Differentiation of IPSC under perfusion conditions

Abstract

The disclosure provides, inter alia, the cultivation of induced pluripotent stem cells (iPSCs) for therapeutic and clinical applications.

Description

Ref. No.: 123828-01-5143-WODIFFERENTIATION OF IPSC UNDER PERFUSION CONDITIONSHELD

[0001] The present disclosure relates to methods of cultivating induced pluripotent stem cells (iPSCs).CROSS-REFERENCE TO REEATED APPEICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 706,955 filed October 14, 2024. the contents of which are incorporated by reference herein in their entirety.BACKGROUND

[0003] Induced pluripotent stem cells (iPSCs) have the power to revolutionize regenerative medicines due to their innate abilities to differentiate into virtually any cell type. Classically, this is done by growing iPSCs on a 2D matrix and subsequently expose them to cocktails of differentiation factors for specific amounts of time to induce their differentiation into the desired cell type. While this 2D process is functional, it is limited in terms of yield, throughput, and manufacturability under GMP conditions. Specifically regenerative medicine which seeks to compensate lost or temporarily unavailable organ functions requires large amounts of effective cells to treat the patient effectively. Hence, it is impractical to generate this in a 2D system.

[0004] There is a need for improved methods for cultivating iPSCs.SUMMARY

[0005] Accordingly, in aspects and embodiments, there is provided a method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system, optionally wherein the system comprises a perfusion means,

[0006] wherein the culture medium comprises effective amounts of a nonionic surfactant and molecular crowder, optionally further comprising one or more of bovine serum albumin (BSA), recombinant albumin, polyvinyl alcohol (PVA), synthetic BSA replacement, and thermally denatured BSA; and

[0007] wherein the method is suitable for differentiation of iPSCs. e.g., to hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells, optionally1DB1 / 162996881.7Ref. No.: 123828-02-5143-WO wherein the method is suitable for differentiation of iPSCs to hepatocytes. [3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0008] In embodiments, the nonionic surfactant is a triblock polymer, optionally wherein the triblock polymer comprises an ethoxylated alcohol.

[0009] In embodiments, the nonionic surfactant is poloxamer 188 (Pl 88).

[0010] In embodiments, the nonionic surfactant is poloxamer 188 at a concentration of up to about: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.00 % (W / V).

[0011] In embodiments, the nonionic surfactant is poloxamer 188 at a concentration of about .05 % - .50 % or about .01% - 1%.

[0012] In embodiments, the molecular crowder is selected from polyethylene glycol (PEG), ficoll, dextran, BSA, methylcellulose, polyvinyl alcohol (PVA), Hydroxyethyl Starch (HES). and a polymer of a vinylpyrrolidone, such as N-vinylpyrrolidone.

[0013] In embodiments, the molecular crowder is a polyvinylpyrrolidone (PVP), optionally PVP360.

[0014] In embodiments, the concentration of the PVP is at least about: 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7. 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 mg / ml, optionally wherein the concentration of PVP is selected from a range of about 0.5 - 8.0 mg / ml, 1.5 - 6.0 mg / ml, or 3.0 - 4.0 mg / ml.

[0015] In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs, optionally differentiation of iPSCs to hepatocytes.

[0016] In embodiments, the differentiation medium is suitable for differentiation of iPSCs to hepatocytes, optionally wherein the differentiation medium comprises a sequence of culture media, further optionally wherein the culture media comprise a series of optionally different base media supplemented with: a. a first media comprising:(a) a GSK3 inhibitor, optionally CHIR99021 , at a concentration of about 0.1 pM - 20 pM or about 2 pM - 10 pM, optionally wherein the concentration of CHIR99021 is between 3 pM - 6 pM, optionally2DB1 / 162996881.8Ref. No.: 123828-02-5143-WO wherein the concentration of CHIR99021 is about 4 pM - 5 pM, optionally wherein the concentration of CHIR99021 is about 5 pM.(b) a TGF-fl family grow th factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(c) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(d) optionally, Bone Morphogenetic Protein 4 (BMP -4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(e) optionally, a PI3K inhibitor, optionally LY294002. at a concentration of about 1 pM - 50 pM. or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM,(1) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g., KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 0.8%, and(g) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0.1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM; b. a second media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) optionally. Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration3DB1 / 162996881.8Ref. No.: 123828-02-5143-WO of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(d) optionally, a PI3K inhibitor, optionally LY294002, at a concentration of about 1 uM - 50 LtM, or about 5 uM - 20 uM. optionally wherein the concentration of LY294002 is about 10 pM.(e) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g., KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%. optionally wherein the concentration of KOSR is about 0.8%, and(f) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0.1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM; c. a third media comprising:(a) a TGF-0 family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g., KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 8%,(d) optionally, Non-Essential Amino Acid Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-prohne, and L-serine, and(e) optionally, B-27 or B-27 plus Supplement; d. a fourth media comprising:DBl / 162996881.8Ref. No.: 123828-02-5143-WO(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 50 ng / mL,(b) optionally, Bone Morphogenetic Protein 4 (BMP -4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / rnL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(c) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g., KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 5%,(d) optionally, a cell protective antioxidant, such as N-Acetyl-Cysteine (NAC) at a concentration of about 0. 1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM,(e) optionally, a biologically active metabolite of vitamin A (retinol) that functions as a regulator of cell growth, differentiation, and development (e.g., All Trans Retinoic Acid (ATRA)) at a concentration of about 0.1 - 4 pM, optionally wherein the concentration of ATRA is about 2 pM and is used for about2 days when the fourth media is being used,(f) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(g) B-27 or B-27 plus Supplement; e. a fifth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e.g., GLUTAMAX),(b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1:50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium,DBl / 162996881.8Ref. No.: 123828-02-5143-WO(e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL, and(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL; and / or f. a sixth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e.g., GLUTAMAX), (b) 1 :50 MEM Non-Essential Amino Acids Solution, optionally wherein theNon-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1:50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium, (e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / ml,(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL, and(g) Forskolin at a concentration of about 2 pM - 10 M or about 100 nM - 100 pM, optionally wherein the concentration of Forskolin is about 5 pM.

[0017] In embodiments, the culture media comprises Essential 6, RPMI-1640, RPMI-1640ATCC Modification, Hepatozyme, Dulbecco's Modified Eagle Medium (DMEM) / F12, DMEM / F 12-base media, William E. supplemented Minimum Essential Medium (MEM).

[0018] In embodiments, the method further comprises evaluating the differentiation state of the iPSCs.

[0019] In embodiments, the iPSCs are differentiated into hepatocytes and the evaluation comprises: a. evaluating the expression of one or more of: cytochrome P450 1 A2 (CYP1 A2), cytochrome P450, family 3, subfamily A (CYP3A), albumin, alpha fetoprotein (AFP),6DB1 / 162996881.8Ref. No.: 123828-02-5143-WO ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), sulfotransferases, carnitine palmitoyltransferase I (CPT1), and alpha-1 antitrypsin (A1AT); b. evaluating ammonia detoxification; c. evaluating ureagenesis; or d. a combination of any of the foregoing.

[0020] In embodiments, the method further comprises preparing a pharmaceutical composition of differentiated iPSCs.

[0021] In aspects and embodiments, there is provided a method comprising administering a therapeutically effective amount of differentiated iPSCs to a subject in need thereof, wherein the differentiated iPSCs are produceable or produced by a method of the present disclosure.

[0022] In aspects and embodiments, there is provided a pharmaceutical composition suitable for administration to a subject comprising a therapeutically effective amount of differentiated iPSCs, wherein the differentiated iPSCs are produceable or produced by a method of the present disclosure.

[0023] In embodiments, the pharmaceutical composition comprises micro encapsulated cells or macro encapsulated cells.

[0024] In embodiments, the subject is a human.

[0025] In embodiments, the differentiated iPSCs are hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary' lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells.

