD, nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), and the like), autoimmune liver disease, Wilson’s disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, hepatic cyst, and/or alcohol-associated liver disease. One skilled in the art will appreciate other liver-related diseases and conditions for which the liver organoids disclosed herein could have relevance.

[0278] For example, the liver organoid can be transplanted into a subject having liver dysfunction and/or failure, where the transplanted liver organoids engraft onto the liver of the subject. Following transplantation, the subject can have reduced serum bilirubin and/or ammonia levels, and/or increased serum protein albumin, and/or improved symptoms of biliary stricture and/or liver regeneration, and can also have increased survival rate.

[0279] For example, these liver organoids can be used an in vitro human model system for studying hepatocyte function and developmental divergence, studying liver-related disease, identifying and/or screening for therapeutic targets, and/or identifying therapeutic compounds and/or compositions effective in treating a liver-related disease or disorder. Accordingly, the liver organoids of the disclosure can allow for new developments in liver disease treatment and study.

Compositions

[0280] In some embodiments, also provided herein are compositions for performing any of the methods disclosed herein. In some embodiments, also provided herein are compositions produced according to processes provided in any of the methods disclosed herein. It is expressly contemplated that, in certain embodiments, any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0281] In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells and/or liver organoids produced using any one or more of the methods provided herein. In some embodiments, provided herein are compositions comprising at least a portion of the posterior foregut cells and/or posterior foregut endoderm cells that have spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure with a single lumen.

[0282] In some embodiments, compositions provided herein are in vitro compositions, created outside of a multicellular living organism. In some embodiments, compositions provided herein may be introduced into a multicellular living organism. In some embodiments, compositions provided herein comprise exogenously provided components, reagents, and/or conditions. In some embodiments, compositions provided herein comprise exogenously provided components, reagents, and/or conditions that mimic in vivo characteristics desirable for inducing specific cellular differentiation and/or organoid organization.

[0283] In some embodiments, provided herein are in vitro compositions comprising: posterior foregut cells and/or posterior foregut endoderm cells, at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator. In some embodiments, compositions may also comprise endogenous TGF-b pathway inhibitors, FGF pathway activators, Wnt pathway activators, and/or VEGF pathway activators. In some embodiments, compositions comprise posterior foregut cells and/or posterior foregut endoderm cells that have been dissociated from a monolayer and/or spheroid. In some embodiments, compositions comprise posterior foregut cells and/or posterior foregut endoderm cells at a cell density of greater than or equal to, exactly or about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities.

[0284] In some embodiments, provided herein are compositions comprising a tissue culture surface that is coated with a basement membrane matrix or component thereof. In some embodiments, a basement membrane matrix or component thereof does not comprise non-human animal components. In some embodiments, a basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans. In some embodiments, a basement membrane matrix or component thereof is not isolated from murine Engelbreth- Holm-Swarm (EHS) sarcoma cells, is not Matrigel®, is not Cultrex®, and/or is not Geltrex®. In some embodiments, a basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

[0285] In some embodiments, provided herein are compositions that include an exogenous TGF-b pathway inhibitor. In some embodiments, an exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of A83-01, RepSox, LY365947, and/or SB431542. In some embodiments, an exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of TGF-b pathway inhibitor A83-01. In some embodiments, a composition comprises a TGF-b pathway inhibitor at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a TGF-b pathway inhibitor at a concentration of, or of about, 500 nM.

[0286] In some embodiments, provided herein are compositions that include an exogenous FGF pathway activator. In some embodiments, a composition comprises an exogenous FGF pathway activator that comprises, consists essentially of, or consists of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and/or FGF23. In some embodiments, an exogenous FGF pathway activator comprises, consists essentially of, or consists of FGF2. In some embodiments, a composition comprises a FGF pathway activator at a concentration of, or of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a FGF pathway activator at a concentration of, or of about 5 ng/mL.

[0287] In some embodiments, provided herein are compositions that include an exogenous Wnt pathway activator. In some embodiments, a composition comprises an exogenous Wnt pathway activator that comprises, consists essentially of, or consists of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and/or TWS119. In some embodiments, a composition comprises an exogenous Wnt pathway activator that comprises, consists essentially of, or consists of CHIR99021. In some embodiments, a composition comprises a Wnt pathway activator at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, In some embodiments, a composition comprises a Wnt pathway activator at a concentration of, or of about, 3 pM.

[0288] In some embodiments, provided herein are compositions that include an exogenous VEGF pathway activator. In some embodiments, a composition comprises an exogenous VEGF pathway activator that comprises, consists essentially of, or consists of VEGF and/or GS4012. In some embodiments, a composition comprises an exogenous VEGF pathway activator that comprises, consists essentially of, or consists of VEGF. In some embodiments, a composition comprises a VEGF pathway activator at a concentration of, or of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a VEGF pathway activator at a concentration of, or of about 10 ng/mL.

[0289] In some embodiments, provided herein are compositions that include an exogenous EGF. In some embodiments, provided herein are compositions that do not include an exogenous EGF. In some embodiments, provided herein are compositions comprising EGF at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising EGF at a concentration of, or of about, 20 ng/mL.

[0290] In some embodiments, provided herein are compositions that include exogenous and/or transgenically produced ascorbic acid. In some embodiments, provided herein are compositions that do not include exogenous and/or transgenically produced ascorbic acid. In some embodiments, provided herein are compositions comprising ascorbic acid at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising ascorbic acid at a concentration of, or of about, 50 pg/mL.

[0291] In some embodiments, provided herein are compositions that include a ROCK inhibitor. In some embodiments, provided herein are compositions that do not include a ROCK inhibitor. In some embodiments, a ROCK inhibitor comprises, consists essentially of, or consists of Y-27632. In some embodiments, provided herein are compositions comprising a ROCK inhibitor at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising a ROCK inhibitor at a concentration of, or of about, 10 pM.

[0292] In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells that have and/or that are being differentiated from stem cells. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells that have and/or that are being differentiated from induced pluripotent stem cells. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells that have been passaged 1 time, 2 times, or 3 times. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells that have been passaged less than 4 times.

[0293] In some embodiments, provided herein are compositions comprising A83-01, FGF2, CHIR99021, VEGF, and/or Y-27632, optionally further comprising iPSCs, PSCs, and/or posterior foregut cells and/or posterior foregut endoderm cells.

[0294] In some embodiments, provided herein are compositions comprising: a) posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids, and b) a medium, wherein the medium optionally comprises hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid, and wherein the composition optionally additionally comprises c) a retinoic acid pathway activator. In some embodiments, compositions provided herein comprise a cMET tyrosine kinase receptor agonist. In some embodiments, compositions provided herein comprise a cMET tyrosine kinase receptor agonist that comprises, consists essentially of, or consists of hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and/or an agonist c-Met antibody (e.g., LMH85).

[0295] In some embodiments, provided herein are compositions comprising an IL-6 family cytokine. In some embodiments, an IL-6 family cytokine comprises, consists essentially of, or consists of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and/or cardiotrophin-like cytokine (CLC).

[0296] In some embodiments, provided herein are compositions comprising a corticosteroid. In some embodiments, a corticosteroid comprises, consists essentially of, or consists of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and/or mometasone.

[0297] In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with HGF, OSM, and/or dexamethasone. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with dexamethasone. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with HGF. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with OSM.

[0298] In some embodiments, provided herein are compositions comprising a retinoic acid pathway activator. In some embodiments, a retinoic acid pathway activator comprises, consists essentially of, or consists of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and/or AM580. In some embodiments, a retinoic acid pathway activator comprises, consists essentially of, or consists of retinoic acid. In some embodiments, compositions comprise a retinoic acid pathway activator at a concentration of, or of about, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, compositions comprise a retinoic acid pathway activator at a concentration of, or of about, 2.0 p M. [0299] In some embodiments, compositions comprise HGF. In some embodiments, compositions comprise HGF at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, compositions comprise HGF at a concentration of, or of about 10 ng/mL.

[0300] In some embodiments, compositions comprise OSM. In some embodiments, compositions comprise OSM at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, compositions comprise OSM at a concentration of, or of about 20 ng/mL.

[0301] In some embodiments, compositions comprise dexamethasone. In some embodiments, compositions comprise dexamethasone at concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, compositions comprise dexamethasone at a concentration of, or of about 100 nM.

[0302] In some embodiments, compositions comprise exogenous bilirubin. In some embodiments, compositions comprise both exogenous bilirubin and endogenous bilirubin. In some embodiments, compositions comprise a low concentration of exogenous bilirubin. In some embodiments, a low concentration of exogenous bilirubin is at or near a human fetal physiological concentration of bilirubin. Human fetal bilirubin levels are thought to be generally around 1 mg/L (0.1 mg/dL), which rises rapidly to 3-10 mg/L (0.3- 1.0 mg/dL) 24 hours after birth. In some embodiments, compositions comprise bilirubin, exogenous and/or endogenous, that is, is about, is less than, or is less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or at any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L. In some embodiments, compositions comprise exogenous bilirubin at a concentration that is, is about, is less than, or is less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or at any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.

[0303] In some embodiments, provided herein are compositions comprising mature liver organoids. In some embodiments, provided herein are compositions comprising mature liver organoids that exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape. In some embodiments, provided herein are compositions comprising mature liver organoids that were produced through contact with a low dose of exogenous bilirubin. In some embodiments, provided are compositions comprising mature liver organoids that express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid not contacted with a low dose of bilirubin. In some embodiments, provided are compositions comprising mature liver organoids expressing increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low dose of bilirubin. In some embodiments, provided are compositions comprising mature liver organoids expressing CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, provided are compositions comprising mature liver organoids that exhibit increased CYP3A4 and/or CYP1A2 activity relative to liver organoids that were not contacted with a low dose of bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids, wherein the cells of the mature liver organoid were contacted with a low dose of exogenous bilirubin, and the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape. In some embodiments, provided herein are compositions comprising mature liver organoids that express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids expressing increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids expressing CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, provided herein are compositions comprising mature liver organoids exhibiting increased CYP3A4 and/or CYP1A2 protein levels and/or enzymatic activity, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin. [0304] In some embodiments, provided herein are compositions comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells were contacted with a high and/or second concentration of bilirubin. In some embodiments, provided herein are compositions comprising hyperbilirubinemia liver organoids, wherein a high/second concentration of bilirubin was, was about, was more than, or was more than about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L. In some embodiments, provided herein are compositions comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

[0305] Also provided herein, in some embodiments, are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that have been engineered to comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are able to synthesize ascorbate. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids engineered to express functional GULO protein, wherein the functional GULO protein is murine GULO (mGULO). In some embodiments, a gene that encodes for a functional GULO protein is conditionally expressed. In some embodiments, a gene that encodes for a functional GULO protein is constitutively expressed. In some embodiments, a gene that encodes for a functional GULO protein is conditionally expressed using a tetracycline inducible system.

[0306] In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that are engineered to comprise a gene that encodes for a functional GULO protein using CRISPR mediated knock-in. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids comprising a functional GULO encoding gene or mRNA, or both, that encodes for a functional GULO protein, wherein the functional gene was introduced to the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids, mature liver organoids, and/or precursor cells by transfection. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that are engineered to comprise a gene that encodes for a functional GULO protein using adenovirus mediated gene transfection. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that are engineered to comprise a gene that encodes for a functional GULO protein using adeno-associated virus mediated gene transfection.

[0307] In some embodiments, compositions provided herein comprise liver organoids and/or mature liver organoids comprising a functional GULO protein, wherein said liver organoids and/or mature liver organoids express increased levels of NRF2 relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein. In some embodiments, compositions provided herein comprise liver organoids and/or mature liver organoids comprising a functional GULO protein, wherein the liver organoids and/or mature liver organoids express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit reduced caspase-3 activity relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising a functional GULO protein express increased levels of ALB relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising a functional GULO protein resemble periportal liver tissue and/or express periportal liver markers. In some embodiments, periportal liver markers comprise or consist of FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit increased CYP3A4 and/or CYP1 A2 protein levels and/or enzymatic activity relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit increased viability in culture relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.

[0308] Also provided herein, in some embodiments, are compositions according to Table 1, Table 2, or Table 3.

[0309] In some embodiments, provided herein are compositions comprising an amino acid supplemented liquid component comprising exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% non-essential amino acid solution by volume (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL glycine.

[0310] In some embodiments, provided herein are compositions comprising an amino acid supplemented liquid component comprising exactly or about 14% non-essential amino acid solution (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 6% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 80% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 20 g/L glycine. In some embodiments, compositions provided herein may comprise a pH of between about pH 6-8, or pH 6.5-7.5, or exactly or about pH 7.0. In some embodiments, compositions provide herein comprise hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid. [0311] In some embodiments, compositions provide herein comprise hepatic lineage committed cells differentiated from definitive endoderm cells using retinoic acid. In some embodiments, compositions provide herein comprise hepatic lineage committed cells that are characterized as liver organoids. In some embodiments, compositions provide herein comprise liver organoids that are characterized as secreting increased levels of albumin and/or urea relative to liver organoids comprised in HCM without amino acid supplementation. In some embodiments, compositions provide herein comprise liver organoids that are characterized as expressing increased levels of hepatic maturation associated gene expression relative to liver organoids comprised in HCM without amino acid supplementation. In some embodiments, compositions provide herein comprise liver organoids that are characterized as expressing reduced levels of Vimentin relative to liver organoids comprised in HCM without amino acid supplementation. In some embodiments, compositions provide herein expressly do not comprise non-human animal basement membrane matrix or components thereof. In some embodiments, compositions provided herein expressly do not comprise murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and/or Geltrex®.

[0312] In some embodiments, also provided herein are hyperbilirubinemia liver organoids comprising a naturally occurring and/or engineered mutation in the UDP glucuronosyltransferase family 1 member Al (UGT1A1) gene. In some embodiments, provided herein are hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid was produced through at least two rounds of contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids to exogenous bilirubin. In some embodiments, provided herein are hyperbilirubinemia liver organoids that were produced from clonally derived cells and/or iPSCs.

[0313] In some embodiments, provided herein are cryopreserved compositions comprising liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT). In some embodiments, provided herein are cryopreserved compositions comprising mature liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT). In some embodiments, provided herein are cryopreserved compositions comprising hyperbilirubinemia liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

[0314] Pharmaceutical compositions in accordance with various embodiments of the disclosure can include one or more additional pharmaceutically acceptable components, which can include carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or earner approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and/or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and/or earners can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0315] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, b- propiolactone, gelatin, cell debris, nucleic acids, peptides, ammo acids, or growth medium components or any combination thereof. The amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w/w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0316] Pharmaceutical compositions can include one or more “pharmaceutically acceptable salts”, which can include relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono- , di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; ammo acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N- methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N- benzylphenethylamine; trihydroxymethyl ammoethane.

[0317] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra- articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0318] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and can refer to a compound, particle, solid, semi- solid, liquid, or diluent that facilitates the passage, delivery and/or incorporation of a compound to cells, tissues and/or bodily organs.

[0319] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and can refer to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.

Dosage and Administration Routes

[0320] Embodiments of the disclosure can include methods of administering or treating an animal, which can involve administering an amount of at least one treatment, that is effective to treat the disease, condition, or disorder that the organism has, or is suspected of having, or is susceptible to, or to bring about a desired physiological effect. In some embodiments, the disease, condition, or disorder can be a liver-related disease or disorder.

