WO2025126208A1 - Media for maintaining, differentiating, or both, pluripotent stem cells - Google Patents

Abstract

The present invention is directed to, inter alia, defined culture media, kits for preparing same, and methods of using same, such as for maintaining, differentiating, or both, pluripotent stem cells.

Description

MEDIA FOR MAINTAINING. DIFFERENTIATING. OR BOTH. PLURIPOTENT

STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/608,838, titled “MEDIA FOR MAINTAINING, DIFFERENTIATING, OR BOTH, PLURIPOTENT STEM CELLS”, filed 12 December 2023, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present invention, in some embodiments, is in the field of culture media for culturing pluripotent stem cells, and more particularly, but not exclusively, to novel culture media which can be used to differentiate pluripotent stem cells into hematopoietic stem cells (HSCs), and to novel culture media which can maintain HSCs in a proliferative, multipotent and undifferentiated state.

BACKGROUND OF THE INVENTION

[003] Stem cells hold immense promise in the field of regenerative medicine and have captured the attention of scientists and medical researchers worldwide. These unique cells possess the remarkable ability to self-renew and differentiate into specialized cell types, making them a critical player in tissue development, repair, and regeneration. Among the diverse types of stem cells, hematopoietic stem cells (HPSCs) stand out for their pivotal role in the formation of blood and immune cells. The process of differentiation, which involves the transformation of a pluripotent stem cell into a specific cell lineage, is a complex and finely orchestrated mechanism. In this context, deciphering stem cells differentiation into hematopoietic stem cells is of paramount importance to harness their therapeutic potential for various blood-related disorders, diseases, and trauma.

[004] There is still a great need for compositions, kits, and methods for specific maintenance, differentiation, and both, of hematopoietic stem cells, to be used such as for, diagnostics and food production, and trauma or therapy for blood-related diseases. SUMMARY OF THE INVENTION

[005] According to the first aspect, there is provided defined culture medium comprising Iscove's Modified Dulbecco Media (IMDM) basal medium and an effective amount of a serum replacement, wherein the defined culture medium is serum-free, and is capable of promoting: (i) differentiation of hematopoietic stem cell (HSC) to descendant cells thereof; (ii) expansion of HSC; or (iii) both (i) and (ii).

[006] According to another aspect, there is provided a method of differentiating PSC into HSC, the method comprising culturing the PSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, IL3, Flt3 ligand, IL6, G-CSF, TPO, UM729, UM171, StemRegenin (SRI), nicotinamide (NAM)and any combination thereof.

[007] According to another aspect, there is provided a method of maintaining HSC in an undifferentiated state, the method comprising culturing the HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: SCF, Flt3 ligand, TPO, and any combination thereof, thereby maintaining HSC in an undifferentiated state.

[008] According to another aspect, there is provided a method of differentiating HSC into erythrocytes, the method comprising culturing the HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, EPO, ITS, holo-transferrin, hydrocortisone, T3, FelII-EDTA, and any combination thereof, thereby differentiating the HSC into erythrocytes.

[009] According to another aspect, there is provided a method of differentiating HSC into megakaryocytes, the method comprising culturing the HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of any one of: ITS, fetal serum, L- ascorbic acid, MTG, and any combination thereof; and (ii) at least one cytokine or agent selected from the group consisting of: TPO, SCF, Flt3 ligand, IL6, IL9, heparin, and any combination thereof, thereby differentiating the HSC into megakaryocytes. [010] According to another aspect, there is provided a kit for use in: (i) the differentiation of PSC to HSC; HSC to descendant cells thereof, or both; (ii) maintenance of HSC in an undifferentiated state; or (iii) both (i) and (ii), the kit comprising IMDM basal medium.

[011] According to another aspect, there is provided a composition comprising any one of: an undifferentiated HSC, a differentiated HSC, a descendent thereof, and any combination thereof, obtained according to the method of the invention.

[012] In some embodiments, the descendant cells comprise erythrocytes.

[013] In some embodiments, the defined culture medium further comprises an effective amount of insulin, transferrin, and Selenium (ITS), and being capable of promoting differentiation of any one of pluripotent stem cells (PSC) to HSC, and of HSC to megakaryocytes.

[014] In some embodiments, the megakaryocytes comprise platelets.

[015] In some embodiments, the differentiation is in two dimensions (2D) configuration or in three dimensions (3D) configuration.

[016] In some embodiments, the 2D configuration comprises culturing on a feeder cell layer, and the 3D configuration comprises culturing in suspension.

[017] In some embodiments, the defined culture medium further comprises at least one first cytokine or agent selected from the group consisting of: stem cell factor (SCF), Fms Related Receptor Tyrosine Kinase 3 (Flt3) ligand, thrombopoietin (TPO), and any combination thereof.

[018] In some embodiments, the defined cultured medium further comprises at least one second cytokine or agent selected from the group consisting of: interleukin (IL) 6, bone morphogenetic protein (BMP) 4, vascular endothelial growth factor (VEGF), IL3, and any combination thereof.

[019] In some embodiments, the defined culture medium further comprises at least one third cytokine or agent selected from the group consisting of: granulocyte colony-stimulating factor (G-CSF), UM729, IL9, heparin, or any combination thereof.

[020] In some embodiments, the defined culture medium comprises: BMP4, SCF, VEGF, G- CSF, FLT3 ligand, IL6, IL3, TPO, and UM729, and is capable of promoting differentiation of PSC to HSC in a 2D configuration.

[021] In some embodiments, the defined culture medium comprises: BMP4, SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, UM729, erythropoietin (EPO), ITS, holo-transferrin, hydrocortisone, T3, and FelII-EDTA, and is capable of promoting differentiation of HSC to erythrocytes in a 3D configuration. [022] In some embodiments, the defined culture medium comprises: TPO, SCF, Flt3 ligand, IL6, IL9, UM729, and heparin, and is capable of promoting differentiation of HSC to megakaryocytes.

[023] In some embodiments, the defined culture medium comprises: TPO, SCF, and FLT3 ligand, and is capable of promoting expansion of HSC.

[024] In some embodiments, the differentiating comprises culturing the PSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of: (a) BMP4; (b) SCF, VEGF, UM171, SRI, and NAM; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, and TPO; and (d) any combination of (a) to (c).

[025] In some embodiments, (a) is for a period of between 1 and 5 days, (b) is for a period of between 1 and 4 days, and (c) is for a period of 2 and 24 days.

[026] In some embodiments, at least 50% of the cultured HSC express CD34.

[027] In some embodiments, the method further comprises a step preceding the maintaining comprising differentiating PSC into the HSC, wherein the differentiating comprises culturing the PSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, IL3, Flt3 ligand, IL6, G-CSF, TPO, UM729, UM171, StemRegenin (SRI), nicotinamide (NAM), and any combination thereof.

[028] In some embodiments, the differentiating comprises culturing the PSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of: (a) BMP4; (b) SCF, VEGF, UM171, SRI, and NAM; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, and TPO; and (d) any combination of (a) to (c).

[029] In some embodiments, the method further comprises a step proceeding the maintaining comprising differentiating the HSC into descendant cells thereof.

[030] In some embodiments, the descendant cells are erythrocytes, and wherein the differentiating comprises culturing the HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, EPO, ITS, holo-transferrin, hydrocortisone, T3, FelII-EDTA, and any combination thereof, thereby differentiating the HSC into erythrocytes.