[0026] In embodiments, the differentiated iPSCs are hepatocytes, [3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0027] In embodiments, the differentiated iPSCs are hepatocytes.

[0028] In embodiments, the method further comprises a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.

[0029] In aspects and embodiments, there is provided a device comprising a therapeutically effective amount of the pharmaceutical composition of the present disclosure.7DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0030] In aspects and embodiments, there is provided a dosage form of a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0031] In aspects and embodiments, there is provided a method of generating differentiated cells for iPSCs substantially as shown in FIG. 1. BRIEF DESCRIPTION OF FIGURESFIG. 1 is a non-limiting schematic diagram of differentiation of iPSC into hepatocytes under perfusion conditions. Cells were seeded as preformed aggregates and subsequent differentiation occurs in the presence of crowding factors.FIG. 2 shows hepatocyte aggregates differentiated under perfusion conditions after 28 days of differentiation.FIG. 3 is a graph demonstrating albumin concentrations determined via Human Albumin ELISA kit for the perfusion process as direct sampling or after 24-hour incubation in a 6-well plate.FIG. 4 is a graph demonstrating Al AT concentrations determined via Human Alpha- 1- Antitrypsin ELISA Kit for the perfusion process as direct sampling or after 24-hour incubation in a 6-well plate.FIG. 5 is a graph demonstrating ammonia detoxification as determined by CedexBio after ammonia stimulus for the perfusion process.FIG. 6 shows morphological change of iPSC during differentiation in the VW-BR over days 1 to 29.FIG. 7 shows iPSC aggregates formed in the 3D vertical wheel bioreactor PBS mini, prior to initiation of differentiation.FIG. 8 shows end of stage lb of the differentiation into hepatocytes with Pl 88 added.FIG. 9 shows middle of stage 3 differentiation. Cells were starting to fall apart (image on the left) and aggregates appeared less healthy. Due to low overall cell density, culture in the VW- BR could not be sustained and they were transferred to 6-well plates.FIG. 10 is a graph demonstrating ammonia detoxification as determined by CedexBio after ammonia stimulus for the non-perfusion process.FIG. 11 is a graph demonstrating albumin concentrations determined via Human Albumin ELISA kit for a perfusion process compared to a representative 6-well plate differentiation after 28 days of differentiation (perfusion process) and 29 days of differentiation (6-well plate), respectively.8DB1 / 162996881.8Ref. No.: 123828-02-5143-WOFIG. 12 is a graph demonstrating A1AT concentrations determined via Human Alpha-1- Antitrypsin ELISA Kit for a perfusion process compared to a representative 6-well plate differentiation after 28 days of differentiation (perfusion process) and 29 days of differentiation (6-well plate). DETAILED DESCRIPTION

[0032] In aspects and embodiments, there is provided a method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system. iPSCs are typically grown on a 2D matrix. Some iPSCS are grown in non-adherent suspension conditions referred to 3D cultivation. While successful differentiation in shaken low-attachment 6-well plates has been achieved for small-scale 3D cultivation, a surface-based cultivation environment still lacks the required scalability. Therefore, a bioreactor emerges as the most promising cultivation system for larger quantities. Despite the potential of bioreactors to yield ample cells, previous attempts have not been successful in generating sufficient differentiated cells from iPSCs. This obstacle may arise from cell fragility during differentiation, the artificial environment lacking, and increased stress from agitation. Two components have been identified to mitigate these effects. Poly oxy ethylene-poly oxy propylene (Poloxamer 188, Pl 88) is a nonionic surfactant that is frequently used in large scale bioreactor cultivation. It increases the tolerance of animal cells to stress-inducing mechanical forces which arise from the stirring but can also be introduced by air bubbles. Polyvinylpyrrolidone acts as a macromolecular crowder, which is introduced to mimic the dense and complex environment surrounding cells where various macromolecules, such as proteins, nucleic acids, and other large molecules are present in high concentrations. This effect is known to have significant physiological effects on biochemical processes. Furthermore, it changes the mediums viscosity which has an impact on flow patterns of cell aggregates and the overall mixing characteristics in the bioreactor.

[0033] In embodiments, there is provided a method of iPSC differentiation under controlled 3D conditions that is superior to classical 2D differentiation in terms of yield and cell functionality. In embodiments, the method uses perfusion to ensure adequate supply of nutrients and differentiation factors to the cells as well as waste product removal. In embodiments, the method uses new media additives supplemented during the differentiation phase that protect cells from shear stress and establish an environment resembling in vivo conditions to enable their 3D differentiation.9DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0034] In aspects and embodiments, there is provided a method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system, optionally wherein the system comprises a perfusion means,

[0035] wherein the culture medium comprises effective amounts of a nonionic surfactant and molecular crowder, optionally further comprising one or more of bovine serum albumin (BSA), recombinant albumin, polyvinyl alcohol (PVA), synthetic BSA replacement, and thermally denatured BSA; and

[0036] wherein the method is suitable for differentiation of iPSCs, e.g., to hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells, optionally wherein the method is suitable for differentiation of iPSCs to hepatocytes, [3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells, neurons, or retinal organoids

[0037] In embodiments, the nonionic surfactant is a triblock polymer, optionally wherein the triblock polymer comprises an ethoxylated alcohol.

[0038] In embodiments, the nonionic surfactant is poloxamer 188 (Pl 88).

[0039] In embodiments, the nonionic surfactant is poloxamer 188 at a concentration of up to about: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.00 % (WAV).

[0040] In embodiments, the nonionic surfactant is poloxamer 188 at a concentration of about .05 % - .50 % or about .01% - 1%.

[0041] In embodiments, the molecular crowder is selected from polyethylene glycol (PEG), ficoll, dextran, BSA, methylcellulose, polyvinyl alcohol (PVA), Hydroxyethyl Starch (HES), and a polymer of a vinylpyrrolidone, such as N-vinylpyrrolidone.

[0042] In embodiments, the molecular crowder is a polyvinylpyrrolidone (PVP), optionally PVP360.

[0043] In embodiments, the concentration of the PVP is at least about: 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7. 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,10DB1 / 162996881.8Ref. No.: 123828-02-5143-WO5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6. 6.7, 6.8, 6.9. 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 mg / ml, optionally wherein the concentration of the PVP is selected from a range of about 0.5 - 8.0 mg / ml, 1.5 - 6.0 mg / ml, or 3.0 - 4.0 mg / ml.

[0044] In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs, optionally differentiation of iPSCs to hepatocytes.