[0321] In some embodiments, at least one treatment can include a composition or pharmaceutical composition, which can be administered to an animal (e.g., mammals, primates, monkeys, or humans) in an amount of about 0.005 to about 50 mg/kg body weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about 15 mg/kg. In regard to some conditions, the dosage can be about 0.5 mg/kg human body weight or about 6.5 mg/kg human body weight. In some instances, some subjects (e.g., mammals, mice, rabbits, feline, porcine, or canine) can be administered a dosage of about 0.005 to about 50 mg/kg body weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg, about 100 mg/kg, or about 150 mg/kg. Of course, those skilled in the art will appreciate that it is possible to employ many concentrations in the methods of the present disclosure, and using, in part, the guidance provided herein, will be able to adjust and test any number of concentrations in order to find one that achieves the desired result in a given circumstance. In some embodiments, a dose or a therapeutically effective dose of a compound disclosed herein will be that which is sufficient to achieve a plasma concentration of the compound or its active metabolite(s) within a range set forth herein, e.g., about 1-10 nM, 10-100 nM, 0.1-1 pM, 1-10 pM, 10-100 pM, 100-200 pM, 200- 500 pM, or even 500-1000 pM, preferably about 1-10 nM, 10-100 nM, or 0.1-1 pM.

[0322] In other embodiments, a treatment can be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

[0323] The compounds and pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disease or disorder, age and body weight of the subject, different daily doses can be used.

[0324] Under certain circumstances, however, higher or lower daily doses can be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.

[0325] A treatment can be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease or disorder and the weight and general state of the subject. Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for treatment of particular disorders.

[0326] Various considerations are described, e. g. , in Langer, 1990, Science, 249: 1527; Goodman and Gilman's (eds.), 1990, Id., each of which is herein incorporated by reference and for all purposes. Dosages for parenteral administration of active pharmaceutical agents can be converted into corresponding dosages for oral administration by multiplying parenteral dosages by appropriate conversion factors. As to general applications, the parenteral dosage in mg/mL times 1.8 = the corresponding oral dosage in milligrams (“mg”). As to oncology applications, the parenteral dosage in mg/mL times 1.6 = the corresponding oral dosage in mg. An average adult weighs about 70 kg. See e.g., Miller-Keane, 1992, Encyclopedia & Dictionary of Medicine, Nursing & Allied Health, 5th Ed., (W. B. Saunders Co.), pp.1708 and 1651.

[0327] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0328] In some embodiments, the administration can include a unit dose of one or more treatments in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. In certain embodiments, the carrier, vehicle or excipient can facilitate administration, delivery and/or improve preservation of the composition. In other embodiments, the one or more carriers, include but are not limited to, saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. Carriers can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. In other embodiments, the one or more excipients can include, but are not limited to water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. [00275] The quantity of active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents. [0329] A treatment can be administered to subjects by any number of suitable administration routes or formulations. The treatment, such as an immunotherapy, can also be used to treat subjects for a variety of diseases. Subjects include but are not limited to mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats. In particular embodiments described herein, the subject is a human.

[0330] The route of administration of the compounds of the treatments described herein can be of any suitable route. Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route. In other embodiments, administration routes can be parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. The choice of administration route can depend on the compound identity (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the particular disease (e.g., type of cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of administration routes can be administered, as desired.

[0331] Some embodiments of the disclosure include a method for providing a subject with a treatment which comprises one or more administrations of one or more compositions; the compositions may be the same or different if there is more than one administration.

Toxicity

[0332] The ratio between toxicity and therapeutic effect for a particular treatment is its therapeutic index and can be expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and ED50 (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from in vitro assays, cell culture assays and/or animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.l, p.l, 1975. The exact formulation, route of administration, and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used. For in vitro formulations, the exact formulation and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used.

Kits

[0333] In some embodiments, also disclosed herein are kits providing means for performing any of the methods described herein. In some embodiments, also disclosed herein are kits comprising any of the compositions or means of producing the compositions described herein.

[0334] In some embodiments, a kit can be prepared from readily available components and reagents. For example, such kits can comprise any one or more of the following components and/or reagents: enzymes, reaction tubes, buffers, detergent, primers, probes, antibodies, cell culture media, differentiation induction reagents, amino acid mixtures/supplements, engineered constructs and/or polynucleotides, transcription induction agents, bilirubin, ascorbic acid, retinoic acid pathway activators, corticosteroids, cMET tyrosine kinase receptor agonists, IL-6 family cytokines, TGF-b pathway inhibitors, FGF pathway activators, Wnt pathway activators, VEGF pathway activators, ROCK inhibitors, and/or cells. In some embodiments, components and reagents may be packaged together in any combination, and/or may be packaged individually. In some embodiments, kits may include components and reagents concentrated above the working concentrations disclosed herein, or at the working concentrations provided herein. In some embodiments, individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. In some embodiments, concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more. In some embodiments, a kit may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0335] In some embodiments, a kit is housed in a container. Kits may further comprise instructions for using the kit for assessing expression and/or differentiation of cells. Agents in a kit for measuring expression and/or determining differentiation may comprise a plurality of PCR probes and/or primers for qRT-PCR and/or a plurality of antibody or fragments thereof for assessing expression of biomarkers appropriate for classifying cell states. [0336] In some embodiments, kits are created using and comply with good manufacturing practice (GMP).

[0337] Having described the embodiments in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the embodiments defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

[0338] The following non-limiting examples are provided to further illustrate embodiments disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the embodiments, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the embodiments disclosed herein.

Example 1. Protocol for freezing liver organoids

Cryopreservation protocol using CELLBANKER® 1

[0339] CELLBANKER® 1 (AMSBIO) containing 50 nM chroman 1, 5 pM emricasan, 1:1000 polyamine, and 7 pM trans-ISRIB (CEPT) was kept on ice or at 4°C.

[0340] Day 20-24 human liver organoids (HLOs) were collected from 6 well plates and filtered through a 150 pM mesh into a 50 mL conical tube (total volume should not exceed approximately 25 mL). Approximately 20 mL of cold PBS was added to reach a final volume of approximately 45 mL. The suspension was pipetted up and down five times to wash the organoids. The tube was then centrifuged at 300xg for 5 minutes at 4°C to pellet the HLOs. The supernatant was aspirated and another wash with 10-20 mL of fresh PBS, centrifugation, and aspiration was repeated. The washed HLOs were resuspended in the CELLBANKER® 1 + CEPT freezing medium into 2 cryovials per well of a 6 well plate, with 0.75 mL of freezing medium suspension per cryovial. The cryovials were then transferred to a freezer container at -80°C for 24 hours, and then to liquid nitrogen for longer storage.

Cryopreservation protocol using Cell Reservoir One Vitrify

[0341] Cell Reservoir One Vitrify (Nacalai) containing CEPT was kept on ice or 4°C. [0342] The same HLO collection and washing steps as described above were performed. The washed HLOs were resuspended in the Cell Reservoir One Vitrify + CEPT freezing medium and transferred quickly to a cryopreservation tube. The tube was immersed at 2/3 height in liquid nitrogen using tweezers for 10 seconds, and then the tube was immersed completely. This step should be performed within 60 seconds or faster. The tube was then transferred to a liquid nitrogen storage tank for long term storage.

Thawing protocol for CELLBANKER® 1

[0343] Standard thawing procedure can be performed. Briefly, the cryovial was submerged at 37 °C water for up to 2 minutes until a small amount of solid medium is left in the vial. Pre-warmed medium is added to the vial and the contents transferred to a conical tube with fresh culture medium. The suspension was pipetted to wash the thawed HLOs, and then centrifuged at 300xg for 5 minutes at room temperature. The medium was aspirated, and the HLOs were resuspended in fresh medium with CEPT (and optionally Matrige®!).

Thawing protocol for Cell Reservoir One Vitrify

[0344] 10 mL of cell culture medium was pre-warmed in a centrifugation tube at

37°C. A sterile plate was also pre-warmed at a 37°C incubator. The cryopreservation tube containing frozen cells was removed from the liquid nitrogen storage tank, the cap was removed, and any liquid nitrogen in the tube was discarded. The HLOs were thawed quickly by adding more than 800 pL of the pre-warmed cell culture medium to the tube and pipetting a few times. The larger the volume used, the quicker the sample thaws. A suitable volume of medium should be added depending on the tube size. The thawed cell suspension was then transferred to a sterile centrifugation tube. Fresh cell culture medium was used to wash the cryopreservation tube and added with the thawed cell suspension. The HLOs were centrifuged at 300xg for 5 minutes at room temperature. The medium was aspirated, and the HLOs were resuspended in fresh medium with CEPT (and optionally Matrigel®).

Example 2. Freeze and thawing of liver organoid cultures

[0345] Human liver organoids (Day 22 of culture) were frozen in CELLBANKER® 1 or Vitrify (24 hours at -80°C and then transferred to liquid nitrogen storage). An exemplary protocol is provided in Example 1.

[0346] For testing, organoids were thawed and immediately put into hepatocyte culture medium. Live/dead staining was done the same day as thawing. In addition, the culture medium of the day of thaw and 48 hours later were collected to detect albumin secretion. A schematic of this is provided in FIG. 1A. [0347] FIG. IB shows the results of live/dead staining of thawed liver organoids. The organoids were stained with Calcein AM (which labels live cells), ethidium homodimer- 1 (which labels dead cells), and NucBlue (which labels nuclei). The human liver organoids showed less damage after short-term freezing, suggesting that freezing is a viable approach to transport the organoids. The survival rate of the organoids subjected to vitrification (e.g., using Cell Reservoir One Vitrify) was slightly higher than that of organoids cryopreserved by slow freezing (e.g., using CELLBANKER® 1).

[0348] FIG. 1C shows the results of measuring albumin (ALB) secretion of liver organoids thawed according to the protocols provided in Example 1, compared to an unfrozen liver organoid control. Liver organoids did not exhibit albumin secretion immediately after thawing after either slow freezing or vitrification cryopreservation. However, liver organoids cryopreserved by vitrification recovered ALB secretion function up to 70% compared to nonfrozen control after 3 days of recovery culture.

Example 3. Enhanced maturation of liver organoids in 2D and 3D

[0349] Approaches for promoting maturation of liver organoids in culture were explored. Previous methods for liver organoid growth have used hepatocyte culture medium, and supplementation of this medium with additional amino acids was tested. Amino acid (AA) supplementation medium was prepared as a mixture of 14% non-essential amino acid solution (lOOx, containing alanine, asparagine, aspartic acid, glycine, serine, proline, and glutamic acid; ThermoFisher, Grand Island, NY), 6% essential amino acid solution (50x, containing arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine; ThermoFisher, Grand Island, NY), and 80% hepatocyte culture medium (HCM) (Hepatocyte Culture Medium BulletKit; Lonza, Walkersville, MD), neutralized to pH 7.0 using NaOH, and further supplemented with 20 g/L glycine. Optionally, the hepatocyte culture medium may be supplemented with hepatocyte growth factors including but not limited to hepatocyte growth factor (HGF), oncostatin M, and/or dexamethasone.

[0350] Liver organoids were prepared by differentiation from definitive endoderm and induction for hepatic lineage commitment using retinoic acid according to conventional approaches, but cultured in AA supplementation medium for 8, 10, or 12 days after retinoic acid induction.

[0351] Expression of the liver- specific genes albumin (ALB), cytochrome P450 family 3 subfamily A member 4 (CYP3A4), phosphoenolpyruvate carboxykinase 1 (PCK1), and glucose-6-phosphate catalytic subunit (G6PC) by these liver organoids were quantified by RT-qPCR, using liver organoids cultured in HCM without AA supplementation as control (FIG. 2A). The liver organoids cultured in AA supplementation medium for 8, 10, and 12 days exhibit enhanced expression of all of these genes relative to control liver organoids.

[0352] Secretion of ALB and production of urea were also quantified in liver organoids cultured in AA supplementation medium for 8, 10, and 12 days (and 14 days for the urea quantification) after retinoic acid induction, compared to liver organoids cultured in HCM without AA supplementation as control (FIG. 2B). Prolonged exposure to AA supplementation medium (12 or 14 days) resulted in enhanced albumin secretion and urea production compared to control liver organoids.

[0353] Pluripotent stem cells were engineered with a luciferase reporter driven by the PCK1 promoter, which enables measurement of gluconeogenesis, and differentiated into liver organoids. An exemplary PCK1 luciferase reporter can be found in PCT Publication WO 2021/262676, which is hereby expressly incorporated by reference in its entirety. PCK1 expression in these liver organoids with this construct can be quantified by luciferase activity, either with live cells, or after lysis. A schematic of this process is provided in FIG. 2C.

[0354] Liver organoids were grown in AA supplementation medium, or HCM without AA supplementation as control. Brightfield images of these organoids are shown in FIG. 2D. Liver organoids grown in AA supplementation medium exhibited increased PCK1 expression compared to control, as measured by luciferase activity in both live cells and after lysis (FIG. 2E).

[0355] The use of AA supplementation medium can also be extended beyond 3D organoids to two-dimensional (2D) hepatocyte culture from pluripotent stem cell differentiation. To test the effects of the medium on 2D hepatocyte cultures, AA supplementation media was used to replace HCM without AA supplementation after hepatic induction at various times of culture (day 12, 14, 16, 18, 21, or 34 of culture, counted from the initial pluripotent stem cell differentiation). A schematic for this process is shown in FIG. 3A, where the A is HCM only, B is HCM + AA starting at day 12, and C-G are replacing HCM with HCM + AA at days 14, 16, 18, 21 and 34, respectively.

[0356] Brightfield images of the 2D hepatocyte cultures grown with AA supplementation medium supplemented at various time points are shown in FIG. 3B. The cells in all conditions exhibit normal hepatocyte morphology.

[0357] Lactate production by the 2D hepatocyte cultures grown in AA supplementation medium or HCM without AA supplementation was compared (FIG. 3C). For each of the time points, fresh medium was replaced the previous day, and the samples for testing were taken after 24 hours. The AA supplementation medium- grown hepatocytes exhibited reduced lactate, suggesting a metabolic switch from a glucose dependent to a pyruvate dependent metabolism relative to cells grown in conventional hepatocyte grown medium.

[0358] Albumin secretion by the 2D hepatocyte cultures following substitution of HCM without AA supplementation with AA supplementation medium at various time points was quantified (FIG. 3D). The hepatocytes where AA supplementation medium was added after 12 days of culture exhibited the greatest albumin secretion. Increased albumin secretion relative to control hepatocytes were also observed for even longer culture durations (FIG. 3E).

[0359] Gene expression of ALB, E-cadherin (E-cad). CYP3A4, G6PC, pyruvate kinase Ml/2 (PKM), and PCK1 was quantified in day 58 2D hepatocytes grown in either HCM without AA supplementation or AA supplementation medium added at various times of culture (FIG. 3F). Overall, AA supplementation enhances hepatic maturation and gene expression. Interestingly, PCK1 and G6PC expression (which is suppressed by insulin normally added in the culture media) was reduced with increasing durations of AA supplementation, suggesting that the addition of AA normalizes the metabolism of liver organoids.

[0360] Pluripotent stem cells were engineered to express mScarlet under a PCK1 promoter, which acts as a reporter for gluconeogenesis, and differentiated to hepatocytes in a 2D culture. The hepatocytes were grown in either HCM without AA supplementation or AA supplementation medium. Prior to fluorescent quantification, the cells were also optionally cultured for 24 hours without insulin (in DMEM/F12 supplemented with 0.2% BSA). FIG. 3G shows the mScarlet fluorescent images of these hepatocytes. The cells grown in AA supplementation medium exhibited increased mScarlet expression driven by the PCK1 promoter after 24 hours of insulin starvation, suggesting that these cells exhibit increased metabolism compared to control.