[031] In some embodiments, the differentiating comprises culturing the HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of: (a) IL3, EPO, and SCF; (b) SCF, holo-transferrin, hydrocortisone, and EPO; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; and (d) any combination of (a) to (c).

[032] In some embodiments, (a) is for a period of between 3 and 10 days, (b) is for a period of between 3 and 10 days, and (c) is for a period of 3 and 10 days.

[033] In some embodiments, the descendant cells are megakaryocytes, and wherein the differentiating comprises culturing the HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of any one of: ITS, fetal serum, L-ascorbic acid, monothioglycerol (MTG), and any combination thereof; and (ii) at least one cytokine or agent selected from the group consisting of: TPO, SCF, Flt3 ligand, IL6, IL9, heparin, and any combination thereof, thereby differentiating the HSC into megakaryocytes.

[034] In some embodiments, the fetal serum is derived from bovine.

[035] In some embodiments, the culturing is for a period of 10 and 20 days.

[036] In some embodiments, the culturing comprises any one of 2D culturing, 3D culturing, and both.

[037] In some embodiments, the kit further comprises any one of: (a) at least one agent promoting health of a blood cell or a precursor thereof, being selected from the group consisting of: serum replacement, insulin, transferrin, Selenium, L-ascorbic acid, MTG, and any combination thereof; (b) at least one agent capable of promoting any of the differentiation and the expansion, being selected from the group consisting of: SCF, Flt3 ligand, TPO, IL6, BMP4, VEGF, IL3, G-CSF, UM729, IL9, heparin, holo-transferrin, hydrocortisone, T3, FelII-EDTA, and any combination thereof; and (c) both (a) and (b).

[038] In some embodiments, the kit further comprises instructions for any one of the differentiations and the maintenance in an undifferentiated state.

[039] In some embodiments, the instructions are for differentiating the PSC into HSC, the instructions comprising: mixing the IMDM basal medium with an effective amount of: (i) the serum replacement and ITS; and (ii) an one of the: (a) BMP4; (b) SCF, VEGF, UM171, SRI, and NAM; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; and (d) any combination of (a) to (c), thereby obtaining HSC differentiation media, and culturing the PSC in the HSC differentiation media.

[040] In some embodiments, die culturing in (a) is for a period of between 1 and 5 days, culturing in (b) is for a period of between 1 and 4 days, and culturing in (c) is for a period of 2 and 24 days.

[041] In some embodiments, the instructions are for maintenance of the HSC in an undifferentiated state, the instructions comprising: mixing the IMDM basal medium with an effective amount of: (i) the serum replacement; and (ii) SCF, Flt3 ligand, TPO, thereby obtaining HSC expansion media, and culturing the HSC in the HSC expansion media.

[042] In some embodiments, the maintenance is for 1-3 weeks, 2 to 4 passages, or both.

[043] In some embodiments, the instructions are for differentiating the HSC into erythrocytes, the instructions comprising: mixing the IMDM basal medium with an effective amount of (i) the serum replacement; and (ii) any one of: (a) IL3, EPO, and SCF; (b) SCF, holo-transferrin, hydrocortisone, and EPO; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; and (d) any combination of (a) to (c), thereby obtaining erythrocytes differentiation media, and culturing the HSC in the erythrocytes differentiation media.

[044] In some embodiments, the culturing in (a) is for a period of between 3 and 10 days, culturing in (b) is for a period of between 3 and 10 days, and culturing in (c) is for a period of 3 and 10 days.

[045] In some embodiments, the at least one agent promoting health of a blood cell or a precursor thereof further comprises fetal serum.

[046] In some embodiments, the instructions are for differentiating the HSC into megakaryocytes, the instructions comprising: mixing the IMDM basal

with an effective amount of: (i) the ITS, fetal serum, L-ascorbic add, and MTG; and (ii) TPO, SCF, Flt3 ligand, IL6, IL9, and heparin, thereby obtaining megakaryocytes differentiation media, and culturing the HSC in the megakaryocyte’s differentiation media.

[047] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and exanqiles are illustrative only and are not intended to be necessarily limiting.

[048] Further embodiments and the foil scope of applicability of the present invention will become qiparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific exanqiles, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become qiparent to those skilled in the art from tins detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[049] Figs.1A-1B include graphs showing expression of CD34 (1A) and CD45 (IB) expression throughout differentiation in adherent culture.

[050] Figs.2A-2B include a graph and scatter plots showing maintenance of CD34+ cells. Flow cytometry analyses of CD34 and CD45 expansion HSCE medium after 11 days in culture was performed. During tins period, the number of cells increased by 10-fold (2A). Staining for CD34 expression after one week of growth in medium as disclosed herein (2B).

[051] F|g.3A-3D include a vertical bar graph, photogrqihs, and micrographs showing a process development of CD34* cells towards red blood cells (RBCs). (3A) Expression of erythroid markers on day 13 in static and dynamic systems from start of Erythroid expansion. (3B) Changes in cell color during erythroid differentiation were observed. Red brownish cell pellets (arrows) strengthened as erythroid maturation progressed. Left - day 13 from starting of Erythroid expansion; Right - day 16 from starting of Erythroid expansion. (3C) May-Grunwald Giemsa staining rqnesentative image of enucleated reticulocytes. Arrows indicate cells without nucleus. Scale bar = 10 μm. (3D) A rqnesentative image of paraffin embedded section stained with hematoxylin and eosin (H&E) and an anti-CD71 antibody, following 13 days of differentiation (from the beginning of the protocol). Arrows indicate CD71 stained cells. Scale bar = 20 μm.

[052] F|g. 4A-4B include photogrqihs showing differentiation of induced PSC (iPSC) towards RBCs in three-dimension culturing (3D). Cell smear and May-Grunwald Giemsa staining were performed. (4A) Day 38 in a static culture; and (4B) day 30 in a dynamic culture. Scale bar = 10 μm. Arrowheads indicate RBCs.

[053] Figs. 5A-5D include fluorescent micrographs showing immunofluorescence staining (IF) of CD235a and Hemoglobin in matured RBCs initiated from iPSCs in three-dimension culturing (3D). Arrows showing positive staining (arrowheads) of CD235a (5A-5B); and of hemoglobin (5C-5D) and negative nuclei staining (with DAPI), thus, indicating that mature erythrocytes were observed (day 55). (5A and SC) Scale bar: 100 μm; (SB and 5D) Scale bar: 25 μm.

[054] Fig. 6 includes a vertical bar graph showing different markers expression (CD34, CD45, CD31, CD71, CD36, and CD235) on day 21 in dynamic culture and day 14 in static culture of human iPSC differentiating to RBCs.

[055] Figs. 7A-7D include graphs showing differentiation of iPSC towards RBCs in a dynamic system. Shown are: total cell concentration (7A); total cell number (7B), percentage of RBCs from total cells (7C); and hemoglobin concentration (7D). Single dots in (7C-7D) reflect calculated values for cells obtained following separation on a Percoll gradient.