[0045] In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs. In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs to hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells. In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs to hepatocytes, 0-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0046] In embodiments, the differentiation medium is suitable for differentiation of iPSCs to hepatocytes, optionally wherein the differentiation medium comprises a sequence of culture media, further optionally wherein the culture media comprise a series of optionally different base media supplemented with: a. a first media comprising:(a) a GSK3 inhibitor, optionally CHIR99021 , at a concentration of about 0.1 pM - 20 pM or about 2 pM - 10 pM, optionally wherein the concentration of CHIR99021 is between 3 pM - 6 pM, optionally wherein the concentration of CHIR99021 is about 4 pM - 5 pM, optionally wherein the concentration of CH1R99021 is about 5 pM.(b) a TGF-0 family grow th factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL, (c) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,11DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(d) optionally, Bone Morphogenetic Protein 4 (BMP -4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(e) optionally, a PI3K inhibitor, optionally LY294002, at a concentration of about 1 pM - 50 pM, or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM,(f) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 0.8%, and(g) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteme (NAC)) at a concentration of about 0.1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM; b. a second media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / rnL,(b) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL.(c) optionally. Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(d) optionally, a PI3K inhibitor, optionally LY294002, at a concentration of about 1 pM - 50 pM. or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM,(e) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,12DB1 / 162996881.8Ref. No.: 123828-02-5143-WOKnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%. optionally wherein the concentration of KOSR is about 0.8%, and(f) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0.1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM; c. a third media comprising:(a) a TGF-p family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 8%,(d) optionally, Non-Essential Amino Acid Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(e) optionally, B-27 or B-27 plus Supplement; d. a fourth media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 50 ng / mL,(b) optionally, Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(c) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,13DB1 / 162996881.8Ref. No.: 123828-02-5143-WOKnockOut™ Serum Replacement (KOSR)) at a concentration of about 0. 1% - 8%, optionally wherein the concentration of KOSR is about 5%,(d) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0. 1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM,(e) optionally, a biologically active metabolite of vitamin A (retinol) that functions as a regulator of cell growth, differentiation, and development (e g., All Trans Retinoic Acid (ATRA)) at a concentration of about 0.1 - 4 pM, optionally wherein the concentration of ATRA is 2 pM and is used for about 2 days when the fourth media is being used,(f) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and (g) B-27 or B-27 plus Supplement; e. a fifth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e g., GLUTAMAX),(b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid. L-glutamic acid. L-proline, and L-serine,(c) 1 :50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium,(e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL, and(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL; and / or f. a sixth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e.g., GLUTAMAX),DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1:50 Chemically defined lipid concentrate, (d) 1 : 100 Insulin-Transferrin-Selenium,(e) Oncostatin M at a concentration of about 10 ng / ml-50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / ml,(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL, and(g) Forskolin at a concentration of about 2 pM - 10 pM or about 100 nM - 100 pM, optionally wherein the concentration of Forskolin is about 5 pM.

[0047] In embodiments, the BMP-4 in the media has a concentration of about 0.01 ng / mL- 100 ng / mL, about 1 ng / mL - 50 ng / mL, or about 5 ng / mL - 15 ng / mL. In embodiments, the BMP-4 in the media has a concentration of about 10 ng / mL.

[0048] In a non-limiting example, the Non-Essential Amino Acid Solution is 100X MEM Non-Essential Amino Acids Solution (Thermo Fisher, 11140050). In embodiments, the 100X MEM Non-Essential Amino Acid Solution is diluted to IX after being added to a medium.

[0049] In a non-limiting example, the B-27 or B-27 plus Supplement is SOX B-27 Supplement (Thermo Fisher, 17504044). In embodiments, the 50X B-27 Supplement is diluted to IX after being added to a medium.

[0050] In embodiments, the differentiation medium is suitable for differentiation of iPSCs to hepatocytes, optionally wherein the method comprises a sequence of culture media, further optionally wherein the culture media comprise a series of optionally different base media supplemented with: a. a first media comprising: (a) a GSK3 inhibitor, optionally CHIR99021 , at a concentration of about 0.1 pM - 20 pM or about 2 pM - 10 pM, optionally wherein the concentration of CHIR99021 is about 3 pM,15DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(b) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(c) Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(d) Bone Morphogenetic Protein 4 (BMP -4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / rnL, optionally wherein the concentration of BMP -4 is about 5 ng / mL - 15 ng / rnL, optionally wherein the concentration of BMP-4 is about 10 ng / mL, and(e) a PI3K inhibitor, optionally LY294002, at a concentration of about 1 11M- 50 pM. or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM; b. a second media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / rnL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP -4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL, and(d) a PI3K inhibitor, optionally LY294002, at a concentration of about 1 pM- 50 pM, or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM; c. a third media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,16DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(b) Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml- 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(d) B-27 Supplement; d. a fourth media comprising: (a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 50 ng / mL,(b) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(c) B-27 Supplement; e. a fifth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e.g., GLUTAMAX), (b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein theNon-Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1:50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium, (e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL, and(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL; f. a sixth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e.g.. GLUTAMAX),17DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1:50 Chemically defined lipid concentrate, (d) 1 : 100 Insulin-Transferrin-Selenium,(e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL,(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / ml, and(g) Forskolin at a concentration of about 2 pM - 10 pM or about 100 nM - 100 pM, optionally wherein the concentration of Forskolin is about 5 pM.

[0051] Non-limiting examples of GSK3 inhibitors include CHIR99021, NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080. SB415286, 6-bromoindirubin-3'- oxime, AR-A014418, Lithium. 9-ING-41 and LY2090314. In embodiments, the GSK3 inhibitor is CHIR99021.

[0052] In embodiments, the culture media comprises Essential 6, RPMI-1640, RPMI-1640ATCC Modification, Hepatozyme, Dulbecco's Modified Eagle Medium (DMEM) / F12, DMEM / F12-base media, William E. supplemented Minimum Essential Medium (MEM).

[0053] In embodiments, the method further comprises evaluating the differentiation state of the iPSCs.

[0054] In embodiments, the iPSCs are differentiated into hepatocytes and the evaluation comprises: a. evaluating the expression of one or more of: cytochrome P450 1 A2 (CYP1 A2), cytochrome P450, family 3, subfamily A (CYP3A), albumin, alpha fetoprotein (AFP), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT). sulfotransferases, carnitine palmitoyltransferase I (CPT1), and alpha- 1 antitrypsin (Al AT); b. evaluating ammonia detoxification;18DB1 / 162996881.8Ref. No.: 123828-02-5143-WO evaluating ureagenesis; or d. a combination of any of the foregoing.In embodiments, the method further comprises preparing a pharmaceutical composition of differentiated iPSCs.

[0055] In aspects and embodiments, there is provided a method comprising administering a therapeutically effective amount of differentiated iPSCs to a subject in need thereof, wherein the differentiated iPSCs are produceable or produced by a method of the present disclosure.

[0056] In aspects and embodiments, there is provided a pharmaceutical composition suitable for administration to a subject comprising a therapeutically effective amount of differentiated iPSCs, wherein the differentiated iPSCs are produceable or produced by a method of the present disclosure.

[0057] In embodiments, the pharmaceutical composition comprises micro encapsulated cells or macro encapsulated cells.

[0058] In embodiments, the subject is a human.

[0059] In embodiments, the differentiated iPSCs are hepatocytes.

[0060] In embodiments, the differentiated iPSCs are hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells. In embodiments, the differentiated iPSCs are hepatocytes, [3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal or organoids.

[0061] In embodiments, the method further comprises a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.

[0062] In aspects and embodiments, there is provided a device comprising a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0063] In aspects and embodiments, there is provided a dosage form of a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0064] In embodiments, the molecular crowder is selected from polyethylene glycol (PEG), ficoll, dextran, BSA. methylcellulose, polyvinyl alcohol (PVA), Hydroxy ethyl Starch (HES), and a polymer of a vinylpyrrohdone, such as N-vinylpyrrolidone.DBl / 162996881.8Ref. No.: 123828-02-5143-WO

[0065] In embodiments, the molecular crowder is a polyvinylpyrrolidone (PVP), optionally PVP360.

[0066] In embodiments, the concentration of the PVP is at least about: 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6. 6.7, 6.8, 6.9. 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 mg / ml, optionally wherein the concentration of thePVP is selected from a range of about 0.5 - 8.0 mg / ml, 1.5 - 6.0 mg / ml, or 3.0 - 4.0 mg / ml.

[0067] In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs, optionally differentiation of iPSCs to hepatocytes.

[0068] In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs. In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs to hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells. In embodiments, the culture medium is a differentiation medium suitable for differentiation of iPSCs to hepatocytes, [3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids..