[0361] Day 57 2D hepatocyte cultures grown in either HCM without AA supplementation or where AA supplementation medium was substituted at day 12 were probed for EpCAM (epithelial marker), Vimentin (mesenchymal marker), and either DAPI or hepatocyte nuclear factor 4 alpha (HNF4a) (nucleus) (FIG. 3H). While hepatocytes differentiated from pluripotent stem cells that were cultured in HCM without AA supplementation exhibited Vimentin positive mesenchymal cells, this Vimentin signal was absent in the cells cultured with AA supplementation medium, suggesting that culturing cells in AA supplementation media potentially reduces mesenchymal cells and enriches hepatocytes. Example 4. Generation of human organoids without Matrigel® embedding

[0362] Culture of 3D human liver organoids without the use of Matrigel®, which contains xenogeneic components, was briefly explored in PCT Publication WO 2018/085615, which is hereby expressly incorporated by reference in its entirety. A schematic for this protocol is depicted in FIG. 4A. Posterior foregut cells and/or posterior foregut endoderm cells differentiated from pluripotent stem cells spontaneously form three dimensional structures after retinoic acid induction and subsequent hepatic culture conditions (e.g., contacting with hepatocyte growth factor, oncostatin M, and dexamethasone). These three-dimensional structures can be pipetted from the original cell layer and further cultured to grow into liver organoids (FIG. 4B). These liver organoids grown in Matrigel®-free conditions exhibit normal organoid morphology (FIG. 4C) and expected hepatic function, such as albumin secretion (FIG. 4D) and expression of ALB, alpha fetoprotein (AFP), HNF4a, retinol binding protein 4 (RBP4). and alpha- 1 antitrypsin (AAF) (FIG. 4E).

[0363] Organoids grown without the use of Matrigel® are advantageous as they are not grown with xenogeneic animal components and so can be used for human purposes. Furthermore, they are able to grow to relatively large sizes that are easier to handle. However, these organoids may have greater size variation than organoids grown in Matrigel®, may exhibit abnormal morphology (as Matrigel® helps maintain the 3D form), and may be less amenable to cryopreservation (which may reduce scaling for manufacturing purposes). In addition, conventional protocols also involve the use of Matrigel® during culture steps other than organoid maturation, such as pluripotent stem cell culture.

[0364] To overcome one or more of these potential disadvantages, alternative approaches for culturing organoids without Matrigel® or other basement membrane matrix with xenogeneic components was explored.

[0365] Methods for culturing foregut cells after initial induction using a combination of FGF2, EGF, VEGF, CHIR99201, and A83-01 were briefly explored in PCT Publication WO 2018/191673. A modified version of this combination with or without EGF may be used to expand foregut cells, which arise from differentiation of definitive endoderm cells with an FGF pathway activator (e.g., FGF4) and a Wnt pathway activator (e.g., CHIR99021).

[0366] A schematic for expanding foregut cells prior to subsequent differentiation to a hepatic lineage is depicted in FIG. 5A. After induction of definitive endoderm to foregut, the cells may be dissociated to single cells using a standard enzymatic dissociation solution such as Accutase and then passaged in EP medium (Advanced DMEM/F12, B27/N2/HEPES/Glutamax, 5 ng/mL FGF2, 10 ng/mL VEGF, 3 pM CHIR99021, 500 nM A83- 01, and 50 pg/mL ascorbic acid) optionally supplemented with 10 pM Y-27632 (ROCK inhibitor).

[0367] The ability for these dissociated foregut cells to grow on substrates other than Matrigel® for expansion purposes was explored. Culture plates were coated with Matrigel® or laminin, which is basement membrane matrix component that can be manufactured and procured without animal products. A basement membrane matrix-like coating is preferred for culture, as the foregut cells will otherwise not properly adhere to the plate for growth. Foregut cells expanded on both Matrigel® and laminin coated plates properly grow and begin forming 3D spheroids over the course of 7 days of culture (FIG. 5B). After additional culture (Days 8- 10), fully formed spheroids, which can be further matured to liver organoids, are observed (FIG. 5C). While there was no significant difference in the rate of initial cell adhesion, the efficiency of spheroid formation was better with the laminin coating over the Matrigel® coating.

[0368] The number of dissociated foregut cells used to seed the plates for expansion was also tested. l.OxlO⁶ or 5.0xl0⁶ foregut cells were seeded onto laminin coated 6-well plates (9.6 cm² surface area per well) and grown for 5 days (FIG. 5D). Spheroid formation begins earlier as the number of seeded cells increases (as seen in the larger spheroids for the 5.0xl0⁶ cell seeded plate at each time point). Accordingly, spheroid formation is dependent on cell density.

[0369] As the expanded foregut cells spontaneously form spheroids, they may be used as a source of spheroids for manufacturing scaling. 3D spheroids that form spontaneously were collected from Day 12 foregut cell culture and use for liver organoid maturation. By continuing the culture of the plates after spheroid collection, additional spheroids formed over the course of 23 additional days (up to Day 35 of culture) (FIG. 5E). These newly formed spheroids exhibited the same capacity to mature into liver organoids.

[0370] After the initial dissociation, plating, and recovery of the foregut cells, additional passages of the proliferative foregut cells may be performed. Four passages (passaging every 10 days) from an initial Day 8 foregut culture differentiated from pluripotent stem cells was tested. Spheroids formed up to the third passage, but not the fourth passage (FIG. 5F). In the fourth passage, mesenchymal cells were predominantly proliferating. Three passages from the initial foregut culture can yield an approximately 200-fold increase in cell number, offering significant advantages for scaling. Gene expression for caudal type homeobox 2 (CDX2), forkhead box A2 (FOXA2), AFP, Vimentin (VIM), SRY-Box transcription factor 17 (SOX 17), HNF4a, and ALB were quantified for foregut cells from each passage (FIG. 5G). Expression of CDX2 and SOX17 was maintained in passage 4. FOXA2 and HNF4a expression peaked in passage 2.

[0371] Overall, to obtain continuous formation of human liver organoids from single cell passaging of foregut cells, the dissociated foregut cells can be grown on a laminin (e.g., Laminin-511) coating in EP medium (comprising FGF4), and with a dense plating number (e.g., 5xl0⁶ cells/6 well plate [9.6 cm²]). The foregut cells should be passaged for a maximum of 3 passages, as mesenchymal cells predominantly grow in subsequent passages and spheroids cease forming. A schematic for liver organoid formation starting from pluripotent stem cells and including foregut cell passaging for scaling is exemplified in FIG. 5H, where “expandable foregut organoid” refers to the spontaneously formed foregut spheroids which are collected for maturation into liver organoids. The “organoid reformation” step represents the continued passage of the foregut cells, and spontaneous formation of additional foregut spheroids, which are collected and matured into liver organoids.

[0372] This process of expanding foregut cells to increase production of liver organoids may be further modified with the use of microwell plates (e.g., an Aggrewell plate (StemCell Technologies)) or other apparatus such as a formed plate that is designed to aggregate foregut cells for more uniform organoid formation. These approaches of using apparatuses for producing uniform organoids is explored in PCT Publication WO 2021/030373, which is hereby expressly incorporated by reference in its entirety. A schematic for liver organoid formation starting from pluripotent stem cells and including foregut cell passaging for scaling as well as the use of an apparatus to aggregate foregut cells is exemplified in FIG. 51.

Example 5. Scalable 3D bioreactor liver organoid formation

[0373] Matrigel®-free liver organoids are difficult to maintain in static culture. The use of 3D rotational culture to maintain freely suspended liver organoids was explored. A schematic for this process is provided in FIG. 6A. Spheroids formed from foregut induction (e.g., those formed on a basement membrane matrix or component thereof that does not comprise non-human animal components as described herein) and culture using methods described in the previous example were collected and grown in hepatocyte culture medium in a rotational culture. The spheroids were able to mature into liver organoids over the course of 15 days in this rotational culture condition (FIG. 6B). Gene expression of AFP, HNF4a, FOXA2, ALB, CDX2, and VIM was quantified in liver organoids grown in rotational culture (3D) and compared with passage 1 (Pl) foregut cells (FIG. 6C). The liver organoids exhibited high expression of ALB and AFP, which is indicative of mature liver cells. The expression of CDX2 and HNF4a was not significantly different from the foregut cells. Accordingly, this demonstrates that liver organoids may be cultured without the need for Matrigel® or other basement membrane matrix when grown in rotational culture.

Example 6. Additional materials and methods

Animals

[0374] All animal experiments were conducted with the approval of an Institutional Review Board and Institutional Animal Care and Use Committee. Adult Gunn (Gunn- Ugtlalj/BluHsdRrrc) rats (breeding pairs, 9-12 weeks old) were obtained from the Rat Resource & Research Center (RRRC, Columbia, MO). Rats were housed in standard rat cages with woodchip bedding, maintained at a temperature of 20-24°C and relative humidity of 45- 55%, under a 12 h:12 h light:dark cycle. All animals had ad libitum access to standard chow (Cincinnati Lab Supply, Cincinnati, OH) before study. All animals were treated in accordance with the guidelines and regulations of the institution.

Maintenance of PSCs

[0375] A human iPSC line, 72.3 (RRID: CVCL_A1BW) was obtained from Cincinnati Children’s Hospital Medical Center (CCHMC) Pluripotent Stem Cell Facility codirected by CN. Mayhew and JM. Wells. Undifferentiated hiPSCs were cultured on Laminin- 511 E8 fragment (Nippi) coated dishes in StemFit medium (Ajinomoto Company) with 100 ng/mL basic fibroblast growth factor (FGF; R&D Systems) at 37°C in 5% CO2 with 95% air.

Human liver organoid (HLO) generation

[0376] Pluripotent stem cells were plated on a 24 well plate coated with Laminin iMatrix-511 Silk at a density of 2xl0⁵ cells/well and maintained with StemFit media with Y- 27632. On Day 2, the media was replaced with fresh StemFit media. The following day, the cells were treated with RPMI media mixed with Activin A and BMP4 to generate definitive endoderm. On Day 4, the media was replaced with RPMI, Activin A and 0.2% dFBS, which was changed to 2% dFBS on Day 5. From Day 6-8, the cells were fed with FGF4 and CHIR99021 in Advanced DMEM (supplemented with B27, N2, 10 mM HEPES, 2 mM L- glutamine, and gentamicin-amphotericin) to induce posterior foregut. On Day 9, the cells were dissociated into a single cell suspension using Accutase treatment. This single cell suspension was then mixed with 50% Matrigel® and 50% EP media (Advanced DMEM/F12, B27/N2/HEPES/Glutamax, 5 ng/mL FGF2, 10 ng/mL VEGF, 3 p M CHIR99021, 500 nM A83- 01, and 50 pg/mL ascorbic acid) and plated as 50 pl drops in a 6-well plate. These cells were fed with EP media every 48 hours for 4 days to generate organoids. These organoids were then treated with Advanced DMEM and retinoic acid (RA) every 48 hours for 4 days to specify the hepatic lineage. The organoids were then fed with hepatocyte culture medium (HCM), hepatocyte growth factor (HGF), Oncostatin M, and Dexamethasone every 3-4 days to generate HLOs.

Maturation of HLOs with low dose bilirubin

[0377] For maturation induction in HLOs, the day 15 organoids were fed with complete HCM with supplements in addition to low dose bilirubin (1 mg/L). This treatment was continued until day 30 when the organoids were harvested. The brightfield images were captured on the KEYENCE BZ-X710 Fluorescence Microscope (Keyence), and the images were analyzed using ImageJ suite. RNA was isolated using the Rneasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) according to manufacturer’s protocol. qPCR was carried out using TaqMan gene expression master mix (Applied Biosystems) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific Inc.). All primers and probe information for each target gene was obtained from the Universal ProbeLibrary Assay Design Center website (available on the World Wide Web at lifescience.roche.com/en_us/brands/universal-probe-library.html). For whole mount immunostains, the organoids were fixed in 4% PFA, permeabilized with 0.1% PBST, blocked with 5% Normal Donkey Serum in 0.1% PBST, and stained with the appropriate primary and secondary antibodies. The images were captured on the Nikon Al inverted confocal microscope.

Bilirubin and drug treatment

[0378] On day 27, the mature organoids were treated with bilirubin (1-10 mg/L), doxycycline (100 ng/mL; added 3 days prior to activate gene expression) and/or additionally with drugs such as hydrocortisone, dexamethasone, ketoconazole and mifepristone (1-2 pM for each) for 5 days and then harvested for downstream assays. Bilirubin assay was performed using a colorimetric kit (ab235627 from Abeam). RNA was isolated using the Rneasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) according to manufacturer’s protocol. qPCR was carried out using TaqMan gene expression master mix (Applied Biosystems) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific Inc.). All primers and probe information for each target gene was obtained from the Universal ProbeLibrary Assay Design Center (available on the World Wide Web at lifescience.roche.com/en_us/brands/universal-probe-library.html). Images were captured on the KEYENCE BZ-X710 Fluorescence Microscope (Keyence).

Organoid transplantation into the portal vein

[0379] HLOs were harvested on Day 27 and dissociated into organoid fragments by repeated pipetting, washed with PBS and resuspended with HCM containing 2% FBS and CEPT cocktail (50 nM chroman 1, 5 pM emricasan, 1:1000 polyamine, and 7 pM trans-ISRIB) to increase viability. The recipient rats were treated with a single dose of retrorsine (5 mg/kg) and tacrolimus (0.8 mg/kg) 4 days prior to the transplantation. A midline incision was drawn, the intestines pushed to one side, and a 32g 1 inch needle was used to inject 3xl0³ organoids (roughly 5xl0⁵ cells) in a 200 pL infusion into the portal vein by clamping it down posteriorly to control bleeding. Excessive blood loss was prevented by application of a SURGICEL SnoW Absorbable Hemostat (Ethicon). The animal was then closed with 5-0 vicryl coated surgical sutures (Ethicon) and GLUture (Zoetis Inc.), and buprenorphine (0.1 mg/kg) was administered as an analgesic. The animal was then maintained on doxycycline (2 mg/kg) and tacrolimus injections every 3-4 days until the day of harvest. Blood was collected regularly by the retro- orbital method as needed.

Live cell imaging and functional assay

[0380] For live imaging of organoids, the Celldiscoverer 7 (Zeiss) was used to image every 30 minutes for 7 days. Visualization of bilirubin conjugation was achieved with 5 pM fluorescent UnaG, which was incubated with HLO media and imaged for 2 days.

RNA sequencing and analysis

[0381] RNA was isolated using the Rneasy mini kit (Qiagen). Reverse transcription was carried out using the High-Capacity cDNA Reverse Transcription Kit for RT-PCR (Applied Biosystems) according to manufacturer protocol. qPCR was carried out using TaqMan gene expression master mix (Applied Biosystems) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). All the samples were amplified with TaqMan Gene Expression Assays and normalized with 18S rRNA Endogenous Control. For RNA sequencing, the extracted RNA quality was evaluated with an Agilent 2100 Bioanalyzer (Agilent). A sequence library was prepared using a TruSeq Stranded mRNA kit (Illumina) and sequenced using NovaSeq 6000 (Illumina). Reads were aligned to human genome assembly hg38 and quantified using the quasi-mapper Salmon (vl.8.0). Gene-expression analysis was performed using the R Bioconductor package DESeq2 (v 1.36.0). The read count matrix was normalized by size factors, and a variance stabilizing transformation (VST) was applied to the normalized expression data. The data was visualized using clusterProfiler (v.4.4.1) andpheatmap (v 1.0.12) packages.