[056] Figs. 8A-8H include a scheme, graphs, micrographs, and scatter plots showing that CD34+ cells cultured in StemSpan™ (SS) + ACS 2 (Accellta™ supplement) express Megakaryocyte (MK) markers. (8A) A scheme showing non-limiting course of experiment. (HDM- hematopoietic differentiation medium). (8B) Left panel: CD34+-derived cell proliferation over 27 days. Right panel: Cell viability overtime. (8C) Fold increase of CD34*- derived cells on day 14 in the different mediums. (8D) Percent of CD34*-derived cells expressing the indicated markers after 14 days in StemSpan™ medium supplemented with ACS 2. (8E) Back-gating showing CD61*CD31* cells out of CD 41a+ cells. (8F) Light microscopy images (x20 magnification) showing the morphology of CD34*-derived cells in different media overtime. (8G) Percent of cells with different diameter range as detected and measured on day 14. (8H) Light microscopy images showing Giemsa-stained cell smear of highly uncertain MKs (black arrows) on day 27.

DETAILED DESCRIPTION OF THE INVENTION

Defined culture medium

[057] According to one aspect, there is provided a defined culture medium comprising basal medium and an effective amount of a serum replacement.

[058] According to one aspect, there is provided a defined culture medium comprising Iscove's Modified Dulbecco Media (IMDM) basal medium and an effective amount of a serum replacement.

[059] In some embodiments, the defined culture medium is serum-free. [060] In some embodiments, the defined culture medium is capable of promoting differentiation of hematopoietic stem cell (HSC) to descendant cells thereof.

[061] In some embodiments, the defined culture medium is capable of promoting expansion of HSC.

[062] In some embodiments, the defined culture medium is capable of promoting differentiation of HSC to descendant cells thereof and expansion of HSC.

[063] In some embodiments, descendant cells comprise: early erythrocytes, reticulocytes, erythrocytes, megakaryocytes, platelets.

[064] In some embodiments, the defined culture medium further comprises an effective amount of insulin, transferrin, Selenium, or a combination thereof (ITS).

[065] In some embodiments, the defined culture medium comprising ITS is capable of promoting differentiation of pluripotent stem cells (PSC) to HSC, HSC to megakaryocytes, or both.

[066] In some embodiments, megakaryocytes comprise platelets.

[067] In some embodiments, differentiation is in two dimensions (2D) configuration.

[068] In some embodiments, a 2D configuration comprises culturing on a feeder cell layer. In some embodiments, culturing on a 2D configuration is feeder free.

[069] In some embodiments, differentiation is in three dimensions (3D) configuration.

[070] In some embodiments, a 3D configuration comprises culturing in suspension, a plate, a spinner flask, or any combination thereof. In some embodiments, a 3D configuration comprises culturing under spinning conditions, means, or both. Means for culturing cells under spinning conditions are common and would be apparent to one of skill in the art. A non-limiting examples for such means includes, but is not limited to a spinner flask.

[071] In some embodiments, differentiation is in a combination of 2D and 3D configurations.

[072] In some embodiments, the defined culture medium of the invention further comprises basic fibroblast growth factor (bFGF).

[073] In some embodiments, the defined culture medium further comprises at least a first cytokine or agent selected from: stem cell factor (SCF), Fms Related Receptor Tyrosine Kinase 3 (Flt3) ligand, thrombopoietin (TPO), or any combination thereof. [074] In some embodiments, the defined culture medium further comprises at least a second cytokine or agent selected from: interleukin (IL) 6, bone morphogenetic protein (BMP) 4, vascular endothelial growth factor (VEGF), IL3, or any combination thereof.

[075] In some embodiments, the defined culture medium further comprises at least a third cytokine or agent selected from: granulocyte colony-stimulating factor (G-CSF), UM729, IL9, heparin, or any combination thereof.

[076] As used herein, the term “UM729” encompasses any one of: a pyrimido-[4,5-b]-indole derivative, a compound represented by Formula I: or a compound

having the CAS no. 1448723-60-1, including any functional analog thereof.

[077] As used herein, the term “functional analog” encompasses any compound having an essentially similar activity to UM729, which is structurally distinct from UM729.

[078] In some embodiments, essentially similar comprises at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the activity of UM729, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[079] In some embodiments, activity of UM729, including any functional analog thereof, comprises: enhancing self-renewal of hematopoietic stem cells, collaboration with an aryl hydrocarbon receptor (AHR) pathway, prevention of differentiation of acute myeloid leukemia (AML) cells, or any combination thereof.

[080] In some embodiments, activity of UM729 does not include or excludes inhibition of AHR. In some embodiments, enhancing (e.g., self-renewal), prevention (e.g., of differentiation of AML), is in vitro.

[081] In some embodiments, a defined culture medium comprising: BMP4, SCF, VEGF, G- CSF, FLT3 ligand, IL6, IL3, TPO, and UM729, is suitable, configured to, or capable of promoting differentiation of PSC to HSC in a 2D configuration, 3D configuration, or both.

[082] In some embodiments, a defined culture medium comprising: BMP4, SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, UM729, erythropoietin (EPO), ITS, holo-transferrin, hydrocortisone, T3, and FeIII-EDTA, is suitable, configured to, or capable of promoting differentiation of HSC to erythrocytes in a 2D configuration, 3D configuration or both [083] In some embodiments, a defined culture medium comprising: TPO, SCF, Flt3 ligand, IL6, IL9, and heparin, is suitable, configured to, or capable of promoting differentiation of HSC to megakaryocytes.

[084] In some embodiment, a defined culture medium comprising: TPO, SCF, and FLT3 ligand, is suitable, configured to, or capable of promoting expansion of HSC.

[085] In some embodiments, the defined culture medium comprises serum replacement in a concentration of between 1-15%, 2-14%, 5-15%, 7-13%, 6-14%, or 8-12%. Each possibility represents a separate embodiment of the invention.

[086] In some embodiments, the defined culture medium comprises insulin in a concentration of between 0.1 and 3%, 0.5 and 3%, 1 and 3%, 2 and 3% or 2.5 and 3%. Each possibility represents a separate embodiment of the invention.

[087] In some embodiments, the defined culture medium comprises transferrin in a concentration of between 0.1 and 3%, 0.5 and 3%, 1 and 3%, 2 and 3% or 2.5 and 3%. Each possibility represents a separate embodiment of the invention.

[088] In some embodiments, the defined culture medium comprises Selenium in a concentration of between 0.1 and 3%, 0.5 and 3%, 1 and 3%, 2 and 3% or 2.5 and 3%. Each possibility represents a separate embodiment of the invention.

[089] In some embodiments, the defined culture medium comprises ITS (e.g., insulin, transferrin, and Selenium combined) in a concentration of between 0.1 and 3%, 0.5 and 3%, 1 and 3%, 2 and 3% or 2.5 and 3%. Each possibility represents a separate embodiment of the invention.

[090] In some embodiments, the defined culture medium comprises SCF in a concentration of between 10 to 100 ng/ml, 20 to 100 ng/ml, 35 to 100 ng/ml, 50 to 100 ng/ml, 70 to 100 ng/ml, 85 to 100 ng/ml, 40 to 80 ng/ml, or 50 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[091] In some embodiments, the defined culture medium comprises Flt3 ligand in a concentration of between 10 to 100 ng/ml, 20 to 100 ng/ml, 35 to 100 ng/ml, 50 to 100 ng/ml, 25 to 90 ng/ml, 35 to 70 ng/ml, 40 to 80 ng/ml, or 40 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[092] In some embodiments, the defined culture medium comprises TPO in a concentration of between 5 to 150 ng/ml, 10 to 120 ng/ml 10 to 100 ng/ml 20 to 100 ng/ml, 15 to 90 ng/ml, 5 to 50 ng/ml, 5 to 100 ng/ml, or 40 to 120 ng/ml. Each possibility represents a separate embodiment of the invention.