[0069] In embodiments, the differentiation medium is suitable for differentiation of iPSCs to hepatocytes, optionally wherein the method comprises a sequence of culture media, further optionally wherein the culture media comprise a series of optionally different base media supplemented with: a. a first media comprising:(a) a GSK3 inhibitor, optionally CHIR99021, at a concentration of about 0. 1 pM - 20 pM or about 2 pM - 10 pM, optionally wherein the concentration of CHIR99021 is about 3 pM, (b) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,20DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(c) Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml- 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(d) Bone Morphogenetic Protein 4 (BMP -4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL, and(e) a PI3K inhibitor, optionally LY294002, at a concentration of about 1 M - 50 pM, or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM; b. a second media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml- 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / rnL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP -4 is about 5 ng / rnL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL, and(d) a PI3K inhibitor, optionally LY294002, at a concentration of about 1 pM - 50 pM, or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM; c. a third media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / rnL,(b) Fibroblast Grow th Factor 2 (FGF2) at a concentration of about 10 ng / ml- 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,21DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(c) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(d) B-27 Supplement; d. a fourth media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 50 ng / mL,(b) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(c) B-27 Supplement; e. a fifth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e.g., GLUTAMAX),(b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1:50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium,(e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL. and(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL; f. a sixth media comprising:(a) 1: 100 L-alanyl-L-glutamine (e g., GLUTAMAX),(b) 1:50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid. L-glutamic acid. L-proline, and L-serine,22DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(c) 1:50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium,(e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL,(I) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL, and (g) Forskolin at a concentration of about 2 M - 10 pM or about 100 nM -100 pM, optionally wherein the concentration of Forskolin is about 5 pM.

[0070] In embodiments, the culture media comprises Essential 6, RPMI-1640, RPMI-1640ATCC Modification, Hepatozyme, Dulbecco's Modified Eagle Medium (DMEM) / F12, DMEM / F12-base media, William E, and supplemented Minimum Essential Medium (MEM).

[0071] In embodiments, the Non-Essential Amino Acid Solution comprises glycine, L- alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. In embodiments, each of glycine, L-alanine. L-asparagine, L-aspartic acid. L-glutamic acid, L- proline, and L-serine are present at a concentration of about 0.01 mM to about 0.2 mM. In embodiments, each of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, and L-serine are present at a concentration of about 0.1 mM.

[0072] In embodiments, the method further comprises evaluating the differentiation state of the iPSCs.

[0073] In embodiments, the iPSCs are differentiated into hepatocytes and the evaluation comprises: a. evaluating the expression of one or more of: cytochrome P450 1A2 (CYP1 A2), cytochrome P450, family 3, subfamily A (CYP3A), albumin, alpha fetoprotein (AFP), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT), sulfotransferases, carnitine palmitoyltransferase I(CPT1), and alpha- 1 antitrypsin (Al AT); b. evaluating ammonia detoxification; c. evaluating ureagenesis; or23DB1 / 162996881.8Ref. No.: 123828-02-5143-WO d. a combination of any of the foregoing.

[0074] In embodiments, the method further comprises preparing a pharmaceutical composition of differentiated iPSCs.

[0075] In aspects and embodiments, there is provided a method comprising administering a therapeutically effective amount of differentiated iPSCs to a subject in need thereof, wherein the differentiated iPSCs are produceable or produced by a method of the present disclosure.

[0076] In aspects and embodiments, there is provided a pharmaceutical composition suitable for administration to a subject comprising a therapeutically effective amount of differentiated iPSCs, wherein the differentiated iPSCs are produceable or produced by a method of the present disclosure.

[0077] In embodiments, the pharmaceutical composition comprises micro encapsulated cells or macro encapsulated cells.

[0078] In embodiments, the subject is a human.

[0079] In embodiments, the differentiated iPSCs are hepatocytes.

[0080] In embodiments, the differentiated iPSCs are hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory’ lineage cells, pulmonary and airway lineage cells, renal and urinary7lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells. In embodiments, the differentiated iPSCs are hepatocytes, [3-cells, cardiomyocytes. endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0081] In embodiments, the method further comprises a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.

[0082] In aspects and embodiments, there is provided a device comprising a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0083] In aspects and embodiments, there is provided a dosage form of a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0084] In aspects and embodiments, there is provided a method of generating differentiated cells for iPSCs substantially as shown in FIG. 1. In embodiments, the differentiated cells are hepatocytes.EXAMPLES24DB1 / 162996881.8Ref. No.: 123828-02-5143-WOExample 1; Differentiation of iPSC under 3D perfusion conditions in bioreactors

[0085] Human iPSCs were cultivated in a suspension bioreactor designed to allow perfusion to achieve high yield differentiation of functional cells. In this example, the goal was to differentiate iPSCs into hepatocytes. The bioreactor used contained an impeller to keep the cells dispersed and to ensure proper mixing. Peristaltic pumps continuously added fresh culture medium to the tank while simultaneously removing spent media. The cells were retained in the bioreactor by a cell retention device, in this instance, a sintered glass sparger, which allowed for the removal of spent media while retaining the cells. The medium flow rates and impeller speed were controlled via the process control system to maintain optimal shear forces and mass transfer of nutrients for cell growth. Dissolved oxygen and pH sensors monitor culture conditions and regulate gas flows or liquid base addition via feedback loops to maintain steady setpoints. The media exchange rates for expansion varied between 0.5 to 1.5 VVD (vessel volume per day) and were kept between 0.5 to 2 VVD for the differentiation phase.

[0086] The process initiated with the thawing of frozen iPSCs, wherein a vial containing approximately 1 million iPSCs was thawed at 37 °C. The ensuing procedure involved diluting the stem cell suspension in a 10-fold volume of cell culture media Essential 8 (E8, Gibco, A1517001) in a 15 mL conical tube, centrifuging the mixture at 200 xg for 5 minutes, discarding the supernatant and resuspending the remaining pellet in 8 mL of E8 medium combined with 10 pM Y27632 ((lR.4r)-4-((R)-l-Aminoethyl)-N-(pyridin-4- yl)cyclohexanecarboxamide) • 2HC1 (Selleckchem, S 1049). This suspension was then transferred to a Vitronectin (Thermo Fisher, A31804) coated T25 flask and incubated at 37 °C and 5 % CO2.

[0087] Approximately 24 hours post-incubation, the old medium was substituted with 8 mL fresh E8 medium and returned to the incubator. Successively, the process involved passaging of cells after 72 - 96 hours since the seeding, which was important in maintaining the cells in beneficial proliferative conditions. The exact timing for passaging was defined by reaching a confluency of 70 - 80 %. Stage-wise, cells were firstly washed with PBS, and the residue of PBS is removed. An addition of 0.1 mL / cm2of Accutase (Innovative Cell Technologies, AT-104) ensues, and the mixture was incubated for 5 minutes at 37 °C. Upon detection of cell detachment via flask tilting, the Accutase w as diluted with an equal volume of E8, transferred to a conical tube, and centrifuged at 200xg for 5 minutes. The supernatant was discarded, and the remaining substance was resuspended in 10 mL E8 supplemented with 10 pM Y27632.25DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0088] Furthermore, the cell quantity was determined via an automated cell counter or a hemocytometer. Subsequently, T175 flask coated with Vitronectin were seeded with approximately 2 million cells per flask in 60 mL E8 supplemented with 10 pM Y27632. The medium was replenished with fresh E8 every 24 hours for 72 - 96 hours and returned to the incubator.

[0089] After reaching 60 - 80 % confluency, cells were passaged at 2 million cells per flask in 60 mL StemScale medium (Thermo Fisher, A4965001). About 3 - 4 days after seeding into StemScale media, the cells had an approximate confluency of 60 - 80 % and were ready for expansion in bioreactors.