[0382] Alternatively, whole-transcriptome RNA sequencing of HLOs (including those treated with bilirubin and those treated with mifepristone) was performed using the services of Novogene Co., Ltd. (Beijing, China) on the Illumina NovaSeq platform from isolated total RNA. RNA sequencing parameters were 150 bp paired-end sequencing at a depth of 20M reads per samples. Fastq read files for each sample were obtained and then aligned using Salmon, a quasi-mapping tool that aligns and quantifies transcripts using RNA-seq data. Raw transcript counts and normalized transcripts per million (TPM) values were obtained and analyzed for differential expression with DESeq2. For differential expression, statistical and biological significance was set at P<0.05, FDR<0.05, log fold-change>l, with a minimum of 3 transcript counts in 3 of the 6 samples. For heatmap visualization and hierarchical clustering analysis, hclust and pheatmap were used respectively. Finally, the pathway analysis was carried out using biomaRt and org.Hs.eg.db in R version 4.0.3. mGULO editing

[0383] The murine GULO (L-gulonolactone oxidase) (mGULO) cDNA sequence was retrieved from NCBI. The 5' linker and Kozak sequence were added to the start of the sequence and HA tags were added to the end of the sequence. Additionally, P2A-mCherry was added after the HA tag and a 3' linker to the very end. The custom gene was then synthesized and cloned into the pAAVSl-Ndi-CRISPRi (Genl) PCSF#117 vector using the restriction sites Aflll and Agel. The vector has a TetON system and a Neo^r selectable marker was then inserted using the Gateway technology. mGULO iPSC generation and maintenance

[0384] The PCSF#117 vector with the modified GULO sequence was then inserted into the AAVS1 locus of a 72.3 iPSC cell line using a lentiviral mediated CRISPR/Cas9. The correct clones were then selected using G418. The surviving clones were then verified for correct insertion, random insertion and copy number using PCR, and verified by DNA sequencing. The edited iPSC was then plated on Laminin iMatrix-511 Silk coated cell culture plates and maintained with StemFit Basic04 Complete Type media with Y-27632. The cells were passaged every 4-7 days with Accutase until passage 40 (p40). mGULO HLOs were generated according to the HLO generation protocol as described herein. The mGULO protein expression was verified using a GLUO ELISA kit (MBS2890737 from MyBioSource, San Diego, CA). mGULO HLO treatment with bilirubin and bilirubin visualization

[0385] On day 24, the mGLUO HLOs were treated with doxycycline (Dox) (100 ng/ml) to induce mGULO expression. Subsequently, on day 27, the mature organoids were treated with bilirubin and Dox for 5 days and then harvested for downstream assays. A bilirubin assay was performed to measure and visualize unconjugated and conjugated bilirubin using a colorimetric kit (ab235627) and UnaG, a green-to-dark photo switching fluorescent protein that only fluoresces upon binding of bilirubin. Images were captured on the KEYENCE BZ-X710 Fluorescence Microscope (Keyence).

ChlP-PCR and ChlP-qPCR

[0386] ChIP experiments were performed using the High Sensitivity ChIP Kit (Abeam). Briefly, organoids were fixed with PFA, and then whole chromatin was prepared and sonicated to an optimal size of 300 bp, which was confirmed by gel electrophoresis. Chromatin was used for immunoprecipitation with either EP300 antibody or IgGl isotype control. DNA fragments were amplified using custom primers for PCR and qPCR, and fold enrichment data were normalized to the immunoprecipitation from IgG controls.

Protein expression assays

[0387] Albumin secretion was measured by collecting 200 pL of the supernatant from HLOs cultured in HCM and stored at -80°C until use. The supernatant was assayed with Human Albumin ELISA Quantitation Set (Bethyl Laboratories) according to manufacturer instructions.

[0388] For murine GULO expression assay, the organoids were dissociated and washed with PBS. The cells were then lysed with RIPA Lysis and Extraction Buffer and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) to extract total protein and assayed with Mouse GULO/L-gulonolactone oxidase ELISA kit (MyBioSource.com) according to manufacturer instructions.

Metabolite assays

[0389] Bilirubin levels were measured by collecting the supernatant from HLOs treated with bilirubin and serum from rats. The supernatant and serum were assayed with Bilirubin Assay Kit (Total and Direct, Colorimetric) (abeam) and Bilirubin Assay Kit (Sigma- Aldrich) according to manufacturer instructions.

[0390] Cellular antioxidant levels were measured by harvesting the HLOs, washing in PBS, and plating them into a 96 well assay plate. The levels were then quantitated using Cellular Antioxidant Assay Kit (abeam) according to manufacturer instructions. Activity assays

[0391] CYP3A4 and CYP1A2 assays were performed by harvesting HLOs, washing in PBS, plating them into a 96 well assay plate, and treating them with rifampicin and omeprazole, respectively, for 24 hours. The assays were then performed using P450-G1O CYP3A4 and CYP1A2 Assay (Promega) and normalized using CellTiter-Glo Luminescent Cell Viability Assay according to manufacturer instructions.

[0392] The apoptosis assay was carried out by lysing HLOs and assaying the lysate with a Caspase-3 Assay Kit (Colorimetric) (abeam) according to manufacturer instructions.

[0393] Rat serum was assayed with the Aspartate Aminotransferase (AST) Activity Assay Kit and Alanine Transaminase (ALT) Activity Assay Kit (Sigma- Aldrich).

Quantification and statistical analysis

[0394] Statistical analyses were mainly performed using R software v4.2.0 with unpaired two-tailed Student’s t-test, Dunn-Holland-Wolfe test, or Welch’s test. Statistical analyses for stiffness measurements were performed using non-parametric Kruskal- Wallis and post hoc Dunn-Holland-Wolfe test. For comparisons between 2 unpaired groups, when groups were independent and the variances were unequal, non-parametric Brunner-Munzel test was performed, unless noted otherwise. P values < 0.05 were considered statistically significant. N- value refers to biologically independent replicates. The image analyses were non-blinded.

Example 7. Low doses of bilirubin promote maturation of fetal-like liver organoids

[0395] The role of bilirubin for liver development was investigated using human liver organoid models. FIG. 7A depicts an exemplary schematic for preparing liver organoids treated with a low concentration of bilirubin (e.g., 1 mg/L) resembling human fetal physiological concentrations (which is approximately 10 times less than the physiological concentrations in adults). The bilirubin is added to early organoids differentiated to a hepatic lineage. Exemplary methods of producing liver organoids have been explored previously in, for example, PCT Publications WO 2018/085615, WO 2018/191673, WO 2018/226267, WO 2019/126626, WO 2020/023245, WO 2020/069285, WO 2020/243613, WO 2021/030373, and WO 2021/262676, each of which is hereby expressly incorporated by references in its entirety. Based on these exemplary methods, the low concentration of bilirubin was added to liver organoids that form after retinoic acid induction of posterior foregut endoderm. The liver organoids may be cultured with bilirubin in a standard hepatocyte culture medium (HCM). For example, the hepatocyte culture medium may be supplemented with hepatocyte growth factors including but not limited to hepatocyte growth factor (HGF), oncostatin M, and/or dexamethasone. The liver organoids were contacted with growth medium containing 1 mg/L bilirubin for at least 5-10 days to promote liver organoid maturation.

[0396] FIG. 7B shows that the liver organoids matured with 1 mg/L bilirubin exhibited luminal projections resembling bile canaliculi, which are natural structures found in liver tissue. The resultant mature liver organoids exhibit lumens with smaller sizes and reduced circularity compared to the lumens of control liver organoids that were not treated with bilirubin (FIG. 7C).

[0397] Gene expression quantification by RT-qPCR of bilirubin-treated liver organoids revealed that these organoids exhibited increased expression of mature liver markers such as albumin (ALB), solute carrier family 4 member 2 (SLC4A2 and heme oxygenase- 1 (HO-1), and reduced expression of immature or fetal liver markers such as alpha fetoprotein (AFP), homeobox protein NANOG, and caudal type homeobox 2 (CDX2) relative to untreated organoids (FIG. 7D).

[0398] The drug metabolic capacity of the bilirubin-treated organoids was assessed by measuring cytochrome P4503A4 (CYP3A4) and cytochrome P450 1A2 (CYP1A2) activity after treatment with rifampicin and omeprazole. The bilirubin-treated organoids exhibited increased cytochrome activity relative to control untreated organoids (FIG. 7E).

[0399] Immunofluorescence microscopy showed that the bilirubin-treated organoids expressed mature liver enzymes and transport proteins, including cytochrome P450 2E1 (CYP2E1), cytochrome P450 7A1(CYP7A1), multidrug resistance protein 1 (MPR1), multidrug resistance associated protein 3 (MRP3), prospero homeobox 1 (PROXI), and organic anion transporting polypeptide 2 (OATP2) (FIGs. 7F-H).

[0400] Therefore, the use of a low concentration of bilirubin resembling physiological conditions induces maturation of liver organoids in culture.

Example 8. Ascorbate promotes liver organoid viability and GULP induces a periportallike identity

[0401] The role of ascorbate (vitamin C) for liver development was investigated using human liver organoid models. Liver organoids cultured in media lacking ascorbate exhibited loss of viability and apoptosis (FIG. 8A). This result was expected, as ascorbate is an essential nutrient that cannot be synthesized by human cells naturally due to a non-functional L-gulonolactone oxidase (GULP) enzyme.

[0402] Unlike humans and some other mammals such as Guinea pigs, many other mammals have a functional GULO gene and are able to enzymatically synthesize ascorbate. As ascorbate is important for liver development, it was hypothesized that expressing exogenous GULO in liver organoids would improve their growth and maturation in culture.

[0403] FIG. 8B depicts an exemplary schematic for genetically engineering human pluripotent stem cells with a GULO expression construct driven by a TetOn conditional expression system using CRISPR/Cas9. It is envisioned that alternative methods of exogenously introducing GULO to cells may be used. Furthermore, although the GULO gene from mouse (mGULO) was used herein, analogous functional GULO genes from other mammals may also be used. FIG. 8C shows an embodiment of a GULO gene operably linked to an mCherry fluorescent reporter for visualization, and the pAAVSl-Ndi-CRISPRi (Genl) vector that was used.

[0404] Pluripotent stem cells were engineered with the mGULO conditional expression construct through conventional approaches, and these mGULO stem cells were differentiated to liver organoids through previously described methods (FIG. 8D). As provided herein, these liver organoids may be cultured with a low dose of bilirubin to promote liver organoid maturation. FIG. 8E depicts brightfield and fluorescence microscopy images of liver organoids expressing the mGULO construct (“mGULO organoids”), where mCherry expression is observed only when doxycycline (Dox) is applied to induce TetOn expression, suggesting the co-expression of mGULO in the liver organoids. When grown in ascorbate- depleted media, the mGULO organoids undergo severe apoptosis, but survivability is rescued when doxycycline is added to induce mGULO expression, which enables the liver organoids to synthesize their own ascorbate (FIG. 8F).The mGULO organoids exhibit a dose dependent correlation between Dox concentration and GULO expression as determined by ELISA and antioxidant levels using the Cellular Antioxidant Assay Kit (abeam), suggesting synthesis of ascorbate (FIG. 8G).

[0405] The expression levels of genes involved in oxidative stress response and inflammation were investigated by RT-qPCR in mGULO organoids cultured in ascorbate- depleted media with or without addition of 100 ng/mL Dox (FIG. 8H). Ascorbate-mediated expression of the cytoprotective protein regulator nuclear factor erythroid 2-related factor 2 (NRF2) was elevated in mGULO organoids treated with Dox relative to control and mGULO organoids not treated with Dox. The inflammatory cytokines interleukin 1 beta (IL1B), interleukin 6 (IL6), and tumor necrosis factor alpha (TNFa) were elevated in ascorbate-starved organoids, suggesting significant oxidative stress, whereas this expression of inflammatory factors was rescued to resemble control in mGULO organoids treated with Dox. Similarly, a caspase-3 activity was increased in ascorbate-starved mGULO organoids, suggesting increased apoptosis, but returned to baseline upon exposure to Dox (FIG. 81).

[0406] RNA sequencing (RNA-seq) of mGULO liver organoids treated with Dox revealed increased expression of markers associated liver maturation and/or with the periportal zone of the liver (FIG. 8J). Increased expression in the mGULO organoids was observed for fumarylacetoacetate hydrolase (FAH), albumin (ALB), phenylalanine hydroxylase (PAH), cytochrome P4503A4 (CYP3A4), carbamoyl-phosphate synthase 1 (CPS1), and homogentisate oxidase (HGD). Generally, expression of genes associated with periportal pathways was observed in mGULO organoids treated with Dox (FIG. 8K).

[0407] Microscopy of mGULO organoids treated with Dox and 1 mg/L of bilirubin showed an increasingly complex and irregular lumen shape compared to control organoids without bilirubin and bilirubin-treated organoids (not expressing mGULO) (FIG. 8L). FIG. 8M shows the relative size and circularity of the lumen of mGULO organoids with or without bilirubin compared to control. Furthermore, albumin secretion is significantly increased in mGULO organoids treated with bilirubin (FIG. 8N). mGULO organoids maintain their gross morphology when treated with different concentrations of Dox (10, 100, 1000 ng/mL) (FIG. 80). The mGULO organoids treated with Dox and low dose bilirubin also exhibit increased CYP3A4 and CYP1A2 activity in response to a rifampicin or omeprazole insult compared to non-mGULO organoids or control (FIG. 8P).

[0408] The protein UnaG binds highly specifically to unconjugated bilirubin to form an apoprotein that fluoresces. Other bilirubin-related compounds, including conjugated bilirubin, biliverdin, or urobilin do not have the same ability to make UnaG fluoresce (FIG. 8Q). Therefore, using UnaG, bilirubin conjugation activity was measured in mGULO organoids with or without Dox. A decrease in UnaG fluorescence was observed in Dox-induced mGULO organoids compared to uninduced, indicating that less bilirubin was being bound to UnaG due to conjugation activity (FIG. 8R). Additional information about UnaG can be found in Kumagai et al. A bilirubin-inducible fluorescent protein from eel muscle. Cell (2013) 153(7): 1602-11, hereby expressly incorporated by reference in its entirety.

[0409] Quantifying the total percentage of organoids that conjugate bilirubin using the UnaG assay as well as organoid viability showed that conjugation activity and viability increased with mGULO expression as represented by the concentration of Dox that was applied (FIG. 8S). Overall, this suggests that significant maturation and morphological specification occurs in human liver organoids when enhanced with GULO expression. Accordingly, intracellular ascorbate promotes maturation of fetal-like liver organoids and the introduction of exogenous GULO can be used to improve liver organoid viability, optionally in conjunction with low dose bilirubin.

Example 9. Hyperbilirubinemia liver organoid models

[0410] Liver organoids were treated with varying concentrations of bilirubin in culture to induce a hyperbilirubinemic phenotype. FIG. 9A depicts a schematic for this process, where bilirubin was applied at 1, 2, 5, or 10 mg/L to liver organoids that were differentiated from pluripotent stem cells. Liver organoids exhibited significant morphological changes and intracellular accumulation of bilirubin upon exposure to increasing concentrations of bilirubin, indicating that these organoids may be used as a model for hyperbilirubinemia (FIG. 9B). RT- qPCR revealed that expression of UDP glucuronosyltransferase family 1 member Al (UGT1A1) and NRF2 increased corresponding to bilirubin dose (FIG. 9C). UGT1A1 is the enzyme that is involved in glucuronic acid conjugation of bilirubin that occurs in the liver, which is needed to render bilirubin water soluble for excretion.

[0411] To further investigate the involvement of UGT1A1 in bilirubin clearance and the use of liver organoids for models of diseases associated with bilirubin dysfunction, cells were obtained from a patient diagnosed with Crigler-Najjar syndrome, which is a genetic disorder characterized by the inability to clear bilirubin from the body (FIG. 9D). Genetic sequencing of the patient’s cells revealed a c.858C>A (p.Cys280X) nonsense mutation in the UGT1A1 gene.

[0412] Induced pluripotent stem cells (iPSCs) that were generated from the patient’s cells through conventional methods were confirmed to express the canonical pluripotency markers Sox2 and Oct4 (FIG. 9E). These iPSCs were successfully differentiated into definitive endoderm and further into liver organoids according to previously described methods (FIG. 9F), thereby resulting in Crigler-Najjar syndrome liver organoids (“CNS organoids” or “CNS HLOs”). Functional liver phenotype of these CNS HLOs was confirmed by the expression of AFP (FIG. 9G).

[0413] Treatment of these CNS HLOs, which are not able to conjugate bilirubin due to the inactive UGT1A1 gene, resulted in liver organoid toxicity when treated with 10 mg/L bilirubin (FIG. 9H). However, this toxicity due to bilirubin was able to be temporarily rescued when exogenous UGT1A1 mRNA was transfected into the CNS HLOs (FIG. 91). Assay quantification of unconjugated bilirubin (UCB) and conjugated bilirubin (CB) showed that CNS HLOs were unable to conjugate bilirubin, while ectopic expression of UGT1A1 by mRNA delivery enabled some level of bilirubin conjugation (FIG. 9J). Similarly, mGULO expression in normal HLOs also increased bilirubin conjugation (FIG. 9K).