[093] In some embodiments, the defined culture medium comprises IL6 in a concentration of between 10 to 100 ng/ml, 20 to 100 ng/ml, 35 to 100 ng/ml, 40 to 90 ng/ml, 25 to 95 ng/ml, 35 to 70 ng/ml, 40 to 80 ng/ml, or 40 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[094] In some embodiments, the defined culture medium comprises BMP4 in a concentration of between 10 to 110 ng/ml, 20 to 120 ng/ml, 15 to 100 ng/ml, 15 to 80 ng/ml, 15 to 40 ng/ml, 5 to 30 ng/ml, or 80 to 120 ng/ml. Each possibility represents a separate embodiment of the invention.

[095] In some embodiments, the defined culture medium comprises T3 in a concentration of between 0.1 to 1 μM, 0.2 to 1 μM, 0.3 to 1 μM, 0.5 to 1 μM, or 0.7 to 1 μM. Each possibility represents a separate embodiment of the invention.

[096] In some embodiments, the defined culture medium comprises VEGF in a concentration of between 10 to 110 ng/ml, 20 to 120 ng/ml, 15 to 100 ng/ml, 15 to 80 ng/ml, 15 to 60 ng/ml, or 5 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[097] In some embodiments, the defined culture medium comprises IL3 in a concentration 10 to 100 ng/ml, 20 to 100 ng/ml, 35 to 100 ng/ml, 40 to 90 ng/ml, 25 to 95 ng/ml, 35 to 70 ng/ml, 40 to 80 ng/ml, or 40 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[098] In some embodiments, the defined culture medium comprises G-CSF in a concentration of between 10 to 100 ng/ml, 20 to 100 ng/ml, 35 to 100 ng/ml, 40 to 90 ng/ml, 25 to 95 ng/ml, 35 to 70 ng/ml, 40 to 80 ng/ml, or 40 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[099] In some embodiments, the defined culture medium comprises UM729 in a concentration of between 200 nM to 2 μM, 250 nM to 1.5 μM, 300 nM to 2 μM, 500 nM to 2 μM, 400 nM to 1 μM, or 220 nM to 1.7 μM. Each possibility represents a separate embodiment of the invention.

[0100] In some embodiments, the defined culture medium comprises IL9 in a concentration of between 5 to 150 ng/ml, 10 to 120 ng/ml, 10 to 100 ng/ml, 15 to 100 ng/ml, 15 to 90 ng/ml, 15 to 50 ng/ml, or 5 to 40 ng/ml. Each possibility represents a separate embodiment of the invention. [0101] In some embodiments, the defined culture medium comprises heparin in a concentration between 1 to 70 U/ml, 1 to 50 U/ml, 1 to 30 U/ml, 5 to 70 U/ml, 5 to 60 U/ml, or 10 to 30 U/ml. Each possibility represents a separate embodiment of the invention.

[0102] In some embodiments, the defined culture medium comprises EPO in a concentration of between 2 U/mL to 10 U/mL, 3 U/mL to 10 U/mL, 4 U/mL to 10 U/mL, 5 U/mL to 10 U/mL, 6 U/mL to 10 U/mL, 7 U/mL to 10 U/mL, 8 U/mL to 10 U/mL, or 9 U/mL to 10 U/mL. Each possibility represents a separate embodiment of the invention.

[0103] In some embodiments, the defined culture medium comprises holo-transferrin or functional analog (such as, but not limited to Lactoferrin or Optiferin) in a concentration of between 20 pg/mL and 1.2 mg/mL, 50 pg/mL and 1.2 mg/mL, 100 pg/mL and 1.2 mg/mL, 200 pg/mL and 1.2 mg/mL, 350 pg/mL and 1.2 mg/mL, 500 pg/mL and 1.2 mg/mL, 750 pg/mL and 1.2 mg/mL, 1,000 pg/mL and 1.2 mg/mL. Each possibility represents a separate embodiment of the invention.

[0104] As used herein, the term “functional analog” encompasses any compound having an essentially similar activity to holo-transferrin, which is structurally distinct from holo-transferrin.

[0105] In some embodiments, essentially similar comprises at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the activity of holo-transferrin, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0106] In some embodiments, the activity of holo-transferrin, including any functional analog thereof, comprises iron transport, delivery protein, or both. In some embodiments, holo- transferrin activity comprises promotion of cell growth, e.g., in vitro.

[0107] In some embodiments, the defined culture medium comprises hydrocortisone in a concentration of between 0.1 μM and 3 μM, 0.5 μM and 3 μM, 1 μM and 3 μM, 1.5 μM and 3 μM, 2 μM and 3 μM, 2.5 μM and 3 μM, 0.1 μM and 2 μM, or 0.5 μM and 2 μM. Each possibility represents a separate embodiment of the invention.

[0108] In some embodiments, the defined culture medium comprises Felll-EDTA in a concentration of between 0.5 μM and 10 μM, 1 μM and 10 μM, 2 μM and 10 μM, 3 μM and 10 μM, 5 μM and 10 μM, 7 μM and 10 μM, 1 μM and 9 μM, or 1 μM and 8 μM. Each possibility represents a separate embodiment of the invention.

[0109] In some embodiments, the defined culture medium comprises L-ascorbic acid. [0110] In some embodiments, the defined culture medium comprises L-ascorbic acid in a concentration of between 10 to 100 ng/ml, 20 to 100 ng/ml, 35 to 100 ng/ml, 40 to 90 ng/ml, 25 to 95 ng/ml, 35 to 70 ng/ml, 40 to 80 ng/ml, or 40 to 70 ng/ml. Each possibility represents a separate embodiment of the invention.

[0111] In some embodiments, the defined culture medium comprises monothioglycerol (MTG).

[0112] In some embodiments, the defined culture medium comprises MTG in a concentration of between 100 to 1,000 μM, 200 to 1,000 μM, 300 to 1,000 μM, 400 to 1,000 μM, 100 to 700 μM, 250 to 600 μM, 300 to 500 μM, or 400 to 600 μM. Each possibility represents a separate embodiment of the invention.

[0113] In some embodiments, the defined culture medium comprises fetal serum.

[0114] In some embodiments, the defined culture medium comprises fetal serum in a concentration of between 1-25%, 2-20%, 5-20%, 7-20%, 6-25%, 10-20%, or 12-16%. Each possibility represents a separate embodiment of the invention.

Methods of use

[0115] According to another aspect, there is provided a method of differentiating PSC into HSC, comprising culturing PSC in a defined culture medium comprising: (i) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum- free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, IL3, Flt3 ligand, IL6, G-CSF, TPO, UM729, UM171, StemRegenin (SRI), nicotinamide (NAM), and any combination thereof.

[0116] In some embodiments, differentiating comprises culturing PSC in a defined culture medium comprising: (i) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of: (a) BMP4; (b) SCF, VEGF, UM171, SRI, and NAM; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, and TPO; and (d) any combination of (a) to (c).

[0117] In some embodiments, the basal medium comprises or is IMDM.

[0118] In some embodiments, culturing PSC in a defined culture medium comprising BMP4 is for a period of between 1 and 2 days, 1 and 3 days, 1 and 4 days, or 1 and 7 days. Each possibility represents a separate embodiment of the invention. [0119] In some embodiments, culturing PSC in a defined culture medium comprising SCF, VEGF, UM171, SRI, and/or NAM, is for a period of between 1 and 2 days, or 1 and 3 days. Each possibility represents a separate embodiment of the invention.