[0090] A DASbox Mini bioreactor system (Eppendorf) was used for cell expansion and differentiation. To prevent cells from sticking to the glass surface, the bioreactor was siliconized by applying Sigmacote (Sigma- Aldrich, SL2-25ML). The previously 2D-expanded cells were dissociated into single cells and used to inoculate the bioreactor at a cell density of 0.6xl06cells / mL. The cells were seeded into a total volume of 120 mL StemScale medium containing 10 LIM Y27632 • 2HC1, and 0.1 % P188 (Sigma, P4894). The temperature was controlled at 37 °C. The pH setpoint was maintained between 7. 1 - 7.2 via CO2 overlay gassing and NaHCOs addition. Dissolved oxygen was maintained at 40 % through a combination of overlay gassing and submerged gassing via an open pipe using O2 after about 96 hours. For the first 12 hours of the cultivation, DO and pH were not controlled. Afterwards, they were maintained at the setpoints of 40 % DO and a pH between 7.1 - 7.2. Media perfusion was initiated 24 hours after inoculation of the bioreactor using a sintered fritted glass microsparger operated in reverse orientation. The perfusion rate varied depending on the day of cultivation and ranged from 0.5 VVD to 1.5 VVD. The medium used was StemScale medium supplemented with 0. 1 % Pl 88, but without Y27632. From 24 to 72 hours post inoculation, the perfusion rate was maintained at 0.5 VVD. From hours 72 to 96, the perfusion rate was set at 1 VVD and an additional 3.6 g / L glucose (Sigma, G7021) was added to the perfusion media. From 96 to 144 hours, the rate w as increased to 1.5 VVD. The culture was regularly sampled, and cell density’ was measured. Additionally, glucose, lactate, LDH and ammonia levels were monitored. After 144 hours of cultivation the cell density was determined. The appropriate number of aggregates were extracted and washed once with pure media. Afterwards, the bioreactor was reseeded to a density of approximately 1.2xl06cells / mL into 120 mL media for the initiation of the differentiation into hepatocytes.26DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0091] Differentiation takes place over 5 stages, namely Stage 1 A, Stage IB, Stage 2, Stage 3, Stage 4, and Stage 5, starting at the definitive endoderm, through the posterior foregut to hepatic progenitors to immature hepatocytes and finally to mature hepatocytes. Each stage utilized a specific media formulation to direct differentiation in a stage-specific manner. To enable successful differentiation under 3D bioreactor conditions, supplementing the differentiation media with Polyvinylpyrrolidone (PVP360, Sigma- Aldrich) to establish the cell protective conditions and simulate a more in vivo-like environment is beneficial. PVP360 was added to all differentiation stage media at a concentration of 3.8 mg / mL. Additionally, 0.1 % Pl 88 (K4894, Sigma- Aldrich) was added to each differentiation stage media to further protect cells from shear stress exerted by the bioreactor system. To ensure adequate supply with nutrients and co-factors for differentiation, the cells were perfused throughout the entire differentiation phase with 0.5 - 1.5 VVD fresh media of the respective stage media. To switch from one stage to the next, a full media exchange was required. For that, the media was aspirated via a vacuum trap through the cell retention device. The cells were washed once with the stage specific base media (without co-factor additions). The wash was then aspirated again, and the media of the next stage was supplied via a feed line to the cells.

[0092] The first stage of differentiation is the definitive endoderm which was induced by switching the StemScale growth medium to Stage 1A medium which consists of Essential 6 media (Thermo Fisher, A1516401), with CHIR99021 (Reprocell, 40004), BMP-4 (R&D Systems. 314-BP). Activin A (R&D Systems. 338-AC), FGF2 (R&D Systems, 233-FB), and LY294002 (Selleckchem, SI 105). CHIR99021 was used in a concentration of 3 pM, Activin A 100 ng / mL, FGF2 80 ng / rnL, BMP -4 10 ng / mL, and LY294002 10 pM. The cells were cultivated in this medium for 24 hours under perfusion conditions with 0.5 VVD. After 24 hours, the medium was switched to Stage IB medium, which was identical to Stage 1 A medium but contained no CHIR99021. The cultivation was continued for 24 hours with active perfusion and 0.5 VVD.

[0093] On day 3 of the cultivation, the medium was fully exchanged to Stage 2 differentiation medium consisting of RPMI-1640 (Thermo Fisher. 21875034) supplemented with lx MEM Non-Essential Amino Acids Solution (Thermo Fisher, 11140050). Activin A 100 ng / mL, FGF2 80 ng / mL, and lx B-27 Supplement (Thermo Fisher, 17504044). The cells were perfused under the same conditions for another 24 hours with 1 VVD.DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0094] On day 4, the medium was exchanged to Stage 3 medium, identical to Stage 2 medium but lacking FGF2. Also, the concentration of Activin A was reduced to 50 ng / mL. The cells were perfused with 1.5 - 2.0 VVD for days 4 - 8 of differentiation.

[0095] On day 9 of the cultivation the medium was switched to Stage 4 medium. Stage 4 medium consisted of HepatoZYME media (Thermo Fisher, 17705021) supplemented with 1 : 100 Glutamax (Thermo Fisher, 35050061), 1:50 MEM Non-Essential Amino Acids Solution, 1:50 Chemically defined lipid concentrate (Thermo Fisher, 1 1905031), E lOO Insulin- Transferrin-Selenium (Thermo Fisher, 41400045), Oncostatin M (R&D Systems, 295-OM), and recombinant Hepatocyte Growth Factor (PeproTech, 100-39H). The concentration of Oncostatin M was 10 ng / mL, while Hepatocyte Growth Factor was used at 50 ng / mL. The perfusion was maintained at 0.5 VVD.

[0096] The switch from differentiation Stage 4 to Stage 5 was done gradually as the required co-factors for this stage were similar. On day 13 of differentiation the perfusion media was switched to Stage 5 media and the perfusion rate was increased to 1.5 VVD to facilitate the media exchange. After 24 hours (day 14) the perfusion was dropped to 0.5 VVD. Stage 5 media was identical to Stage 4 media but with added Forskohn (PeproTech, 40025) in a concentration of 5 pM.

[0097] On day 19, the Stage 5 media was exchanged to Stage 5 media minus Forskohn. The perfusion rate was increased to 1.5 VVD and dropped to 0.5 VVD on day 20 to facilitate the washout of Forskohn. Cells were then matured in these cultivation conditions until day 28 of differentiation, which w as when they reach full functionality.

[0098] Throughout the process, cells were monitored via microscopy regarding their morphology and with supporting assays that measured the secretion of certain biomarkers that express throughout the differentiation into hepatocytes. In contrast to previous unsuccessful attempts of differentiation in bioreactors with regular media exchanges, differentiation under perfusion conditions with the additions of Pl 88 and PVP as described above were successful yielding morphologically well-defined aggregates as seen in FIG. 2.

[0099] In terms of yield, the perfusion differentiation yielded approximately 13320 aggregates from 120 mL of bioreactor volume. As the extracellular matrix of hepatocytes made the aggregates stick together tightly, it was difficult to get the aggregates apart to do single cell counting. Therefore, the effectiveness of perfusion differentiation was currently based on the ability' to create aggregates in general and to analyze them based on their function. After successful differentiation, hepatocyte aggregates were round and dark under the brightfield28DB1 / 162996881.8Ref. No.: 123828-02-5143-WO microscope. They should form clusters of roughly 150 - 400 pm. The darker color indicated tightly packed aggregates without cyst formation or ballooning which would appear more translucent in the brightfield image. Ballooned hepatocytes were usually associated with inflammatory reactions and steatosis inhibiting the desired function of the hepatocytes.

[0100] While yield and morphology were important criteria for the success of the cultivation, functionality was equally as important. Hepatocytes were assessed in different ways regarding their functionality. Expression of certain cellular markers, such as CYP3A or secreted proteins, such as albumin or Al AT characterize well defined hepatocytes. A major function however was the detoxification of ammonia into urea or other metabolites.