[0414] Accordingly, bilirubin is conjugated by UGT1 Al and expression of functional UGT1A1 in a liver organoid model of Crigler-Najjar Syndrome restored bilirubin conjugation function and improved liver organoid surviv ability. This suggests that these liver organoids may be used to study bilirubin dysfunctions.

Example 10. UGT1A1 is negatively regulated by glucocorticoid signaling

[0415] Glucocorticoids have been implicated in increased serum bilirubin levels in human. Therefore, the effect of glucocorticoid signaling modulation was investigated using liver organoid models.

[0416] Regular liver organoids (not conditionally expressing mGULO) were cultured in 10 mg/L bilirubin to induce a hyperbilirubinemic phenotype and treated with the glucocorticoid agonists hydrocortisone or dexamethasone, which caused additional organoid toxicity (FIG. 10A) and decreased bilirubin conjugation ability (FIG. 10B). Conversely, hyperbilirubinemic organoids treated with the glucocorticoid antagonists ketoconazole and mifepristone showed normal organoid morphology (FIG. 10C) and increased bilirubin conjugation (FIG. 10D). RT-qPCR revealed that UGT1A1 expression was depressed upon treatment with hydrocortisone or dexamethasone, and elevated upon treatment with ketoconazole or mifepristone (FIG. 10E). NRF2 expression was also improved by ketoconazole or mifepristone treatment. Accordingly, the glucocorticoid pathway was shown to have a role in bilirubin metabolism and clearance in liver organoids.

[0417] RNA sequencing and comparison of gene expression between control organoids and those treated with mifepristone showed enrichment of many genes involved in liver function (FIG. 10F). These enriched genes were further categorized as either those that are involved in oxidative stress and/or xenobiotic metabolism (FIG. 10G).

[0418] ChlP-PCR and CHIP-qPCR of hyperbilirubinemic organoids treated with mifepristone or dexamethasone showed that methyl CpG binding protein 2 (MECP2) was involved in silencing of the UGT1A1 gene, and treatment with mifepristone reverses this silencing (FIG. 10H). Example 11. Orthotopic transplantation of HLOs alleviates hyperbilirubinemia in rodents

[0419] The effect of liver organoid composition administration into an in vivo hyperbilirubinemic rat model was investigated. Gunn rats, which are deficient in all members of the UGT1A family of bilirubin conjugating enzymes, exhibit lifelong unconjugated hyperbilirubinemia. FIG. 11A depicts an exemplary schematic (also described in Example 6) for using human liver organoid compositions to restore bilirubin conjugation ability to Gunn rat models.

[0420] FIG. 11B shows elevated albumin production in Gunn rats transplanted with HLOs, suggesting increased liver function. FIG. 11C shows decreased serum bilirubin concentration in HLO-transplanted rats, suggesting an improvement in bilirubin conjugation and clearance. FIG. 11D show decreased serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in HLO-transplanted rats, where increased levels of AST and ALT is indicative of liver damage, suggesting that the HLO-transplanted rats exhibit healthier liver function.

Example 12. Large scale production of HLOs using a 3D bioreactor

[0421] The ability to culture and expand HLOs in a 3D bioreactor, with or without Matrigel®, was investigated. The expanded HLOs were then characterized, and reconstructed and expanded.

[0422] A scalable culture of HLOs was achieved with a 3D Bioreactor. FIG. 12A shows an exemplary process for subjecting HLOs induced by conventional methods to expansion culture under conditions with (FIG. 12B) or without (FIG. 12C) Matrigel®. A dynamic method for mass production of functional HLOs was established using a 3D bioreactor culture with 50% less Matrigel® (5%) than static cultures. FIG. 12B shows that after 15 days of 3D bioreactor culture, the HLOs grew significantly larger, and compared to conventional static cultures, HLOs with 3-5 times larger diameters could be obtained. FIG. 12C shows that even in the group without Matrigel® addition, large HLOs were obtained. The 3D bioreactor culture effectively captured high proliferative and expansive capabilities compared to static cultures.

[0423] The HLOs expanded in the 3D bioreactor were characterized. Immunohistochemical staining was then used to compare HLOs induced by static culture with those obtained from 3D bioreactor culture, regardless of Matrigel® addition. As shown in FIG. 13, similar expression levels of E-cadherin, Vimentin, and Proxl were observed, indicating that HLOs obtained from 3D bioreactor culture have equivalent properties to those obtained from static culture.

[0424] The HLOs were then reconstructing and expanded in a 3D Bioreactor FIG. 14A shows an exemplary process for dissociating HLOs induced by conventional methods into single cells, and performing passaging and reconstruction of HLOs using a 3D bioreactor. Initially, HLOs induced by conventional static culture were dissociated into single cells using enzyme treatment. The single-cell HLOs were then reconstructed in the 3D bioreactor using HLO formation medium containing FGF2, VEGF, EGF, A83-01, and CHIR99021. FIG. 14B shows that, remarkably, uniform HLOs were induced after 6 days. This reconstruction of HLOs in the 3D bioreactor allowed for more than 3 passages and reconstructions under Matrigel® addition conditions.

Example 13. Treatment of mice with acetaminophen acute liver injury by transplantation of AA-treated HLOs

[0425] The therapeutic effect of HLO transplantation was tested in a mouse model of acetaminophen (APAP)-induced acute liver injury.

[0426] The acetaminophen (APAP) solution was prepared as follows: A fresh batch of APAP solution was prepared one day before each experiment, as 25 mL of APAP solution, which is sufficient for injection into 12 adult mice. Then, 375 mg of APAP (25 mL * 15 mg/mL) was weighed and transferred into a 50 mL tube containing 25 mL of 0.9% normal saline (NaCl). The tube was vortexed briefly to mix the APAP powder with the saline solution, and the tube was then placed in a water bath or bead bath set to 37°C. Steps 3 and 4 were then repeated to completely dissolve the APAP powder. The solution was sterilzed with APAP completely dissolved by filtration using a 0.2 pm filter. The materials and kits used included pharmaceutical grade acetaminophen: Sigma Aldrich Cat# A5000-100G; DPBS: Gibco Cat# AT104-500; Alanine Transaminase Activity Assay Kit: abeam Cat#abl05134. Because APAP is difficult to dissolve, it was diluted to 15 mg/mL with 0.9% saline. To dissolve APAP, the solution was heated to 37°C and vortexed frequently (~3-4 hours required for complete dissolution).

[0427] The transplantation utilized HLO culture medium (HCM), HLO, and AA_HLO as controls. The experimental method was to transplant 5,000 HLO into one mouse with acute liver injury induced by APAP, and the therapeutic effect was evaluated after 7 days of follow-up. The number of transplants was 22 mice for medium-only control, 29 for HLO, and 24 for AA_HLO. [0428] For the overnight fasting NSG mice, one day before the transplantation, the animals were marked and weighed, removing all food, and leaving only water available in the cage. The cage card was labeled indicating overnight fasting as per the protocol. Overnight fasting is important for the APAP overdose model because fasting allows for depletion of hepatic glutathione, which is needed for liver damage via APAP overdose. Typically, fasting was initiated between 4-6 pm to avoid excessively long fasting periods (e.g., to avoid fasting in the morning).

[0429] For the APAP overdose in fasting NSG mice, the animals were weighed, and the amount of APAP solution to inject for each mouse was calculated. A dose of 700 mg/kg has been found to generate severe acute liver failure (ALF) for survival curve studies and to demonstrate the therapeutic benefit of HLO transplantations. The mice were placed under anesthesia (3% isoflurane for induction, 2% for maintenance) for baseline blood draw.

[0430] For the 0-hour blood collection, 20 pL of blood was obtained using heparinized capillary tubes and transferred 20 pL of blood to 180 pL of saline in tube to make a 200 pL 1:10 diluted blood sample. An appropriate volume of APAP solution was drawn for the corresponding mouse in a 3mL syringe, and a 27G needle was used to inject intraperitoneally into mouse in right lower quadrant. Regular chow was then placed back into the cages, and HydroGel and nutritional supplement were supplied to support hydration and nutrition for the mice, who were very sick for a few days after APAP overdose.

[0431] For the intraperitoneal HLO transplant, HLOs were transplanted 6 hrs after APAP injection. The mice were placed in anesthesia for blood draw at 6 hr time point. The mice were already sick by this point and needed less anesthesia (~1.5 to 2%); because using too high % of isoflurane can lead to death, 1.5-2% isoflurane was used for the rest of the experiments.

[0432] For pre-transplant blood collection, 20 pL of blood was obtained using heparinized capillary tubes, and 20 pL of blood was transferred to 180 pL of saline in the tube to make a 200 pL 1:10 diluted blood sample. Then, 300 pL of HLO suspension was drawn into a ImL syringe using an 18G needle. The needle was switched to a 23G needle, and the suspension was injected into the right lower quadrant of mice, without penetrating too deeply, in order to avoid injuring other organs. The mice were then placed back in the cages. The control was 300 pL of regular HCM without HLO.

[0433] For post-transplant follow-up, each mouse was weighted daily. For posttransplant blood collection, 20 pL of blood was obtained using heparinized capillary tubes, and then 20 pL of blood was transferred to 180 pL of saline in a tube to make a 200 pL 1:10 diluted blood sample. The collected blood was then centrifuged at 4C° 1000g for 10 minutes to separate red blood cells and other cells. The supernatant fraction was then transferred to a new Eppendorf tube and stored at -80°C until analysis. Blood sampling was performed after 48, 72, and 168 hours.

[0434] For determination of liver damage and ammonia levels, the Alanine Transaminase Activity Assay Kit was used to measure APAP-induced liver injury, measuring according to the kit instructions. 20uL of blood sample was used for ALT measurement. A dilution of 1:10 will give a value within the range of the kit.

[0435] Dry chemistry methods (mammalian liver panel from Schubert Research Clinic for ALT and our portable ammonia machine for ammonia) can be used. These should be performed immediately after specimen collection (specimens cannot be cryopreserved, only temporarily stored at 4°C for 1-2 hours).

[0436] The results showed that the HLO transplant group helped mice with acute liver injury survive compared to medium alone. In particular, the AA_HLO transplant group significantly improved the survival rate compared to the medium alone (Log-rank test p=0.0267).

[0437] FIG. 15A shows a schematic diagram of a model of acetaminophen acute liver injury rescue by HLO transplantation. FIG. 15B shows an image of an HLO used for transplantation. FIG. 15C shows Kaplan-Meier survival curves for acute liver injury rescue by HLO transplantation.

[0438] In at least some of the previously described and following embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0439] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0440] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0441] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [0442] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed herein. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0443] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0444] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

RECITATION OF EMBODIMENTS

[0445] Exemplary embodiments of the present disclosure are provided in the following numbered embodiments:

1. An embodiment for expanding posterior foregut cells and/or posterior foregut endoderm cells, comprising: a) dissociating a posterior foregut and/or posterior foregut endoderm cell monolayer to posterior foregut cells and/or posterior foregut endoderm cells; b) seeding the posterior foregut cells and/or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and/or posterior foregut endoderm cells with a TGF-b pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

2. Embodiment 1, wherein the posterior foregut cell monolayer is dissociated to the posterior foregut cells and/or posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation.

3. Any of embodiments 1 or 2, wherein the posterior foregut cells and/or posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density of, or of about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities.

4. Any of embodiments 1-3, wherein the tissue culture surface is coated with a basement membrane matrix or component thereof.

5. Embodiment 4, wherein the basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®.

6. Any of embodiments 4 or 5, wherein the basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

7. Any one of embodiments 1-6, wherein the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids are formed spontaneously, optionally wherein the 3D spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

8. Any one of embodiments 1-7, wherein the posterior foregut cells and/or posterior foregut endoderm cells are cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days.

9. Any one of embodiments 1-8, wherein the TGF-b pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542, optionally A83-01. 10. Any one of embodiments 1-9, wherein the TGF-b pathway inhibitor is provided at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the TGF-b pathway inhibitor is provided at a concentration of, or of about, 500 nM.

11. Any one of embodiments 1-10, wherein the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally FGF2.

12. Any one of embodiments 1-11, wherein the FGF pathway activator is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is provided at a concentration of, or of about, 5 ng/mL.

13. Any one of embodiments 1-12, wherein the Wnt pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119, optionally CHIR99021.

14. Any one of embodiments 1-13, wherein the Wnt pathway activator is provided at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is provided at a concentration of, or of about, 3 pM.

15. Any one of embodiments 1-14, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally VEGF.

16. Any one of embodiments 1-15, wherein the VEGF pathway activator is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is provided at a concentration of, or of about, 10 ng/mL. 17. Any one of embodiments 1-16, wherein the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises EGF, or cultured in a media that does not comprise EGF.

18. Embodiment 17, wherein the EGF is provided at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the EGF is provided at a concentration of, or of about, 20 ng/mL.

19. Any one of embodiments 1-18, wherein the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises ascorbic acid, or cultured in a media that does not comprise ascorbic acid.

20. Embodiment 19, wherein the ascorbic acid is provided at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the ascorbic acid is provided at a concentration of, or of about, 50 pg/mL.

21. Any one of embodiments 1-20, wherein the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises a ROCK inhibitor, or cultured in a media that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632.

22. Embodiment 21, wherein the ROCK inhibitor is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally where the ROCK inhibitor is provided at a concentration of, or of about, 10 pM.

23. Any one of embodiments 1-22, further comprising passaging the cells of step c) one or more times.

24. Embodiment 23, wherein the cells of step c) are passaged until the posterior foregut cells and/or posterior foregut endoderm cells do not form spheroids spontaneously.

25. Any of embodiment 23 or 24, wherein the cells of step c) are passaged not more than 3 times. 26. Any one of embodiments 23-25, further comprising collecting the posterior foregut cells and/or posterior foregut endoderm cells and differentiating the posterior foregut cells and/or posterior foregut endoderm cells to liver organoids.

27. Embodiment 26, wherein the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three-dimension (3D) spheroids are formed spontaneously, and the posterior foregut cells and/or posterior foregut endoderm cells are collected from the spheroids, optionally further comprising dissociating the spheroids into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

28. Embodiment 26, wherein the posterior foregut cells and/or posterior foregut endoderm cells are collected from the posterior foregut cell monolayer by dissociating the posterior foregut cell monolayer into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step.

29. An embodiment including a method of differentiating posterior foregut cells and/or posterior foregut endoderm cells to liver organoids, comprising: i) contacting posterior foregut cells and/or posterior foregut endoderm cells, optionally in the form of spheroids, optionally in the form of individual cells or cell clusters dissociated from spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells, and/or optionally cells aggregated in a microwell or other apparatus as described herein, with a retinoic acid pathway activator; and ii) contacting the cells of step i) with a medium for a period of time thereby differentiating the posterior foregut cells and/or posterior foregut endoderm cells to liver organoids, optionally wherein the medium is hepatocyte culture medium.

30. Embodiment 29, wherein the medium is supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid.

31. Embodiment 30, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85. 32. Embodiment 30 or 31, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIE), cardiotrophin-1, ciliary neurotrophic factor (CTNE), and cardiotrophin-like cytokine (CLC).

33. Any one of embodiments 30-32, wherein the corticosteroid is selected from a group consisting of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and mometasone.

34. Embodiment 29, wherein the medium is supplemented with HGE, OSM, and dexamethasone.

35. Embodiment 29, wherein the medium is supplemented with dexamethasone.

36. Any one of embodiments 29-35, wherein the posterior foregut cells and/or posterior foregut endoderm cells are the posterior foregut cells and/or posterior foregut endoderm cells produced by any one of embodiments 1-28.