[0120] In some embodiments, culturing PSC in a defined culture medium comprising SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, and TPO is for a period of 2 and 7 days, 2 and 10 days, -2 and 14 days, 2 and 20 days, 2 and 24 days, 2 and 30 days, 6 and 12 days, 6 and 18 days, 6 and 24 days, 10 and 14 days, 10 and 18 days, or 10 and 30 days. Each possibility represents a separate embodiment of the invention.

[0121] According to another aspect, there is provided a method of maintaining HSC in an undifferentiated state, comprising culturing HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from: SCF, Flt3 ligand, TPO, or any combination thereof.

[0122] In some embodiments, at least: 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cultured HSC express CD34 (e.g., CD34⁺), or any value and range therebetween. In some embodiments, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, or 99-100% of the cultured HSC express CD34 (e.g., CD34⁺). Each possibility represents a separate embodiment of the invention.

[0123] Methods and means for determining expression in a cell, e.g., HSC, or a gene or a protein product thereof are common and would be apparent to one of ordinary skill in the art. Non- limiting examples of such methods include, but are not limited to, PCR, quantitative PCR (e.g., real-time RT-PCR), western blot, flow cytometry, and others, such as exemplified herein below, e.g., fluorescence associated cell sorting (FACS).

[0124] In some embodiments, the method further comprises a step preceding or before the maintaining (step) comprising differentiating PSC into HSC, wherein the differentiating comprises culturing the PSC in a defined culture medium comprising: (i) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum- free; and (ii) at least one cytokine or agent selected from: BMP4, SCF, VEGF, IL3, Flt3 ligand, IL6, G-CSF, TPO, UM729, UM171, StemRegenin (SRI), nicotinamide (NAM), or any combination thereof.

[0125] In some embodiments, the differentiating comprises culturing PSC in a defined culture medium comprising: (i) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free and (ii): (a) BMP4; (b) SCF and VEGF; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, mbination of (a) to (c). [0126] In some embodiments, the culturing in a defined culture medium comprising BMP4 is for a period of between 1 and 2 days, 1 and 3 days, 1 and 4 days, or 1 and 5 days. Each possibility represents a separate embodiment of the invention.

[0127] In some embodiments, the culturing in a defined culture medium comprising SCF, VEGF, UM171, SRI, and/or NAM is for a period of between 1 and 2 days, or 1 and 3 days. Each possibility represents a separate embodiment of the invention.

[0128] In some embodiments, the culturing in a defined culture medium comprising SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, and TPO is for a period of 2 and 7 days, 2 and 10 days, 2 and 14 days, 2 and 20 days, 2 and 24 days, 2 and 30 days, 6 and 12 days, 6 and 18 days, 6 and 24 days, 10 and 14 days, 10 and 18 days, or 10 and 30 days. Each possibility represents a separate embodiment of the invention.

[0129] In some embodiments, the method further comprises a step proceeding or after the maintaining (step) comprising differentiating the HSC into descendant cells thereof.

[0130] In some embodiments the descendant cells comprise or are erythrocytes, and the differentiating comprises culturing HSC in a defined culture medium comprising: (i) basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from: BMP4, SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, UM729, EPO, ITS, holo-transferrin, hydrocortisone, T3, FelII-EDTA, or any combination thereof.

[0131] In some embodiments, differentiating comprises culturing HSC in a defined culture medium comprising: (i) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii): (a) IL3, EPO, and SCF; (b) SCF, holo-transferrin, hydrocortisone, and EPO; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; and (d) or any combination of (a) to (c).

[0132] In some embodiments, culturing in a defined culture medium comprising IL3, EPO, and SCF is for a period of between 3 and 5 days, 3 and 7 days, 3 and 10 days, 4 and 8 days, 4 and 10 days, 5 and 7 days, 5 and 10 days, 6 and 10 days, or 7 and 10 days. Each possibility represents a separate embodiment of the invention.

[0133] In some embodiments, culturing in a defined culture medium comprising SCF, holo- transferrin, hydrocortisone, and EPO is for a period of between 3 and 5 days, 3 and 7 days, 3 and 10 days, 4 and 8 days, 4 and 10 days, 5 and 7 days, 5 and 10 days, 6 and 10 days, or 7 and 10 days. Each possibility represents a s ion. [0134] In some embodiments, culturing in a d d" culture medium comprising SCF, ILS, Flt3 ligand, IL6, G-CSF, and TPO is for a period of between 3 and 5 days, 3 and 7 days, 3 and 10 days, 4 and 8 days, 4 and 10 days, 5 and 7 days, 5 and 10 days, 6 and 10 days, or 7 and 10 days. Each possibility represents a separate embodiment of the invention.

[0135] In some embodiments, the descendant cells are or comprise megakaryocytes, and the differentiating comprises culturing HSC in a defined culture medium co

mprising (i) IMDM basal medium and an effective amount of: ITS, fetal serum, L-ascorbic acid, monothioglycerol (MTG), or any combination thereof; and (ii) at least one cytokine or agent selected from: TPO, SCF, Flt3 ligand, IL6, IL9, heparin, or any combination thereof.

[0136] In some embodiments, fetal serum is derived from a livestock animal. In some embodiments, fetal serum is derived from bovine.

[0137] In some embodiments, the culturing is for a period of 10 and 12 days, 10 and 15 days, 10 and 17 days, 10 and 20 days, 10 and 30 days, 14 and 28 days, 15 and 20 days, 15 and 25 days, 16 and 24 days, or 16 and 30 days. Each possibility represents a separate embodiment of the invention.

[0138] According to another aspect, there is provided a method of differentiating HSC into erythrocytes, comprising culturing HSC in a defined culture

medium comprising: (i) basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: SCF, VEGF, Flt3 ligand, IL6, ILS, TPO, EPO, ITS, holo-transferrin, hydrocortisone, T3, Felll-EDTA, and any combination thereof.

[0139] In some embodiments, differentiating comprises culturing HSC in a defined culture medium comprising: (i) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii): (a) ILS, EPO, and SCF; (b) SCF, holo-transferrin, hydrocortisone, and EPO; (c) SCF, ILS, Flt3 ligand, IL6, G-CSF, and TPO; and (d) or any combination of (a) to (c).

[0140] In some embodiments, culturing in a defined culture medium comprising ILS, EPO, and SCF is for a period of between 3 and 5 days, 3 and 7 days, 3 and 10 days, 4 and 8 days, 4 and 10 days, 5 and 7 days, 5 and 10 days, 6 and 10 days, or 7 and 10 days. Each possibility represents a separate embodiment of the invention.

[0141] In some embodiments, culturing in a defined culture medium comprising SCF, holo- transferrin, hydrocortisone, and EPO is for a period of between 3 and 5 days, 3 and 7 days, 3 and 10 days, 4 and 8 days, 4 and 10 days, 5 and 7 days, 5 and 10 days, 6 and 10 days, or 7 and 10 days. Each possibility represents a separate embodiment of the invention.

[0142] In some embodiments, culturing in a defined culture medium comprising SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO is for a period of between 3 and 5 days, 3 and 7 days, 3 and 10 days, 4 and 8 days, 4 and 10 days, 5 and 7 days, 5 and 10 days, 6 and 10 days, or 7 and 10 days. Each possibility represents a separate embodiment of the invention.