[0101] FIG. 3 shows the production of albumin by the hepatocytes after 28 days (direct measurement from the bioreactor) or 29 days (transfer of about 4000 aggregates to one well of a 6 well plate and incubation for 24 hours). The values were determined via ELISA using the Human Albumin ELISA Kit (Bethyl Laboratories, E88129) in a 1 :10 dilution. FIG. 4 shows the production of Al AT measured in a 1:40 dilution with the Human Alpha- 1 -Antitry psin ELISA Kit (Bethyl Laboratories, E88122). Values were either measured directly from the bioreactor after 28 days or after a 24-hour incubation period (29 days after start of differentiation) of about 4000 aggregates in one well of a 6 well plate. Both proteins were well expressed in this differentiation and showed that the generated cells express ty pical proteins of hepatocytes. The value measured directly from the bioreactor was generally lower which was a result of the perfusion and the continuous dilution / removal of proteins. FIGs. 11 and 12 show the production of albumin and Al AT, respectively, by hepatocytes derived from perfusion process-based differentiation of iPSCs and hepatocytes derived from a standard 6- well plate-based differentiation of iPSCs, demonstrating that the methods disclosed herein produce cells (e.g.. hepatocytes) of similar or higher function compared to a more traditional way of differentiation.

[0102] To determine the capability7of ammonia detoxification, about 4000 aggregates were transferred to one well of a 6 well plate. Afterwards, 10 mM NH4CI and 4 rnM L-Omithine were added, and the starting ammonia concentration was measured by taking a sample of the well using the NH3B ammonia test with the CedexBio analyzer (Roche). The cells were incubated for 24 hours in a controlled incubator after which another CedexBio reading was done. The difference in ammonia concentration represented the detoxification over 24 hours which was then converted to a nmol / min value. FIG. 5 shows the ammonia detoxification of the perfusion-derived hepatocytes. Due to the difficulty of dissociating the aggregates, the29DB1 / 162996881.8Ref. No.: 123828-02-5143-WO detoxification was based on nmol / min / 4000 aggregates. A detoxification of about 12 nm / min for one 6 well was within the higher end of the expectations indicating that the generated cells were well functioning.

[0103] FIG. 11 shows albumin concentrations determined via Human Albumin ELISA kit for the perfusion process compared to a representative 6 well differentiation after 28 days of differentiation (Perfusion) and 29 days of differentiation (6-well).

[0104] FIG. 12 shows A1AT concentrations determined via Human Alpha- 1 -Antitrypsin ELISA Kit for the perfusion process compared to a representative 6 well differentiation after 28 days of differentiation (Perfusion) and 29 days of differentiation (6-well). Example 2; Differentiation of iPSC under 3D non-perfusion conditions in bioreactors

[0105] As an alternative system to compare perfusion differentiation to a more classical 3D system with daily media changes, cells were differentiated in the PBS Mini (PBS Biotech) system. The rotational speed per minute (rpm) was maintained between 25 to 60, based on the sedimentation tendencies of the aggregates, which varied depending on the stage of differentiation. The PBS Mini was placed in an incubator set to maintain a temperature of 37°C and 5 % CO2 exposure. Cells were seeded as single cells at a density of 5xl05cells / mL in a working volume of 100 mL. Media was exchanged daily or every other day by stopping the impeller, leading to sedimentation of the cell aggregates. The spent media was aspirated, and fresh media was supplied, before the vessel was returned to the incubator. The timings and the differentiation media per stage were identical to those used for perfusion differentiation. The only other difference was the addition of 2 % BSA (Sigma- Aldrich, A9418) as a supplement to each differentiation media. The differentiation process was initiated about 72 hours post seeding, to allow the single cells to form aggregates.

[0106] Previous 3D differentiations under non-perfusion conditions have used Pl 88 additions to the differentiation media to increase the protection against shear. The cells have been differentiated as described in this experiment (in this case only with addition of Pl 88 to the differentiation media and no other supplements).

[0107] FIG. 7 shows the iPSC aggregates as they have formed in the vertical wheel bioreactor (VW-BR) prior to the start of differentiation. FIG. 8 and FIG. 9 show some morphological changes during the differentiation phase. The cells became less stable and break apart and by day 7 of the differentiation have dissolved so much, that no viable cells could be recovered.30DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0108] With the addition of 2 % BSA, Pl 88, and PVP however, successful differentiation in the VW-BR was possible. A non-perfusion system relied on daily media exchanges to replenish nutrients and co-factors. Without being bound to a particular theory, with the added shear forces of the vertical wheel, it is hypothesized that the BSA further protects the cells and potentially stabilizes some of the differentiation co-factors for longer.

[0109] FIG. 6 shows the morphological changes during the transition of the iPSCs towards hepatocytes. In comparison to cells that were generated in the same system without the addition PVP and BSA (FIG. 9), cells did survive the entire differentiation process with the two additional media supplements. While the overall morphology looks less defined compared to a perfusion differentiation (FIG. 2), the cells still were functional, proving that media additions enabled successful differentiation.

[0110] Similarly to the perfusion process, the non-perfusion derived cells were also tested for functionality. However, only ammonia detoxification was assessed in this case. The cells from the bioreactor were distributed equally to 3 wells of a 6 well plate and measured according to the previously described protocol. FIG. 10 shows the ammonia detoxification after 24 hours in nmol / min. While the cells detoxify ammonia, the actual detoxification rate was lower, as compared to the perfusion process. Without being bound to a particular theory, this implies that the perfusion generated cells were likely more functional compared to the non-perfusion 3D- derived hepatocytes. Example 3: Differentiation of iPSC under 3D perfusion conditions in larger scale bioreactors

[0111] Human iPSCs are cultivated in a suspension bioreactor designed to allow perfusion to achieve high yield differentiation of functional cells. In this embodiment, the goal was to differentiate iPSCs into hepatocytes. The bioreactor used contained an impeller to keep the cells dispersed and to ensure proper mixing. Peristaltic pumps continuously added fresh culture medium to the tank while simultaneously removing spent media. The cells were retained in the bioreactor by a cell retention device, in this instance, a sintered sparger, which allowed for the removal of spent media while retaining the cells. The medium flow rates and impeller speed were controlled via the process control system to maintain optimal shear forces and mass transfer of nutrients for cell growth. Dissolved oxygen and pH sensors monitor culture conditions and regulate gas flows or liquid base addition via feedback loops to maintain steady setpoints. The media exchange rates for expansion varied between 0.5 to 1.5 VVD (vessel volume per day) and were kept between 0.5 to 2 VVD for the differentiation phase.31DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0112] In one embodiment, the process was initiated with the thawing of frozen iPSCs, wherein a vial containing approximately 1 million iPSCs is thawed at 37 °C. The ensuing procedure involved diluting the stem cell suspension in a 10-fold volume of cell culture media Essential 8 (E8, Gibco, A1517001) in a 15 mL conical tube, centrifuging the mixture at 200xg for 5 minutes, discarding the supernatant and resuspending the remaining pellet in 8 mL of E8 medium combined with 10 pM Y27632 • 2HC1 (Selleckchem. S1049). This suspension was then transferred to a Vitronectin (Thermo Fisher, A31804) coated T25 flask and incubated at 37 °C and 5 % CO2.

[0113] Approximately 24 hours post-incubation, the old medium was substituted with 8 mL fresh E8 medium and returned to the incubator. Successively, the process involved passaging of cells after 72 - 96 hours after the seeding, which is pivotal in maintaining the cells in optimal proliferative conditions. The exact timing for passaging is defined by reaching a confluency of 70 - 80 %. Stage-wise, cells were firstly washed with PBS, and the residue of PBS was removed. An addition of 0. 1 mL / cm2of Accutase (Innovative Cell Technologies, AT- 104) ensued, and the mixture was incubated for 5 minutes at 37 °C. Upon detection of cell detachment via flask tilting, the Accutase was diluted with an equal volume of E8, transferred to a conical tube, and centrifuged at 200xg for 5 minutes. The supernatant was discarded, and the remaining substance was resuspended in 10 mL E8 supplemented with 10 pM Y27632.

[0114] Furthermore, the cell quantity was determined via an automated cell counter or a Hemocytometer. Subsequently. T175 flask coated with Vitronectin were seeded with approximately 2 million cells per flask in 60 mL E8 supplemented with 10 pM Y27632. The medium was replenished with fresh E8 every' 24 hours for 72 - 96 hours and returned to the incubator.