37. Any one of embodiments 29-36, wherein the posterior foregut cells and/or posterior foregut endoderm cells are in the form of spheroids or individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells derived from dissociating the spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

38. Any one of embodiments 29-37, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally retinoic acid.

39. Any one of embodiments 29-38, wherein the retinoic acid pathway activator is provided at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator is provided at a concentration of, or of about, 2.0 pM.

40. Embodiment 34, wherein the HGF is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the HGF is provided at a concentration of, or of about 10 ng/mL. 41. Embodiment 34, wherein the OSM is provided at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the OSM is provided at a concentration of, or of about 20 ng/mL.

42. Any one of embodiments 34 or 35, wherein the dexamethasone is provided at a concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone is provided at a concentration of, or of about 100 nM.

43. Any one of embodiments 29-42, wherein the cells of step i) and/or step ii) are not contacted with EGF.

44. Any one of embodiments 29-43, wherein the cells of step ii) are cultured in a growth medium supplemented with non-essential amino acids, essential amino acids, and glycine.

45. Embodiment 44, wherein the growth media after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% non-essential amino acids by total volume, or a range defined by any two to the preceding values, optionally wherein the growth medium after supplementation is about 4-10%, 6-12%, 10-16%, 12-15%, 13-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% non-essential amino acids by total volume.

46. Embodiment 44 or 45, wherein the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or a range defined by any two to the preceding values, optionally wherein the growth medium after supplementation is about 4- 10%, 6- 12%, 10-16%, 12-15%, 13-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% essential amino acids by total volume.

47. Any one of embodiments 44-46, wherein the supplemented glycine is provided at a concentration of, or of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the supplemented glycine is provided at a concentration of, or of about, 18-22 mg/mL or 20 mg/mL. 48. Any one of embodiments 29-47, wherein the cells of step ii) are further contacted with a low/first concentration of bilirubin, wherein the liver organoids that are formed are mature liver organoids.

49. Embodiment 48, wherein the low/first concentration of bilirubin is a human fetal physiological concentration of bilirubin.

50. Embodiment 48 or 49, wherein the low/first concentration of bilirubin is, is about, is less than, or is less than about: a) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or b) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.

51. Any one of embodiments 48-50, wherein the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells.

52. Any one of embodiments 48-51, wherein the mature liver organoids express reduced levels of ALP, CDX2, NANOG, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin.

53. Any one of embodiments 48-52, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin.

54. Any one of embodiments 48-53, wherein the mature liver organoids express CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof.

55. Any one of embodiments 48-54, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with the low/first dose of bilirubin.

56. Any one of embodiments 29-55, wherein the cells of step ii) are further contacted with a high/second concentration of bilirubin, wherein the liver organoids that are formed are hyperbilirubinemia liver organoids. 57. Embodiment 56, wherein the high/second concentration of bilirubin is, is about, is more than, or is more than about: a) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L.

58. Embodiment 56 or 57, wherein the hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRE2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

59. Any one of embodiments 29-58, wherein the liver organoids comprise a functional L- gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the liver organoids are able to synthesize ascorbate.

60. Embodiment 59, wherein the functional GULO protein is murine GULO (mGULO).

61. Embodiment 59 or 60, wherein the gene that encodes for the functional GULO protein is conditionally expressed, optionally using a tetracycline inducible system.

62. Any one of embodiments 59-61, wherein the liver organoids are engineered with the gene that encodes for the functional GULO protein using CRISPR.

63. Any one of embodiments 59-62, wherein the gene or mRNA, or both, that encodes for the functional GULO protein is introduced to the liver organoids by transfection.

64. Any one of embodiments 59-63, wherein the liver organoids comprising the functional GULO protein express increased levels of NRE2 relative to liver organoids that do not comprise the functional GULO protein.

65. Any one of embodiments 59-64, wherein the liver organoids comprising the functional GULO protein expresses reduced levels of IL1B, IL6, or TNEa, or any combination thereof, relative to liver organoids that do not comprise the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate.

66. Any one of embodiments 59-65, wherein the liver organoids comprising the functional GULO protein exhibits reduced caspase-3 activity relative to liver organoids that do not comprise the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate.

67. Any one of embodiments 59-66, wherein the liver organoids comprising the functional GULO protein express increased levels of ALB relative to liver organoids that do not comprise the functional GULO protein.

68. Any one of embodiments 59-67, wherein the liver organoids comprising the functional GULO protein resemble periportal liver tissue and express periportal liver markers.

69. Embodiment 68, wherein the periportal liver markers comprise FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

70. Any one of embodiments 59-69, wherein the liver organoids comprising the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that do not comprise the functional GULO protein.

71. Any one of embodiments 59-70, wherein the liver organoids comprising the functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids that do not comprise the functional GULO protein.

72. Any one of embodiments 59-71, wherein the liver organoids comprising the functional GULO protein exhibit increased viability in culture relative to liver organoids that do not comprise the functional GULO protein.

73. Any one of embodiments 59-72, wherein the liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.

74. Any one of embodiments 59-73, wherein the liver organoids comprise an inactive UGT1A1 gene, wherein the liver organoids are a model for Crigler-Najjar Syndrome.

75. Any one of embodiments 29-74, further comprising aggregating the posterior foregut cells and/or posterior foregut endoderm cells in a microwell or other apparatus (e.g., Aggrewell) prior to step i), wherein aggregating the posterior foregut cells and/or posterior foregut endoderm cells results in more uniformly sized liver organoids.

76. Any one of embodiments 29-75, wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof that is xenogeneic to humans, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of step i) and/or step ii) are not contacted with Matrigel®, Cultrex®, or Geltrex®.

77. Any one of embodiments 29-76, wherein the cells of step i) and/or step ii), and/or the liver organoids formed therefrom, are cultured in a static or non- static bioreactor, optionally a rotational bioreactor, optionally a 3D bioreactor, optionally a 3D rotational bioreactor.

78. Embodiment 77, wherein after culturing in a static or non-static bioreactor, the liver organoids are dissociated into single cells, and are subsequently reconstructed and/or expanded via an additional culturing step in a static or non-static bioreactor, optionally a 3D bioreactor, optionally a 3D rotational bioreactor.

79. Any one of embodiments 29-78, further comprising cry opreserving the liver organoids.

80. Embodiment 79, wherein cryopreserving the liver organoids comprises slow-freezing or vitrification cryopreservation, optionally wherein the liver organoids are cryopreserved with chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

81. Any one of embodiments 1-80, wherein the posterior foregut cells and/or posterior foregut endoderm cells have been derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

82. Any one of embodiments 1-81, wherein the posterior foregut cells and/or posterior foregut endoderm cells have been derived from a subject, optionally a subject having a liver- related disease or disorder.

83. Any one of embodiments 1-82, wherein the method can be used in a good manufacturing practice (GMP) compliant process.

84. An embodiment including posterior foregut cells and/or posterior foregut endoderm cells or liver organoids produced by any one of embodiments 1-83.

85. An embodiment including an in vitro composition comprising: pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types, and at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

86. Embodiment 85, wherein the composition comprises posterior foregut cells and/or posterior foregut endoderm cells, and the posterior foregut cells and/or posterior foregut endoderm cells are dissociated posterior foregut cells and/or posterior foregut endoderm cells.

87. Embodiment 85 or 86, wherein the posterior foregut cells and/or posterior foregut endoderm cells are at a cell density of greater than or equal to, exactly or about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities.

88. Any one of embodiments 85-87, wherein the tissue culture surface is coated with a basement membrane matrix or component thereof.

89. Embodiment 88, wherein the basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®.

90. Embodiment 88 or 89, wherein the basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

91. Any one of embodiments 85-90, wherein at least a portion of the posterior foregut cells and/or posterior foregut endoderm cells are spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure with a single lumen.

92. Any one of embodiments 85-91, wherein the TGF-b pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542; optionally wherein the TGF-b pathway inhibitor comprises or is A83-01.

93. Any one of embodiments 85-92, wherein the TGF-b pathway inhibitor is at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the TGF-b pathway inhibitor is at a concentration of, or of about, 500 nM. 94. Any one of embodiments 85-93, wherein the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally wherein the FGF pathway activator comprises or is FGF2.

95. Any one of embodiments 85-94, wherein the FGF pathway activator is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is at a concentration of, or of about, 5 ng/mL.

96. Any one of embodiments 85-95, wherein the Wnt pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS1 19, optionally wherein the Wnt pathway activator comprises or is CHIR99021.

97. Any one of embodiments 85-96, the Wnt pathway activator is at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is at a concentration of, or of about, 3 pM.

98. Any one of embodiments 85-97, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally wherein the VEGF pathway activator comprises or is VEGF.

99. Any one of embodiments 85-98, wherein the VEGF pathway activator is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is at a concentration of, or of about, 10 ng/mL.

100. Any one of embodiments 85-99, wherein the composition further comprises exogenous EGF, or wherein the composition does not comprise exogenous EGF.

101. Any one of embodiments 85-100, wherein the EGF is at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the EGF is at a concentration of, or of about, 20 ng/mL.

102. Any one of embodiments 85-101, wherein the composition further comprises exogenous and/or transgenically produced ascorbic acid, or wherein the composition does not comprise exogenous and/or transgenically produced ascorbic acid.

103. Any one of embodiments 85-102, wherein the ascorbic acid is at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the ascorbic acid is at a concentration of, or of about, 50 pg/mL.

104. Any one of embodiments 85-103, further comprising a ROCK inhibitor, or cultured in a media that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor comprises or is Y-27632.

105. Any one of embodiments 85-104, wherein the ROCK inhibitor is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally where the ROCK inhibitor is at a concentration of, or of about, 10 pM.

106. Any one of embodiments 85-105, wherein the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm, and/or downstream liver cells are differentiated from stem cells, optionally wherein the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm, and/or downstream liver cells are differentiated from induced pluripotent stem cells.

107. Any one of embodiments 85-107, wherein the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm, and/or downstream liver cells have been passaged less than 4 times.

108. Embodiment 108, wherein the cells comprise or consist essentially of posterior foregut cells and/or posterior foregut endoderm cells.

109. Any one of embodiments 85-108, wherein the TGF-b pathway inhibitor is A83-01, the FGF pathway activator is FGF2, the Wnt pathway activator is CHIR99021, the VEGF pathway activator is VEGF, and the ROCK inhibitor is Y-27632. 110. An embodiment including the liver organoid produced by any one of embodiments 29- 83.

111. An embodiment including an in vitro composition comprising, a) posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids, and b) a medium, wherein the medium optionally comprises hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid, and wherein the composition optionally additionally comprises c) a retinoic acid pathway activator.

112. Embodiment 111, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85.

113. Any one of embodiments 111-112, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC).

114. Any one of embodiments 111-113, wherein the corticosteroid is selected from a group consisting of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and mometasone.

115. Any one of embodiments 111-114, wherein the medium is supplemented with HGF, OSM, and dexamethasone.

116. Any one of embodiments 111-115, wherein the medium is supplemented with dexamethasone.

117. Any one of embodiments 111-116, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally wherein the retinoic acid pathway activator comprises or is retinoic acid.

118. Any one of embodiments 111-117, wherein the retinoic acid pathway activator is at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator is at a concentration of, or of about, 2.0 pM. 119. Any one of embodiments 115-118, wherein the HGF is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the HGF is at a concentration of, or of about 10 ng/mL.

120. Any one of embodiments 115-119, wherein the OSM is at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the OSM is at a concentration of, or of about 20 ng/mL.

121. Any one of embodiments 115-120, wherein the dexamethasone is at a concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone is at a concentration of, or of about 100 nM.

122. Any one of embodiments 111-121, wherein the composition does not comprise exogenous EGF.

123. Any one of embodiments 111-122, further comprising a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin is at or near a human fetal physiological concentration of bilirubin.

124. Embodiment 123, wherein the bilirubin is, is about, is less than, or is less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.

125. Embodiment 124, wherein the composition comprises mature liver organoids, and wherein the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells.

126. Embodiment 125, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid not contacted with a low dose of bilirubin. 127. Embodiment 125 or 126, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low dose of bilirubin.

128. Any one of embodiments 125-127, wherein the mature liver organoids express CYP2E1, CYP7A1, PR0X1, MRP3, MRP3, or OATP2, or any combination thereof.

129. Any one of embodiments 125-128, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with a low dose of bilirubin.

130. An embodiment including an in vitro composition comprising mature liver organoids, wherein the cells of the mature liver organoid were contacted with a low dose of bilirubin, optionally wherein the low dose of bilirubin was exogenously provided, and the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells.

131. Embodiment 130, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

132. Embodiment 130 or 131, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

133. Any one of embodiments 130-132, wherein the mature liver organoids express CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof.

134. Any one of embodiments 130-133, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

135. Any one of embodiments 130-134, further comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells were contacted with a high and/or second concentration of bilirubin.

136. Embodiment 135, wherein the high/second concentration of bilirubin was, was about, was more than, or was more than about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L.

137. Embodiment 135 or 136, wherein the hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

138. Any one of embodiments 111-137, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids comprise a functional L- gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are able to synthesize ascorbate.

139. Embodiment 138, wherein the functional GULO protein is murine GULO (mGULO).

140. Embodiment 138 or 139, wherein the gene that encodes for the functional GULO protein is conditionally expressed, optionally using a tetracycline inducible system.

141. Any one of embodiments 138-139, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are engineered to comprise a gene that encodes for the functional GULO protein using CRISPR.

142. Any one of embodiments 138-141, wherein the gene or mRNA, or both, that encodes for the functional GULO protein was introduced to the liver organoids by transfection.

143. Any one of embodiments 138-142, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein express increased levels of NRF2 relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

144. Any one of embodiments 138-143, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein expresses reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein. 145. Any one of embodiments 138-144, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit reduced caspase-3 activity relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

146. Any one of embodiments 138-145, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein express increased levels of ALB relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

147. Any one of embodiments 138-146, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein resemble periportal liver tissue and express periportal liver markers.

148. Embodiment 147, wherein the periportal liver markers comprise FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

149. Any one of embodiments 138-148, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids an/dor mature liver organoids that do not comprise the functional GULO protein.

150. Any one of embodiments 138-149, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

151. Any one of embodiments 138-150, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit increased viability in culture relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

152. Any one of embodiments 138-151, wherein the liver organoids and/or mature liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate. 153. An embodiment including a method comprising administering the liver organoid or composition of any one of embodiments 110-152 to a subject in need thereof.

154. An embodiment including a method for treating a liver-related disease or disorder in a subject in need thereof, comprising administering one or more liver organoids or compositions of embodiments 110-152 to the subject.

155. Embodiment 153 or 154, wherein the liver organoid has been produced from cells derived from the subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells.

156. Any one of embodiments 154-155, wherein administering comprises transplanting the liver organoid or composition into the subject.

157. Any one of embodiments 154-156, wherein the liver-related disease or disorder comprises one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug- induced cholestasis, metabolic disease, autoimmune liver disease, Wilson’s disease, metabolic- associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, metabolic dysfunction-associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, hepatic cyst, acetaminophen acute liver injury, and/or alcohol-associated liver disease.

158. Any one of embodiments 154-157, wherein the liver dysfunction and/or failure comprises hyperammonemia and/or hyperbilirubinemia; or wherein the metabolic disease comprises nonalcoholic fatty liver disease (NAFLD); or wherein the nonalcoholic fatty liver disease (NAFLD) comprises metabolic dysfunction-associated steatohepatitis (MASH); or wherein the hepatitis comprises hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and/or autoimmune hepatitis.

159. Any one of embodiments 154-158, wherein the subject has reduced serum bilirubin and/or ammonia levels, and/or increased serum protein albumin following transplantation.

160. Any of embodiments 154-159, wherein the subject has improved symptoms of biliary stricture and/or liver regeneration following transplantation. 161. Any of embodiments 154-160, wherein the subject has an increased survival rate following transplantation.