[0143] According to another, there is provided a method of differentiating HSC into megakaryocytes, comprising culturing HSC in a defined culture medium comprising: (i) basal medium and an effective amount of: ITS, fetal serum, L-ascorbic acid, MTG, or any combination thereof; and (ii) at least one cytokine or agent selected from: TPO, SCF, Flt3 ligand, IL6, IL9, heparin, or any combination thereof.

Kits

[0144] According to another aspect, there is provided a kit for use in: (i) the differentiation of PSC to HSC; HSC to descendant cells thereof, or both; (ii) maintenance of HSC in an undifferentiated state; or (iii) both (i) and (ii), the kit comprising basal medium.

[0145] In some embodiments, the basal medium comprises or is IMDM.

[0146] In some embodiments, the kit further comprises at least one agent being capable of promoting health of a blood cell or a precursor thereof.

[0147] In some embodiments, the kit further comprises at least one agent being capable of promoting differentiation of PSC to HSC; HSC to descendant cells thereof, or both, expansion or maintenance of HSC in an undifferentiated state, or both.

[0148] As used herein, the term “agent promoting health of a blood cell or a precursor thereof’ refers to any compound being capable of increasing, maintaining, sustaining, required for survival, activity, or both, of a blood cell or a precursor thereof. Such compound(s) may increase differentiation, proliferation, DNA synthesis or replication, RNA transcription, protein translation, survival rate, hemoglobin production, or any combination thereof, of a blood cell or a precursor thereof.

[0149] In some embodiments, a precursor cell comprises embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell, hematopoietic stem cell, or any combination thereof. [0150] In some embodiments, an agent promoting health of a blood cell or a precursor thereof is selected from: serum replacement, insulin, transferrin, Selenium, L-ascorbic acid, MTG, or any combination thereof.

[0151] In some embodiments, the at least one agent capable of promoting differentiation, expansion, or both, as disclosed herein, is selected from: SCF, Flt3 ligand, TPO, IL6, BMP4, VEGF, ILS, G-CSF, UM729, IL3, heparin, holo-transferrin, hydrocortisone, T3, FelII-EDTA, or any combination thereof.

[0152] In some embodiments, the kit further comprises instructions for the differentiation, the maintenance in an undifferentiated state, or both.

[0153] In some embodiments, the instructions for differentiating PSC into HSC comprises: mixing basal medium with an effective amount of: (i) serum replacement and ITS; and (ii): (a) BMP4; (b) SCF, VEGF UM171, SRI, or NAM; (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; or (d) any combination of (a) to (c).

[0154] In some embodiments, the instructions for maintenance of HSC in an undifferentiated state comprise: mixing basal medium with an effective amount of: (i) serum replacement; and (ii) SCF, FltS ligand, TPO.

[0155] In some embodiments, the instructions for differentiating HSC into erythrocytes comprise: mixing basal medium with an effective amount of (i) serum replacement; and (ii): (a) IL3, EPO, and SCF; (b) SCF, holo-transferrin, hydrocortisone, and EPO; (c) SCF, IL3, FltS ligand, IL6, G-CSF, and TPO; or (d) any combination of (a) to (c).

[0156] In some embodiments, the at least one agent promoting health of a blood cell or a precursor thereof further comprises fetal serum.

[0157] In some embodiments, the instructions for differentiating HSC into megakaryocytes comprise: mixing basal medium with an effective amount of: (i) ITS, fetal serum, L-ascorbic acid, and MTG; and (ii) TPO, SCF, FltS ligand, IL6, IL3, and heparin.

[0158] In some embodiments, serum replacement comprises or is knockout serum (Ko-SR).

Cells, compositions, and methods of using same

[0159] According to another aspect, there is provided an undifferentiated HSC expanded or maintained according to the method of the invention.

[0160] According to another aspect, there is provided an HSC resulting from a PSC differentiated according to the method of the inve [0161] According to another aspect, there is provided an erythrocyte resulting from an HSC differentiated according to the method of the invention.

[0162] According to another aspect, there is provided a megakaryocyte resulting from an HSC differentiated according to the method of the invention.

[0163] According to another aspect, there is provided a composition comprising any one of the afore-mentioned cdls, obtained according to the method of the invention.

[0164] In some embodiments, the composition further comprises an accqitable carrier.

[0165] In some embodiments, the carrier comprises a pharmaceutically accqitable carrier.

[0166] The term “pharmaceutically accqitable carrier" as used herein refers to any of the standard pharmaceutical camera known in the fidd such as sterile solutions, tablets, coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, sugar; certain types of clay, gdatin, stearic adds or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such camera may also indude flavor and color additives or other ingredients. Examples of pharmaceutically accqitable camera indude, but are not limited to, the following: water, saline, buffers, inert, nontoxic solids (e.g., mamritol, talc). Compositions comprising such camera are formulated by well-known conventional methods. Depending on the intended mode of administration and the intended use, the compositions may be in the form of solid, semi-solid, or liquid dosage forms, such, for example, as powders, granules, crystals, liquids, suspensions, liposomes, nano-partides, nanoemulsions, pastes, creams, salves, etc., and may be in unit-dosage forms suitable for administration of rdativdy precise dosages.

[0167] The term "stem cell" is used herein to refer to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to sdf-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298). Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.

[0168] The term “pluripotent stem cell” refers to cdls capable of differentiating, or being differentiated by means known to one ordinary in the art, into cells of any lineage. The term “embryonic stem cdl” refers to stem cdls derived firm the undifferentiated inner mass of an embryo. Such cdls are pluripotent, and capable of differentiating, or being differentiated by means known to one ordinary in the art, into cdls of any lineage. In order for a ESC to be considered undifferentiated, it must continue to express stem cell markers or not express markers of differentiated cells.

[0169] The term PSC refers to pluripotent stem cells regardless of then: derivation, the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC. PSCs may be in the form of an established cell line, they may be obtained directly from primary embryonic tissue, or they may be derived from a somatic cell. PSCs can be target, cells of the methods described herein.

[0170] ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES- 5, HES-6 (ES Cell International); Miz-hESl (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and HI, H7, H9, HI 3, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells may be obtained from any mammalian species, e.g. human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flat colonies with large nudeo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA- 1-81, and Alkaline Phosphatase, but not SSEA-1. Examples of methods of generating and characterizing ESCs may be found in, for example, US Patent No. 7,029,913, US Patent No. 5,843,780, and US Patent No. 6,200,806. Methods for proliferating hESCs in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920.

[0171] By "embryonic germ stem cell" (EGSC) or "embryonic germ cell" or "EG cell" is meant a PSC that is derived from germ cells and/or germ cell progenitors, e.g., primordial germ cells, i.e. those that would become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, US Patent No. 7,153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Nad. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., etal. (1996) Development, 122:1235.

[0172] By "induced pluripotent stem cell" or "iPSC" it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells.

[0173] In some embodiments, there is provided a use of the composition of the invention.

[0174] In some embodiments, the use is selected from: therapeutics, diagnosis, or drug screening.

[0175] In some embodiments, therapeutics comprise treating blood loss or hemorrhage.

[0176] According to another aspect, there is provided a method of treating a subject afflicted with blood loss or hemorrhage, comprising administering to the subject a therapeutically effective amount of a composition comprising an erythrocyte resulting from an HSC differentiated according to the method of the invention.