[0115] After reaching 60 - 80 % confluency, cells were passaged at 2 - 4 million cells per flask in 50 mL StemScale medium (Thermo Fisher, A4965001) with 10 pM Y27632 • 2HC1 (Selleckchem, SI 049).

[0116] To achieve the required cell densities for inoculation of a larger scale bioreactor, the cells were then passaged into a 5-stack CellSTACK® Culture Chamber (Coming. 3319). For that, the CellSTACK® was coated with Vitronectin and 90 million cells were seeded into 800 mL StemScale media supplemented with 10 pM Y27632 • 2HC1.

[0117] About 3 days after seeding into StemScale media, cells had an approximate confluency of 60 - 80 % and were ready for expansion in bioreactors.32DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0118] In this embodiment, a Sartorius Biostat B-DCU with a 2 L glass Univessel (Sartorius) was used for cell expansion and differentiation. To prevent cells from sticking to the glass surface, the bioreactor was siliconized by applying Sigmacote (Sigma- Aldrich, SL2- 25ML). The previously 2D-expanded cells were dissociated into single cells and used to inoculate the bioreactor at a cell density' of 0.65xl06cells / mL. The cells were seeded into a total volume of 1000 mL StemScale medium containing 10 pM Y27632 • 2HC1, and 0.1 % Pl 88 (Sigma, P4894). The temperature was controlled at 37 °C. The pH setpoint was maintained at 7.15 with a 0.15 pH deadband via CO2 overlay gassing and NaHCCh addition. Dissolved oxygen was maintained at 40 % through a combination of overlay gassing and submerged gassing via a ring sparger using O2 after about 96 hours. Media perfusion was initiated 12 hours after inoculation of the bioreactor using a sintered fritted microsparger operated in reverse orientation. The perfusion rate varied depending on the day of cultivation and ranged from 1 VVD to 1.5 VVD. The medium used was StemScale medium supplemented with 0.1 % Pl 88, but without Y27632. From 12 to 72 hours post inoculation, the perfusion rate was maintained at 1 VVD. From hours 72 to 96 and above, the perfusion rate was set at 1.5 VVD. The culture was regularly sampled, and cell density was measured. Additionally, glucose, lactate, LDH and ammonia levels were monitored. After 120 hours of cultivation the cell density was determined and reached around 7.82xl06viable cells / mL. The impeller was stopped to allow for cell settling and the supernatant media was carefully removed via the microsparger. Afterwards, prewarmed differentiation media was introduced into the bioreactor.

[0119] In this embodiment, differentiation takes place over 5 stages, namely Stage 1A, Stage IB, Stage 2, Stage 3, Stage 4, and Stage 5, starting at the definitive endoderm, through the posterior foregut to hepatic progenitors to immature hepatocytes and finally to mature hepatocytes. Each stage utilizes a specific media formulation to direct differentiation in a stage- specific manner. To enable successful differentiation under 3D bioreactor conditions, supplementing the differentiation media with Polyvinylpyrrolidone (PVP360, Sigma- Aldrich) to establish the cell protective conditions and simulate a more in vivo-like environment is critical. PVP360 is added to all differentiation stage media at a concentration of 3.8 mg / mL. Additionally, 0. 1 % Pl 88 (K.4894, Sigma- Aldrich) is added to each differentiation stage media to further protect cells from shear stress exerted by the bioreactor system. To ensure adequate supply with nutrients and co-factors for differentiation, the cells are perfused throughout the entire differentiation phase with 0.5 - 1.5 VVD fresh media of the respective stage media. To switch from one stage to the next, a full media exchange is required in most cases. For that, the33DB1 / 162996881.8Ref. No.: 123828-02-5143-WO impeller is switched off and the supernatant media is aspirated via the microsparger and the fresh media is supplied via a feed line. This is not required for transitions to stages 3 and 5 where simply the feed media is exchanged and the media change is performed gradually via perfusion.

[0120] The first stage of differentiation is the definitive endoderm which is induced by switching the StemScale growth medium to Stage 1 A medium which consists of RPMI-ATCC media (Thermo Fisher, 21875034), with CHIR99021 (Reprocell, 40004), Activin A (R&D Systems, 338-AC), and N-Acetyl-L-cysteine (Sigma, A91 5). In this embodiment, CHIR99021 was used in a concentration of 5 pM, Activin A 100 ng / mL, and N-Acetyl-L- cysteine 2 mM. The cells were cultivated in this medium for 24 hours under perfusion conditions with 0.5 VVD. The pH is allowed to drop down to 6.7 and maintained at that level with bicarbonate. After 24 hours, the medium was switched to Stage IB medium, which was identical to Stage 1A medium but instead of CHIR99021 contains 0.8 % KOSR (Thermo Fisher, 10828028). The cultivation was continued for 24 hours with active perfusion and 0.5 VVD.

[0121] On day 3 of the cultivation, the medium was fully exchanged to Stage 2 differentiation medium which has the same composition as Stage IB medium except an increase in KOSR to 8.0 %. The cells were perfused under the same conditions for another 24 hours with 1 VVD.

[0122] On day 4, the medium was exchanged to Stage 3 medium by perfusing the media instead of a full media exchange. Stage 3 media consist of RPMI-ATCC media as a base but is supplemented with MEM NEAA (Thermo Fisher, 11140050) and B27 supplement (Thermo Fisher, 17504044). The concentration of Activin A is reduced to 50 ng / mL. Also, KOSR is excluded in Stage 3 media. The cells were perfused with 1 VVD for days 4 - 8 of differentiation. From here onwards the pH is kept at 7. 15 with a deadband of 0. 15.

[0123] On day 9 of the cultivation the medium w as switched to Stage 4 medium. Stage 4 medium consists of HepatoZYME media (Thermo Fisher, 17705021) supplemented with 1: 100 Glutamax (Thermo Fisher. 35050061), 1:50 MEM Non-Essential Amino Acids Solution, 1:50 Chemically defined lipid concentrate (Thermo Fisher, 11905031), 1 : 100 Insulin-Transferrin- Selenium (Thermo Fisher, 41400045), Oncostatin M (R&D Systems, 295-OM), and recombinant Hepatocyte Growth Factor (PeproTech, 100-39H). The concentration of Oncostatin M is 10 ng / mL, while Hepatocyte Grow th Factor is used at 50 ng / mL. The perfusion was maintained at 0.5 VVD.34DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0124] The differences between Stage 4 and Stage 5 media are only in the addition of 5 pM Forskolin (Peprotech, 40025). Therefore, the Stage 4 perfusion media is switched to Stage 5 media and the bioreactor is supplemented with a pulse of concentrated Forskolin to bring the concentrations in the bioreactor up to 5 pM. The perfusion rate stays steady at 0.5 VVD until the end of differentiation, usually day 28, when they reach their peak functionality.

[0125] It should be understood that for all numerical bounds describing some parameter in this application, such as “about.” ‘‘at least,” "less than,” and ‘"more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1 - 2, 1 - 3, 1 - 4, 1 - 5, 2 - 3, 2 - 4, 2 - 5, 3 - 4, 3 - 5, and 4 - 5, et cetera.

[0126] For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g.. PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety.

[0127] Preferred features of each of the aspects provided by the disclosure are applicable to all of the other aspects of the disclosure mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplar}7embodiments) of particular embodiments and aspects of the disclosure, including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed disclosure piecemeal without departing from the disclosure. For example, for materials that are disclosed, while specific reference of each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B. and C are disclosed as well as a class of elements D, E. and F and an example35DB1 / 162996881.8Ref. No.: 123828-02-5143-WO of a combination of elements A-D is disclosed, then, even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C;D, E, and F; and the example combination A-D. This concept applies to all aspects of this application, including elements of a composition of matter and steps of method of making or using the compositions.