162. Any of embodiments 154-161, wherein the liver organoid engrafts onto the liver of the subject.

163. Any of embodiments 154-162, wherein the liver organoid has been treated with amino acid (AA) supplementation.

164. Any of embodiments 154-163, wherein the liver organoid has been treated with amino acid (AA) supplementation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to transplantation.

165. Any of embodiments 154-164, wherein the liver-related disease or disorder comprises acetaminophen acute liver injury.

166. Any of embodiments 154-165, wherein the method can be used in a good manufacturing practice (GMP) compliant process.

167. An embodiment including a method for screening, comprising contacting the liver organoid of any one of embodiments 84-152 with a candidate compound or composition, and assessing the effects of the candidate compound or composition on the liver organoid.

168. Embodiment 167, wherein the liver organoid is a model for a liver-related disease or disorder, and assessing the effects of the candidate compound or composition on the liver organoid comprises assessing the effects of the candidate compound or composition on the liver-related disease or disorder.

169. Embodiment 167 or 168, wherein the liver organoid has been produced from cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells.

170. Embodiment 169, wherein the subject has a liver-related disease or disorder.

171. Any of embodiments 167-170, wherein the method can be used in a good manufacturing practice (GMP) compliant process.

172. An embodiment including a composition comprising an amino acid supplemented liquid component according to Table 3.

173. An embodiment including a composition comprising a cocktail of growth factors according to an embodiment of Table 1 or Table 2. 174. An embodiment including a composition comprising an amino acid supplemented liquid component comprising exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% non-essential amino acid solution by volume (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL glycine.

175. Any of embodiments 172-174, comprising an amino acid supplemented liquid component comprising exactly or about 14% non-essential amino acid solution (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 6% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 80% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 20 g/L glycine.

176. Any one of embodiments 172-175, wherein the pH is between about pH 6-8, or pH 6.5- 7.5, or exactly or about pH 7.0.

177. Any one of embodiments 172-176, further comprising hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid.

178. Any one of embodiments 172-177, further comprising hepatic lineage committed cells differentiated from definitive endoderm cells using retinoic acid.

179. Embodiment 178, wherein the hepatic lineage committed cells are characterized as liver organoids. 180. Embodiment 179, wherein the liver organoids are characterized as secreting increased levels of albumin and urea relative to liver organoids comprised in HCM without amino acid supplementation.

181. Embodiment 179 or 180, wherein the liver organoids are characterized as expressing increased levels of hepatic maturation associated gene expression relative to liver organoids comprised in HCM without amino acid supplementation.

182. Any one of embodiments 179-181, wherein the liver organoids are characterized as expressing reduced levels of Vimentin relative to liver organoids comprised in HCM without amino acid supplementation.

183. Any one of embodiments 172-182, wherein the composition does not comprise nonhuman animal components such that the basement membrane matrix or component thereof is xenogeneic to humans.

184. A embodiment including a composition of embodiment 182, wherein the composition does not comprise murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and/or Geltrex®.

185. An embodiment including an in vitro hyperbilirubinemia liver organoid comprising a naturally occurring and/or engineered mutation in the UDP glucuronosyltransferase family 1 member Al UGTIAI) gene.

186. An embodiment including an in vitro hyperbilirubinemia liver organoid, wherein the hyperbilirubinemia liver organoid was produced through at least two rounds of contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids to exogenous bilirubin.

187. An embodiment including an in vitro hyperbilirubinemia liver organoid of embodiment 185 or 186, wherein the hyperbilirubinemia liver organoid was clonally derived and/or derived from iPSCs.

188. An embodiment including a cryopreserved composition, comprising liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

189. An embodiment including a cryopreserved composition, comprising mature liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT). 190. An embodiment including a cryopreserved composition, comprising hyperbilirubinemia liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

191. An embodiment including a kit comprising means for conducting the method according to any one of embodiments 1-83, or 153-171.

192. An embodiment including a kit comprising the compositions or means for generating the compositions according to any one of embodiments 84-152, 172-184, or 188-190, or for generating the liver organoid of any one of embodiments 185-187.

193. An embodiment using the method, composition, or kit according to any one of embodiments 1-192, as a medicament, means for treatment and/or prevention of a disease, means of diagnosis, and/or medical research.

Claims

CLAIMS What is claimed is:

1. A method for expanding posterior foregut cells and/or posterior foregut endoderm cells, comprising: a) dissociating a posterior foregut and/or posterior foregut endoderm cell monolayer to posterior foregut cells and/or posterior foregut endoderm cells; b) seeding the posterior foregut cells and/or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and/or posterior foregut endoderm cells with a TGF-b pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

2. The method of claim 1, wherein the posterior foregut cell monolayer is dissociated to the posterior foregut cells and/or posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation.

3. The method of claim 1 or 2, wherein the posterior foregut cells and/or posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density of, or of about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities.

4. The method of any one of claims 1-3, wherein the tissue culture surface is coated with a basement membrane matrix or component thereof.

5. The method of claim 4, wherein the basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®.

6. The method of claim 4 or 5, wherein the basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

7. The method of any one of claims 1-6, wherein the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids are formed spontaneously, optionally wherein the 3D spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

8. The method of any one of claims 1-7, wherein the posterior foregut cells and/or posterior foregut endoderm cells are cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days.

9. The method of any one of claims 1-8, wherein the TGF-b pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542, optionally A83-01.

10. The method of any one of claims 1-9, wherein the TGF-b pathway inhibitor is provided at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the TGF-b pathway inhibitor is provided at a concentration of, or of about, 500 nM.

11. The method of any one of claims 1-10, wherein the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally FGF2.

12. The method of any one of claims 1-11, wherein the FGF pathway activator is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is provided at a concentration of, or of about, 5 ng/mL.

13. The method of any one of claims 1-12, wherein the Wnt pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119, optionally CHIR99021.

14. The method of any one of claims 1-13, wherein the Wnt pathway activator is provided at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is provided at a concentration of, or of about, 3 pM.

15. The method of any one of claims 1-14, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally VEGF.

16. The method of any one of claims 1-15, wherein the VEGF pathway activator is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is provided at a concentration of, or of about, 10 ng/mL.

17. The method of any one of claims 1-16, wherein the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises EGF, or cultured in a media that does not comprise EGF.

18. The method of claim 17, wherein the EGF is provided at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the EGF is provided at a concentration of, or of about, 20 ng/mL.

19. The method of any one of claims 1-18, wherein the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises ascorbic acid, or cultured in a media that does not comprise ascorbic acid.

20. The method of claim 19, wherein the ascorbic acid is provided at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the ascorbic acid is provided at a concentration of, or of about, 50 pg/mL.

21. The method of any one of claims 1-20, wherein the posterior foregut cells and/or posterior foregut endoderm cells of step c) are cultured in a media that further comprises a ROCK inhibitor, or cultured in a media that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632.

22. The method of claim 21, wherein the ROCK inhibitor is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally where the ROCK inhibitor is provided at a concentration of, or of about, 10 pM.

23. The method of any one of claims 1-22, further comprising passaging the cells of step c) one or more times.

24. The method of claim 23, wherein the cells of step c) are passaged until the posterior foregut cells and/or posterior foregut endoderm cells do not form spheroids spontaneously.

25. The method of claim 23 or 24, wherein the cells of step c) are passaged not more than 3 times.

26. The method of any one of claims 23-25, further comprising collecting the posterior foregut cells and/or posterior foregut endoderm cells and differentiating the posterior foregut cells and/or posterior foregut endoderm cells to liver organoids.

27. The method of claim 26, wherein the posterior foregut cells and/or posterior foregut endoderm cells are cultured until three-dimension (3D) spheroids are formed spontaneously, and the posterior foregut cells and/or posterior foregut endoderm cells are collected from the spheroids, optionally further comprising dissociating the spheroids into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

28. The method of claim 26, wherein the posterior foregut cells and/or posterior foregut endoderm cells are collected from the posterior foregut cell monolayer by dissociating the posterior foregut cell monolayer into individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells prior to the differentiating step.

29. A method of differentiating posterior foregut cells and/or posterior foregut endoderm cells to liver organoids, comprising: i) contacting posterior foregut cells and/or posterior foregut endoderm cells, optionally in the form of spheroids, optionally in the form of individual cells or cell clusters dissociated from spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells, and/or optionally cells aggregated in a microwell or other apparatus as described herein, with a retinoic acid pathway activator; and ii) contacting the cells of step i) with a medium for a period of time thereby differentiating the posterior foregut cells and/or posterior foregut endoderm cells to liver organoids, optionally wherein the medium is hepatocyte culture medium.

30. The method of claim 29, wherein the medium is supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid.

31. The method of claim 30, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85.

32. The method of claim 30 or 31, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC).

33. The method of any one of claims 30-32, wherein the corticosteroid is selected from a group consisting of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and mometasone.

34. The method of claim 29, wherein the medium is supplemented with HGF, OSM, and dexamethasone.

35. The method of claim 29, wherein the medium is supplemented with dexamethasone.

36. The method of any one of claims 29-35, wherein the posterior foregut cells and/or posterior foregut endoderm cells are the posterior foregut cells and/or posterior foregut endoderm cells produced by the method of any one of claims 1-28.

37. The method of any one of claims 29-36, wherein the posterior foregut cells and/or posterior foregut endoderm cells are in the form of spheroids or individual posterior foregut cells and/or posterior foregut endoderm cells and/or clumps of posterior foregut cells and/or posterior foregut endoderm cells derived from dissociating the spheroids, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

38. The method of any one of claims 29-37, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally retinoic acid.

39. The method of any one of claims 29-38, wherein the retinoic acid pathway activator is provided at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator is provided at a concentration of, or of about, 2.0 pM.

40. The method of claim 34, wherein the HGF is provided at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the HGF is provided at a concentration of, or of about 10 ng/mL.

41. The method of claim 34, wherein the OSM is provided at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the OSM is provided at a concentration of, or of about 20 ng/mL.

42. The method of claims 34 or 35, wherein the dexamethasone is provided at a concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone is provided at a concentration of, or of about 100 nM.

43. The method of any one of claims 29-42, wherein the cells of step i) and/or step ii) are not contacted with EGF.

44. The method of any one of claims 29-43, wherein the cells of step ii) are cultured in a growth medium supplemented with non-essential amino acids, essential amino acids, and glycine.

45. The method of claim 44, wherein the growth media after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% non-essential amino acids by total volume, or a range defined by any two to the preceding values, optionally wherein the growth medium after supplementation is about 4- 10%, 6- 12%, 10-16%, 12-15%, 13-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% non-essential amino acids by total volume.

46. The method of claim 44 or 45, wherein the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or a range defined by any two to the preceding values, optionally wherein the growth medium after supplementation is about 4- 10%, 6- 12%, 10-16%, 12-15%, 13-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% essential amino acids by total volume.

47. The method of any one of claims 44-46, wherein the supplemented glycine is provided at a concentration of, or of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the supplemented glycine is provided at a concentration of, or of about, 18-22 mg/mL or 20 mg/mL.

48. The method of any one of claims 29-47, wherein the cells of step ii) are further contacted with a low/first concentration of bilirubin, wherein the liver organoids that are formed are mature liver organoids.

49. The method of claim 48, wherein the low/first concentration of bilirubin is a human fetal physiological concentration of bilirubin.

50. The method of claim 48 or 49, wherein the low/first concentration of bilirubin is, is about, is less than, or is less than about: a) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or b) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.

51. The method of any one of claims 48-50, wherein the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells.

52. The method of any one of claims 48-51, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin.

53. The method of any one of claims 48-52, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid that is not contacted with the low/first dose of bilirubin.

54. The method of any one of claims 48-53, wherein the mature liver organoids express CYP2E1, CYP7A1, PR0X1, MRP3, MRP3, or 0ATP2, or any combination thereof.

55. The method of any one of claims 48-54, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with the low/first dose of bilirubin.

56. The method of any one of claims 29-55, wherein the cells of step ii) are further contacted with a high/second concentration of bilirubin, wherein the liver organoids that are formed are hyperbilirubinemia liver organoids.

57. The method of claim 56, wherein the high/second concentration of bilirubin is, is about, is more than, or is more than about: a) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L.

58. The method of claim 56 or 57, wherein the hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

59. The method of any one of claims 29-58, wherein the liver organoids comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the liver organoids are able to synthesize ascorbate.

60. The method of claim 59, wherein the functional GULO protein is murine GULO (mGULO).

61. The method of claim 59 or 60, wherein the gene that encodes for the functional GULO protein is conditionally expressed, optionally using a tetracycline inducible system.

62. The method of any one of claims 59-61, wherein the liver organoids are engineered with the gene that encodes for the functional GULO protein using CRISPR.

63. The method of any one of claims 59-62, wherein the gene or mRNA, or both, that encodes for the functional GULO protein is introduced to the liver organoids by transfection.

64. The method of any one of claims 59-63, wherein the liver organoids comprising the functional GULO protein express increased levels of NRF2 relative to liver organoids that do not comprise the functional GULO protein.

65. The method of any one of claims 59-64, wherein the liver organoids comprising the functional GULO protein expresses reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids that do not comprise the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate.

66. The method of any one of claims 59-65, wherein the liver organoids comprising the functional GULO protein exhibits reduced caspase-3 activity relative to liver organoids that do not comprise the functional GULO protein, optionally when cultured in ascorbate-depleted medium or in the absence of ascorbate.

67. The method of any one of claims 59-66, wherein the liver organoids comprising the functional GULO protein express increased levels of ALB relative to liver organoids that do not comprise the functional GULO protein.

68. The method of any one of claims 59-67, wherein the liver organoids comprising the functional GULO protein resemble periportal liver tissue and express periportal liver markers.

69. The method of claim 68, wherein the periportal liver markers comprise FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

70. The method of any one of claims 59-69, wherein the liver organoids comprising the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that do not comprise the functional GULO protein.

71. The method of any one of claims 59-70, wherein the liver organoids comprising the functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids that do not comprise the functional GULO protein.

72. The method of any one of claims 59-71, wherein the liver organoids comprising the functional GULO protein exhibit increased viability in culture relative to liver organoids that do not comprise the functional GULO protein.

73. The method of any one of claims 59-72, wherein the liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.

74. The method of any one of claims 59-73, wherein the liver organoids comprise an inactive UGT1A1 gene, wherein the liver organoids are a model for Crigler-Najjar Syndrome.

75. The method of any one of claims 29-74, further comprising aggregating the posterior foregut cells and/or posterior foregut endoderm cells in a microwell or other apparatus (e.g., Aggrewell) prior to step i), wherein aggregating the posterior foregut cells and/or posterior foregut endoderm cells results in more uniformly sized liver organoids.

76. The method of any one of claims 29-75, wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof that is xenogeneic to humans, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of step i) and/or step ii) are not contacted with Matrigel®, Cultrex®, or Geltrex®.

77. The method of any one of claims 29-76, wherein the cells of step i) and/or step ii), and/or the liver organoids formed therefrom, are cultured in a static or non- static bioreactor, optionally a rotational bioreactor, optionally a 3D bioreactor, optionally a 3D rotational bioreactor.

78. The method of claim 77, wherein after culturing in a static or non-static bioreactor, the liver organoids are dissociated into single cells, and are subsequently reconstructed and/or expanded via an additional culturing step in a static or non-static bioreactor, optionally a 3D bioreactor, optionally a 3D rotational bioreactor.

79. The method of any one of claims 29-78, further comprising cry opreserving the liver organoids.

- ISO-

80. The method of claim 79, wherein cryopreserving the liver organoids comprises slow- freezing or vitrification cryopreservation, optionally wherein the liver organoids are cryopreserved with chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

81. The method of any one of claims 1-80, wherein the posterior foregut cells and/or posterior foregut endoderm cells have been derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

82. The method of any one of claims 1-81, wherein the posterior foregut cells and/or posterior foregut endoderm cells have been derived from a subject, optionally a subject having a liver-related disease or disorder.