[0177] In some embodiments, administering comprises transfusing.

[0178] In some embodiments, the subject is afflicted with trauma. In some embodiments, the blood loss or hemorrhage results from or is induced by trauma.

[0179] According to another aspect, there is provided a method for treating or preventing a blood-related disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a differentiated blood cell resulting from an HSC differentiated according to the method of the invention.

[0180] In some embodiments, the differentiated blood cell comprises an erythrocyte, megakaryocyte, or both. General

[0181] Any number range recited herein relating to any physical feature, such as sequence homology or identity, are to be understood to include any integer within the recited range, unless otherwise indicated.

[0182] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0183] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0184] About refers to ±10%.

[0185] As used herein, the terms “comprises” or “comprising” comprise “consists of’ or “consisting of’.

[0186] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

[0187] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0188] In the description and claims of the present application, each of the verbs, “comprise", “include" and “have” and conjugates thereof are used to indicate that the obj ect or obj ects of the verb are not necessarily a complete listing of components, dements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be d

d by their well-known meanings in the art

[0189] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separatdy or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those dements.

[0190] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

[0191] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, bioengineering, bioprocessing, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrodk et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubd, R. M., ed. (1994); Ausubd et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Binen et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); Molecular Cell Biology Berk A. et al. 8^th edition; Molecular Biotechnology : Principles and Applications of Recombinant DN, Glick BR. 5^th edition; Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications Freshney IR, 7^th edition;; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and Methods

Differentiation to HPSCsfrom PSC in adherent culture

[0192] Fifty thousand (50,000) cells per well were seeded 4-6 days prior to the start of differentiation on a 6-well plate coated with Matrigel™ in mTeSRl medium. At the beginning of differentiation, medium was changed to the basal medium (BM1) which contained: Iscove Modified Dulbecco Media (IMDM), Knockout serum, ITS, L-Glutamine and penicillinstreptomycin (Pen/Step). Different cytokines were added to the basal medium on the following days of differentiation: Days 0-4: BMP4; Days 4-6: SCF + VEGF; Day 6-onwards: (Cyto Mix): SFC+ C-GSC + Flt3 + IL6 + IL3, + TPO, + -UM729. From Day 6 supernatant was collected, centrifuged, resuspended in fresh medium, and added back to the adherent cells. From Day 6 the medium with the Cyto Mix was changed every 3-4 days. Flow cytometer analysis was performed from day 6 every 3 days to assess the percentage of CD34-positive cells in the adherent and suspension cells.

Differentiation to erythrocytes in suspension or dynamic culture

[0193] Suspension differentiation: 2xl0⁶ cells of PSC were seeded in a 6 cm low attachment plate and grown in basal BM1 medium for 14 days with different cytokines that were changed every 7 days. Days 0-7: SCF+VEGF+BMP4. Days 7-14: SCF +G-CSF + FLT3 +IL6 + IL3 + TPO and UM729. Medium was refreshed every 3 days. [0194] On days 14 to 21, the BM1 medium was change to the commercial StemSpan™ (SCT) medium with the following cytokines SCF + IL3 + EPO. Cells were divided during the experiments to ensure cell concentration did not exceed 0.8-lxl0⁷ cells/plate.

[0195] On day 21, the medium was changed back to BM1 without ITS, and the cells were exposed to different cytokines and proteins that are changed every 7 days.

[0196] Days 21-28: SCF + Holotransferrin + Hydrocortisone + EPO. Days 28-32: Holo transferrin + T3 + Fein EDTA + Mifepristone + EPO. Day 32-onwards: EPO. Medium in plates was refreshed every 3 days. The final maturation stage of an erythrocyte can take up to 18 days (from day 32 onwards).

[0197] Dynamic differentiation: The course of differentiation is as described for suspension differentiation and performed in a spinner flask or bioreactor. There were two modifications from the suspension differentiation method: (1) In the dynamic culture, the cells were seeded in approximately 2xl0⁶cell/mL in 40 mL; and (2) The volume of the spinner flask or bioreactor had to be adjusted according to cell concentration.

[0198] Flow cytometer analysis, immunofluorescent staining (IF) and histological sections, were performed at different points to assess the progression of the differentiation.

Maintenance of HPSCs

[0199] CD34-positive cells were thawed and maintained in HSCE medium which contained: Iscove Modified Dulbecco Media (IMDM), Knockout serum, ITS, L-Glutamine and Pen/Step. The medium was supplemented with TPO + SCF and Flt3 -in suspension culture.

Differentiation of CD34+ to megakaryocytes in suspension culture

[0200] Bone marrow CD34+ cells were thawed on a 6 cm plate and grown for 14 days in Accellta™ maintenance medium. On day 14 the medium was changed to HDM (hematopoietic differentiation medium) with addition of differentiation cytokines (such as TPO, SCF, Heparin etc.) The cells were culuted for an additional 14 days in 6cm low attachment plates or a dwell plate. Flow cytometer analysis, immunofluorescent staining (IF) and histological sections, were performed at different points to assess the progression of the differentiation.

EXAMPLE 1

Differentiation of PSC to HPSCs in adherent culture [0201] iPSCs were plated in 6-weU plates in the presence of BM1 medium +TTS (xl) and with the addition of certain cytokines combinations . Flow cytometry analysis was performed from day 6 every 2-3 days to assess the expression of CD34 marker on the cells. It was shown that some cell lines express the CD34 marker at an early stage (from day 8-9) and some at a later stage (from day 12). The CD34-positive cell population was observed to be between 20-50%. CD34- positive cells were observed both in adherent cells and in the cell suspension fraction (Fig. 2A).

EXAMPLE 2

Differentiation of PSC to Erythrocytes in Static or Dynamic system

[0202] Two million cells per ml (2x10⁶) cell/mL were seeded in a static or a dynamic system in the same condition as detailed above. Flow cytometry analysis demonstrated the following: between days 0-14 an increase in the expression of CD45, CD31 and CD34 (not shown). From day 14 onwards, a decrease in the expression of CD45, CD31 and an increase in CD71, CD36 was seen as the cell differentiation continued (Fig. 7). During the final maturation stages, an increase in CD235a expression was observed (Figs. 6A-6B). The results show that the cells express the correct markers profile, at the different stages of differentiation till mature erythrocytes are received, as was described in the literature.

[0203] At the final mutation stages of the differentiated erythrocytes, a red pellet was observed after cell centrifugation. Uris was an indication that the cells expressed hemoglobin (not shown). Moreover, similar to endogenous erythrocytes, the cells were found to undergo proper erythroid enucleation (Figs. 5A-5B;). The differentiated cells were also determined to be expressing hemoglobin from day 42 onwards (Figs.6C-6D). All in all, the results show that under the current protocol cells RBC maturation was successfully achieved, as demonstrated erythroid enucleation, and hemoglobin expression.

EXAMPLES

Differentiation of CD34+ to erythrocytes

[0204] The differentiation was performed in a static and a dynamic system, as described in Example 2. At the starting point cells were CD34+ cells, thus, the protocol was initiated on day 14.

[0205] The results show that all required markers, protein expression, and enucleation were observed on the expected days according to the protocol (Fig. 4A). The cells showed, towards the final mutation steps, CD71 staining (using a specific Ab), high expression of CD235a (Fig. 4A) and of hemoglobin, exhibiting red pellet throughout the maturation (Fig. 4B), and enucleation was determined via May-Grunwald Giemsa staining (Fig. 4C).

EXAMPLE 4

Differentiation of CD34+ to Megakaryocytes

[0206] One million (IxlO⁶) bone marrow CD34+ cells were thawed on a 6 cm plate and cultured in HSCE medium supplemented with different cytokines (TPO, SCF, FLT3, etc.). The cells were cultured for 14 days. The cells were transferred to three different differentiation mediums: StemSpan™ (SS) with the addition of Accellta™ supplement (ACS) 1, SS + ACS2 or hematopoietic differentiation medium (HDM) + ACS1. Cells were cultured in these media in 6 cm plate or a 6 well plate. Cells were maintained in this meaner for 14 days with periodical medium changes. During the differentiation cells were examined for Accumulated cell number and viability- cell counts and morphology images were performed (Figs. 8B-8C, and 8F). Flow cytometry was performed throughout the differentiation to assess progression of the cells towards megakaryocytes (Figs. 8D-8E). On day 14 cell diameter was assessed in the different differentiation mediums (Fig. 8G). May-Grunwald staining cell smear was performed on day 27 and light microscopy images show highly uncertain megakaryocytes (Fig. 8H).

[0207] While the present invention has been particularly described, people skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims which follow.

Claims

CLAIMS What is claimed:

1. A method of differentiating pluripotent stem cells (PSC) into hematopoietic stem cell (HSC), the method comprising culturing said PSC in a defined culture medium comprising: (i) Iscove's Modified Dulbecco Media (IMDM) basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, IL3, Flt3 ligand, IL6, G-CSF, TPO, UM729, UM171, StemRegenin (SRI), nicotinamide (NAM), and any combination thereof.

2. The method of claim 1, wherein said differentiating comprises culturing said PSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of:

(a) BMP4;

(b) SCF, VEGF, UM171, SRI, and NAM;

(c) SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, and TPO; and

(d) any combination of (a) to (c).

3. The method of claim 2, wherein: (a) is for a period of between 1 and 5 days, (b) is for a period of between 1 and 4 days, and (c) is for a period of 2 and 24 days.

4. A method of maintaining HSC in an undifferentiated state, the method comprising culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: SCF, Flt3 ligand, TPO, SRI, NAM and any combination thereof, thereby maintaining HSC in an undifferentiated state.

5. The method of claim 4, wherein at least 50% of said cultured HSC express CD34.

6. The method of claim 4 or 5, further comprising a step preceding said maintaining comprising differentiating PSC into said HSC wherein said differentiating comprises culturing said PSC in a defined c MDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, IL3, Flt3 ligand, IL6, G-CSF, TPO, UM729, UM171, SRI, NAM and any combination thereof.

7. The method of claim 6, wherein said differentiating comprises culturing said PSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of:

(a) BMP4;

(b) SCF, VEGF, SRI, and NAM ;

(c) SCF, IL3, Flt3 ligand, IL6, G-CSF, UM729, UM171 and TPO; and

(d) any combination of (a) to (c).

8. The method of claim 7, wherein: (a) is for a period of between 1 and 5 days, (b) is for a period of between 1 and 4 days, and (c) is for a period of 2 and 24 days.

9. The method of any one of claims 4 to 8, further comprising a step proceeding said maintaining comprising differentiating said HSC into descendant cells thereof.

10. The method of claim 9, wherein said descendant cells are erythrocytes, and wherein said differentiating comprises culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: BMP4, SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, EPO, ITS, holo-transferrin, hydrocortisone, T3, FelII-EDTA, and any combination thereof, thereby differentiating said HSC into erythrocytes.

11. The method of claim 10, wherein said differentiating comprises culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of:

(a) IL3, EPO, and SCF;

(b) SCF, holo-transfe (c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; and

(d) any combination of (a) to (c).

12. The method of claim 11, wherein: (a) is for a period of between 3 and 10 days, (b) is for a period of between 3 and 10 days, and (c) is for a period of 3 and 10 days.

13. The method of claim 9, wherein said descendant cells are megakaryocytes, and wherein said differentiating comprises culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of any one of: ITS, fetal serum, L-ascorbic acid, monothioglycerol (MTG), and any combination thereof; and (ii) at least one cytokine or agent selected from the group consisting of: TPO, SCF, Flt3 ligand, IL6, IL9, heparin, and any combination thereof, thereby differentiating said HSC into megakaryocytes.

14. The method of claim 13, wherein said fetal serum is derived from bovine.

15. The method of claim 14, wherein said culturing is for a period of 10 and 20 days.

16. A method of differentiating HSC into erythrocytes, the method comprising culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of serum replacement, wherein the defined culture medium is serum-free; and (ii) at least one cytokine or agent selected from the group consisting of: SCF, VEGF, Flt3 ligand, IL6, IL3, TPO, EPO, ITS, holo-transferrin, hydrocortisone, T3, Felll-EDTA, and any combination thereof, thereby differentiating said HSC into erythrocytes.

17. The method of claim 16, wherein said differentiating comprises culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of: serum replacement and ITS, wherein the defined culture medium is serum-free, and (ii) any one of:

(a) IL3, EPO, and SCF;

(b) SCF, holo-transferrin, hydrocortisone, and EPO;

(c) SCF, IL3, Flt3 ligand, IL6, G-CSF, and TPO; and

(d) any combination of (a) to (c).

18. The method of claim 17, wherein: (a) is for a period of between 3 and 10 days, (b) is for a period of between 3 and 10 and 10 days.

19. A method of differentiating HSC into megakaryocytes, the method comprising culturing said HSC in a defined culture medium comprising: (i) IMDM basal medium and an effective amount of any one of: ITS, fetal serum, L-ascorbic acid, MTG, and any combination thereof; and (ii) at least one cytokine or agent selected from the group consisting of: TPO, SCF, Flt3 ligand, IL6, IL9, heparin, and any combination thereof, thereby differentiating said HSC into megakaryocytes.

20. The method of claim 19, wherein said culturing is for a period of 10 and 20 days.

21. The method of any one of claims 1 to 20, wherein said culturing comprises any one of 2D culturing, 3D culturing, and both.

22. A defined culture medium comprising IMDM basal medium and an effective amount of a serum replacement, wherein the defined culture medium is serum-free, and is capable of promoting: (i) differentiation of HSC to descendant cells thereof; (ii) expansion of HSC; or (iii) both (i) and (ii).

23. A kit for use in: (i) the differentiation of PSC to HSC ; HSC to descendant cells thereof, or both; (ii) maintenance of HSC in an undifferentiated state; or (iii) both (i) and (ii), the kit comprising IMDM basal medium.

24. The kit of claim 23, further comprising any one of:

(a) at least one agent promoting health of a blood cell or a precursor thereof, being selected from the group consisting of: serum replacement, insulin, transferrin, Selenium, L-ascorbic acid, MTG, and any combination thereof;

(b) at least one agent capable of promoting any of said differentiation and said expansion, being selected from the group consisting of: SCF, Flt3 ligand, TPO, IL6, BMP4, VEGF, IL3, G-CSF, UM729, IL9, heparin, holo-transferrin, hydrocortisone, T3, FelII-EDTA, SRI, NAM, UM171 and any combination thereof; and

(c) both (a) and (b).