83. The method of any one of claims 1-82, wherein the method can be used in a good manufacturing practice (GMP) compliant process.

84. The posterior foregut cells and/or posterior foregut endoderm cells or liver organoids produced by the method of any one of claims 1-83.

85. An in vitro composition comprising: pluripotent stem cells, definitive endoderm, posterior foregut, posterior foregut endoderm, and/or downstream liver cell types, and at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

86. The in vitro composition of claim 85, wherein the composition comprises posterior foregut cells and/or posterior foregut endoderm cells, and the posterior foregut cells and/or posterior foregut endoderm cells are dissociated posterior foregut cells and/or posterior foregut endoderm cells.

87. The in vitro composition of claim 85 or 86, wherein the posterior foregut cells and/or posterior foregut endoderm cells are at a cell density of greater than or equal to, exactly or about, IxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, IxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, or 5xl0⁶ cells/cm² of surface area of the tissue culture surface, or any cell density with a range defined by any two of the aforementioned cell densities.

88. The in vitro composition of any one of claims 85-87, wherein the tissue culture surface is coated with a basement membrane matrix or component thereof.

89. The in vitro composition of claim 88, wherein the basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth- Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel®, Cultrex®, or Geltrex®.

90. The in vitro composition of claim 88 or 89, wherein the basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.

91. The in vitro composition of any one of claims 85-90, wherein at least a portion of the posterior foregut cells and/or posterior foregut endoderm cells are spontaneously formed three- dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure with a single lumen.

92. The in vitro composition of any one of claims 85-91, wherein the TGF-b pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542; optionally wherein the TGF-b pathway inhibitor comprises or is A83-01.

93. The in vitro composition of any one of claims 85-92, wherein the TGF-b pathway inhibitor is at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the TGF-b pathway inhibitor is at a concentration of, or of about, 500 nM.

94. The in vitro composition of any one of claims 85-93, wherein the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally wherein the FGF pathway activator comprises or is FGF2.

95. The in vitro composition of any one of claims 85-94, wherein the FGF pathway activator is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mE, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is at a concentration of, or of about, 5 ng/mE.

96. The in vitro composition of any one of claims 85-95, wherein the Wnt pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR- A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119, optionally wherein the Wnt pathway activator comprises or is CHIR99021.

97. The in vitro composition of any one of claims 85-96, the Wnt pathway activator is at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is at a concentration of, or of about, 3 pM.

98. The in vitro composition of any one of claims 85-97, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally wherein the VEGF pathway activator comprises or is VEGF.

99. The in vitro composition of any one of claims 85-98, wherein the VEGF pathway activator is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is at a concentration of, or of about, 10 ng/mL.

100. The in vitro composition of any one of claims 85-99, wherein the composition further comprises exogenous EGF, or wherein the composition does not comprise exogenous EGF.

101. The in vitro composition of any one of claims 85-100, wherein the EGF is at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the EGF is at a concentration of, or of about, 20 ng/mL.

102. The in vitro composition of any one of claims 85-101, wherein the composition further comprises exogenous and/or transgenically produced ascorbic acid, or wherein the composition does not comprise exogenous and/or transgenically produced ascorbic acid.

103. The in vitro composition of any one of claims 85-102, wherein the ascorbic acid is at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the ascorbic acid is at a concentration of, or of about, 50 g/mL.

104. The in vitro composition of any one of claims 85-103, further comprising a ROCK inhibitor, or cultured in a media that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor comprises or is Y -27632.

105. The in vitro composition of any one of claims 85-104, wherein the ROCK inhibitor is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally where the ROCK inhibitor is at a concentration of, or of about, 10 pM.

106. The in vitro composition of any one of claims 85-105, wherein the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm, and/or downstream liver cells are differentiated from stem cells, optionally wherein the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm, and/or downstream liver cells are differentiated from induced pluripotent stem cells.

107. The in vitro composition of any one of claims 85-107, wherein the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm, and/or downstream liver cells have been passaged less than 4 times.

108. The in vitro composition of claim 108, wherein the cells comprise or consist essentially of posterior foregut cells and/or posterior foregut endoderm cells.

109. The in vitro composition of any one of claims 85-108, wherein the TGF-b pathway inhibitor is A83-01, the FGF pathway activator is FGF2, the Wnt pathway activator is CHIR99021, the VEGF pathway activator is VEGF, and the ROCK inhibitor is Y-27632.

110. The liver organoid produced by the method of any one of claims 29-83.

111. An in vitro composition comprising, a) posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids, and b) a medium, wherein the medium optionally comprises hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid, and wherein the composition optionally additionally comprises c) a retinoic acid pathway activator.

112. The in vitro composition of claim 111, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and an agonist c-Met antibody, optionally LMH85.

113. The in vitro composition of any one of claims 111-112, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIL), cardiotrophin-1, ciliary neurotrophic factor (CTNL), and cardiotrophin- like cytokine (CLC).

114. The in vitro composition of any one of claims 111-113, wherein the corticosteroid is selected from a group consisting of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and mometasone.

115. The in vitro composition of any one of claims 111-114, wherein the medium is supplemented with HGL, OSM, and dexamethasone.

116. The in vitro composition of any one of claims 111-115, wherein the medium is supplemented with dexamethasone.

117. The in vitro composition of any one of claims 111-116, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally wherein the retinoic acid pathway activator comprises or is retinoic acid.

118. The in vitro composition of any one of claims 111-117, wherein the retinoic acid pathway activator is at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 pM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator is at a concentration of, or of about, 2.0 pM.

119. The in vitro composition of any one of claims 115-118, wherein the HGL is at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the HGL is at a concentration of, or of about 10 ng/mL.

120. The in vitro composition of any one of claims 115-119, wherein the OSM is at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the OSM is at a concentration of, or of about 20 ng/mL.

121. The in vitro composition of any one of claims 115-120, wherein the dexamethasone is at a concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone is at a concentration of, or of about 100 nM.

122. The in vitro composition of any one of claims 111-121, wherein the composition does not comprise exogenous EGF.

123. The in vitro composition of any one of claims 111-122, further comprising a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin is at or near a human fetal physiological concentration of bilirubin.

124. The in vitro composition of claim 123, wherein the bilirubin is, is about, is less than, or is less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.

125. The in vitro composition of claim 124, wherein the composition comprises mature liver organoids, and wherein the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells.

126. The in vitro composition of claim 125, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid not contacted with a low dose of bilirubin.

127. The in vitro composition of claim 125 or 126, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low dose of bilirubin.

128. The in vitro composition of any one of claims 125-127, wherein the mature liver organoids express CYP2E1, CYP7A1, PR0X1, MRP3, MRP3, or OATP2, or any combination thereof.

129. The in vitro composition of any one of claims 125-128, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids that are not contacted with a low dose of bilirubin.

130. An in vitro composition comprising mature liver organoids, wherein the cells of the mature liver organoid were contacted with a low dose of bilirubin, optionally wherein the low dose of bilirubin was exogenously provided, and the mature liver organoids exhibit luminal projections that resemble bile canaliculi, and/or a structure having a single lumen and generally a spherical shape, and/or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells.

131. The in vitro composition of claim 130, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

132. The in vitro composition of claim 130 or 131, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

133. The in vitro composition of any one of claims 130-132, wherein the mature liver organoids express CYP2E1, CYP7A1, PROXI, MRP3, MRP3, or OATP2, or any combination thereof.

134. The in vitro composition of any one of claims 130-133, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity, relative to a liver organoid where the cells were not contacted with a low dose of bilirubin.

135. The in vitro composition of any one of claims 130-134, further comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells were contacted with a high and/or second concentration of bilirubin.

136. The in vitro composition of claim 135, wherein the high/second concentration of bilirubin was, was about, was more than, or was more than about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 2 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L; or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 4 to 20 mg/L, 2 to 10 mg/L, 10 to 20 mg/L, 5 to 15 mg/L, or 8 to 12 mg/L.

137. The in vitro composition of claim 135 or 136, wherein the hyperbilirubinemia liver organoids express elevated levels of UGT1 Al or NRF2, or both, relative to liver organoids not treated with a high/second concentration of bilirubin.

138. The in vitro composition of any one of claims 111-137, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are able to synthesize ascorbate.

139. The in vitro composition of claim 138, wherein the functional GULO protein is murine GULO (mGULO).

140. The in vitro composition of claim 138 or 139, wherein the gene that encodes for the functional GULO protein is conditionally expressed, optionally using a tetracycline inducible system.

141. The in vitro composition of any one of claims 138-139, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are engineered to comprise a gene that encodes for the functional GULO protein using CRISPR.

142. The in vitro composition of any one of claims 138-141, wherein the gene or mRNA, or both, that encodes for the functional GULO protein was introduced to the liver organoids by transfection.

143. The in vitro composition of any one of claims 138-142, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein express increased levels of NRF2 relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

144. The in vitro composition of any one of claims 138-143, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein expresses reduced levels of IL1B, IL6, or TNFa, or any combination thereof, relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

145. The in vitro composition of any one of claims 138-144, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit reduced caspase-3 activity relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

146. The in vitro composition of any one of claims 138-145, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein express increased levels of ALB relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

147. The in vitro composition of any one of claims 138-146, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein resemble periportal liver tissue and express periportal liver markers.

148. The in vitro composition of claim 147, wherein the periportal liver markers comprise FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

149. The in vitro composition of any one of claims 138-148, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids an/dor mature liver organoids that do not comprise the functional GULO protein.

150. The in vitro composition of any one of claims 138-149, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

151. The in vitro composition of any one of claims 138-150, wherein the liver organoids and/or mature liver organoids comprising the functional GULO protein exhibit increased viability in culture relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.

152. The in vitro composition of any one of claims 138-151, wherein the liver organoids and/or mature liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.

153. A method comprising administering the liver organoid or composition of any one of claims 110-152 to a subject in need thereof.

154. A method for treating a liver-related disease or disorder in a subject in need thereof, comprising administering one or more liver organoids or compositions of claims 110-152 to the subject.

155. The method of claim 153 or 154, wherein the liver organoid has been produced from cells derived from the subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells.

156. The method of any one of claims 154-155, wherein administering comprises transplanting the liver organoid or composition into the subject.

157. The method of any one of claims 154-156, wherein the liver-related disease or disorder comprises one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug- induced cholestasis, metabolic disease, autoimmune liver disease, Wilson’s disease, metabolic- associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, metabolic dysfunction-associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, hepatic cyst, acetaminophen acute liver injury, and/or alcohol-associated liver disease.

158. The method of any one of claims 154-157, wherein the liver dysfunction and/or failure comprises hyperammonemia and/or hyperbilirubinemia; or wherein the metabolic disease comprises nonalcoholic fatty liver disease (NAFLD); or wherein the nonalcoholic fatty liver disease (NAFLD) comprises metabolic dysfunction-associated steatohepatitis (MASH); or wherein the hepatitis comprises hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and/or autoimmune hepatitis.

159. The method of any one of claims 154-158, wherein the subject has reduced serum bilirubin and/or ammonia levels, and/or increased serum protein albumin following transplantation.

160. The method of any of claims 154-159, wherein the subject has improved symptoms of biliary stricture and/or liver regeneration following transplantation.

161. The method of any of claims 154-160, wherein the subject has an increased survival rate following transplantation.

162. The method of any of claims 154-161, wherein the liver organoid engrafts onto the liver of the subject.

163. The method of any of claims 154-162, wherein the liver organoid has been treated with amino acid (AA) supplementation.

164. The method of any of claims 154-163, wherein the liver organoid has been treated with amino acid (AA) supplementation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to transplantation.

165. The method of any of claims 154-164, wherein the liver-related disease or disorder comprises acetaminophen acute liver injury.

166. The method of any of claims 154-165, wherein the method can be used in a good manufacturing practice (GMP) compliant process.

167. A method for screening, comprising contacting the liver organoid of any one of claims 84-152 with a candidate compound or composition, and assessing the effects of the candidate compound or composition on the liver organoid.

168. The method of claim 167, wherein the liver organoid is a model for a liver-related disease or disorder, and assessing the effects of the candidate compound or composition on the liver organoid comprises assessing the effects of the candidate compound or composition on the liver-related disease or disorder.

169. The method of claim 167 or 168, wherein the liver organoid has been produced from cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells.

170. The method of claim 169, wherein the subject has a liver-related disease or disorder.

171. The method of any of claims 167-170, wherein the method can be used in a good manufacturing practice (GMP) compliant process.

172. A composition comprising an amino acid supplemented liquid component according to Table 3.

173. A composition comprising a cocktail of growth factors according to an embodiment of Table 1 or Table 2.

174. A composition comprising an amino acid supplemented liquid component comprising exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% non-essential amino acid solution by volume (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg/mL glycine.

175. The composition of any of claims 172-174, comprising an amino acid supplemented liquid component comprising exactly or about 14% non-essential amino acid solution (containing exactly or about alanine 890 mg/L, asparagine 1320 mg/L, aspartic acid 1330 mg/L, glycine 750 mg/L, serine 105 mg/L, proline 1150 mg/L, and glutamic acid 1470 mg/L), exactly or about 6% essential amino acid solution by volume (containing exactly or about arginine 6320 mg/L, cysteine 1200 mg/L, histidine 2100 mg/L, isoleucine 2620 mg/L, leucine 2620 mg/L, lysine 3625 mg/L, methionine 755 mg/L, phenylalanine 1650 mg/L, threonine 2380 mg/L, tryptophan 510 mg/L, tyrosine 1800 mg/L, and valine 2340 mg/L), and exactly or about 80% hepatocyte culture medium (HCM) by volume, and further supplemented with exactly or about 20 g/L glycine.

176. The composition of any one of claims 172-175, wherein the pH is between about pH 6- 8, or pH 6.5-7.5, or exactly or about pH 7.0.

177. The composition of any one of claims 172-176, further comprising hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid.

178. The composition of any one of claims 172-177, further comprising hepatic lineage committed cells differentiated from definitive endoderm cells using retinoic acid.

179. The composition of claim 178, wherein the hepatic lineage committed cells are characterized as liver organoids.

180. The composition of claim 179, wherein the liver organoids are characterized as secreting increased levels of albumin and urea relative to liver organoids comprised in HCM without amino acid supplementation.

181. The composition of claim 179 or 180, wherein the liver organoids are characterized as expressing increased levels of hepatic maturation associated gene expression relative to liver organoids comprised in HCM without amino acid supplementation.

182. The composition of any one of claims 179-181, wherein the liver organoids are characterized as expressing reduced levels of Vimentin relative to liver organoids comprised in HCM without amino acid supplementation.

183. The composition of any one of claims 172-182, wherein the composition does not comprise non-human animal components such that the basement membrane matrix or component thereof is xenogeneic to humans.

184. The composition of claim 182, wherein the composition does not comprise murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and/or Geltrex®.

185. An in vitro hyperbilirubinemia liver organoid comprising a naturally occurring and/or engineered mutation in the UDP glucuronosyltransferase family 1 member Al UGT1A1) gene.

186. An in vitro hyperbilirubinemia liver organoid, wherein the hyperbilirubinemia liver organoid was produced through at least two rounds of contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids to exogenous bilirubin.

187. The in vitro hyperbilirubinemia liver organoid of claim 185 or 186, wherein the hyperbilirubinemia liver organoid was clonally derived and/or derived from iPSCs.

188. A cryopreserved composition, comprising liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

189. A cryopreserved composition, comprising mature liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

190. A cryopreserved composition, comprising hyperbilirubinemia liver organoids, chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

191. A kit comprising means for conducting the method according to any one of claims 1- 83, or 153-171.

192. A kit comprising the compositions or means for generating the compositions according to any one of claims 84-152, 172-184, or 188-190, or for generating the liver organoid of any one of claims 185-187.

193. Use of the method, composition, or kit according to any one of claims 1-192, as a medicament, means for treatment and/or prevention of a disease, means of diagnosis, and/or medical research.