[0128] The forgoing aspects of the disclosure, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art — thus, to the extent an element is described in one or more references known to the person having ordinary skill in the art. they may be excluded from the claimed disclosure by, inter alia, a negative proviso or disclaimer of the feature or combination of features.EQUIVALENTS

[0129] While the invention has been described in connection with embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0130] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.INCORPORATION BY REFERENCE

[0131] All patents and publications referenced herein are hereby incorporated by reference in their entireties.DB1 / 162996881.8Ref. No.: 123828-02-5143-WO

[0132] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0133] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.DB1 / 162996881.8

Claims

Ref. No.: 123828-02-5143-WOCLAIMSWhat is claimed is:1 . A method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system, optionally wherein the system comprises a perfusion means, wherein the culture medium comprises effective amounts of a nonionic surfactant and molecular crowder, optionally further comprising one or more of bovine serum albumin (BSA), recombinant albumin, polyvinyl alcohol (PVA), synthetic BSA replacement, and thermally denatured BSA; and wherein the method is suitable for differentiation of iPSCs, e g., to hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells, optionally wherein the method is suitable for differentiation of iPSCs to hepatocytes, (3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

2. The method of claim 1 , wherein the nonionic surfactant is a triblock polymer, optionally wherein the triblock polymer comprises an ethoxylated alcohol.

3. The method of claim 1 or 2, wherein the nonionic surfactant is poloxamer 188 (P188).

4. The method of any one of the preceding claims, wherein the nonionic surfactant is poloxamer 188 at a concentration of up to about: 0.01, 0.02, 0.03, 0.

04. 0.

05. 0.06, 0.07, 0.08, 0.

09. 0.10, 0.11, 0.12, 0.13, 0.14, 0.

15. 0.

16. 0.

17. 0.18, 0.19, 0.20, 0.25, 0.30, 0.

35. 0.

40. 0.

45. 0.50, 0.55, 0.60, 0.65 , 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.00 % (W / V).

5. The method of claim 4, wherein the nonionic surfactant is poloxamer 188 at a concentration of about .05 % - .50 % or about .01% - 1%.

6. The method of any one of the preceding claims, wherein the molecular crowder is selected from polyethylene glycol (PEG), ficoll, dextran, BSA, methylcellulose, polyvinyl alcohol (PVA), Hydroxyethyl Starch (HES), and a polymer of a vinylpyrrolidone, such as N- vinylpyrrolidone.

7. The method of any one of the preceding claims, wherein the molecular crowder is a polyvinylpyrrolidone (PVP), optionally PVP360.38DB1 / 162996881.8Ref. No.: 123828-02-5143-WO8. The method of any one of the preceding claims, wherein a concentration of the PVP is at least about: 0.5, 0.

6. 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.

3. 1.4, 1.5, 1.

6. 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 mg / ml, optionally wherein the concentration of the PVP is selected from a range of about 0.5 - 8.0 mg / ml, 1.5 - 6.0 mg / ml, or 3.0 - 4.0 mg / ml,9. The method of any one of the preceding claims, wherein the culture medium is a differentiation medium suitable for differentiation of iPSCs, optionally differentiation of iPSCs to hepatocytes, optionally differentiation of iPSCs to hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary' lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, or epithelial and barrier lineage cells, optionally differentiation of iPSCs to hepatocytes, {3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

10. The method of any one of the preceding claims, wherein the differentiation medium is suitable for differentiation of iPSCs to hepatocytes, optionally wherein the differentiation medium comprises a sequence of culture media, further optionally wherein the culture media comprise a series of optionally different base media supplemented with: a. a first media comprising:(a) a GSK3 inhibitor, optionally CHIR99021, at a concentration of about 0.1 pM - 20 pM or about 2 pM - 10 pM, optionally wherein the concentration of CHIR99021 is between 3 pM - 6 pM, optionally wherein the concentration of CHIR99021 is about 4 pM - 5 pM, optionally wherein the concentration of CHIR99021 is about 5 pM,(b) a TGF-f) family grow th factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(c) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(d) optionally, Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / rnL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(e) optionally, a PI3K inhibitor, optionally LY294002, at a concentration of about 1 pM - 50 pM, or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM,(f) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g., KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%. optionally wherein the concentration of KOSR is about0.8%, and(g) optionally, a cell protective antioxidant (e g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0. 1 rnM - 4 mM, optionally wherein the concentration of NAC is about 2 mM; b. a second media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) optionally, Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,DB1 / 162996881.8Ref. No.: 123828-02-5143-WO(d) optionally, a PI3K inhibitor, optionally LY294002, at a concentration of about 1 LI.M - 50 pM, or about 5 pM - 20 pM, optionally wherein the concentration of LY294002 is about 10 pM,(e) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%. optionally wherein the concentration of KOSR is about 0.8%, and(f) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0. 1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM; c. a third media comprising:(a) a TGF-0 family grow th factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 100 ng / mL,(b) optionally, Fibroblast Growth Factor 2 (FGF2) at a concentration of about 10 ng / ml - 100 ng / ml or about 0.1 ng / ml - 150 ng / ml, optionally wherein the concentration of FGF2 is about 80 ng / mL,(c) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 8%,(d) optionally, Non-Essential Amino Acid Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and(e) optionally, B-27 or B-27 plus Supplement; d. a fourth media comprising:(a) a TGF-P family growth factor, optionally Activin A at a concentration of about 10 ng / ml - 200 ng / ml or about 0.5 ng / ml - 500 ng / ml, optionally wherein the concentration of Activin A is about 50 ng / mL,(b) optionally, Bone Morphogenetic Protein 4 (BMP-4) at a concentration of about 0.01 ng / mL - 100 ng / mL, optionally wherein the concentration41DB1 / 162996881.8Ref. No.: 123828-02-5143-WO of BMP-4 is about 1 ng / mL - 50 ng / mL, optionally wherein the concentration of BMP-4 is about 5 ng / mL - 15 ng / mL, optionally wherein the concentration of BMP-4 is about 10 ng / mL,(c) optionally, fetal bovine serum (FBS) or a defined, serum-free supplement used in cell culture to replace fetal bovine serum (FBS) (e.g.,KnockOut™ Serum Replacement (KOSR)) at a concentration of about 0.1% - 8%, optionally wherein the concentration of KOSR is about 5%,(d) optionally, a cell protective antioxidant (e.g., N-Acetyl-Cysteine (NAC)) at a concentration of about 0. 1 mM - 4 mM, optionally wherein the concentration of NAC is about 2 mM,(e) optionally, a biologically active metabolite of vitamin A (retinol) that functions as a regulator of cell growth, differentiation, and development (e.g., All Trans Retinoic Acid (ATRA)) at a concentration of about 0.1 - 4 pM, optionally wherein the concentration of ATRA is 2 pM and is used for about 2 days when the fourth media is being used,(f) Non-Essential Amino Acid Solution, optionally wherein the Non- Essential Amino Acid Solution comprises glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, and (g) B-27 or B-27 plus Supplement; e. a fifth media comprising:(a) 1 : 100 L-alanyl-L-glutamine (e.g., GLUTAMAX),(b) 1 :50 MEM Non-Essential Amino Acids Solution, optionally wherein the Non-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1 :50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium,(e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about 0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / mL, and(f) recombinant Hepatocyte Grow th Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the42DB1 / 162996881.8Ref. No.: 123828-02-5143-WO concentration of recombinant Hepatocyte Growth Factor is about 50 ng / mL; and / or f. a sixth media comprising:(a) 1 : 100 L-alanyl-L-glutamine (e.g., GLUTAMAX), (b) 1 :50 MEM Non-Essential Amino Acids Solution, optionally wherein theNon-Essential Amino Acid Solution comprises glycine, L-alanine. L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine,(c) 1 :50 Chemically defined lipid concentrate,(d) 1 : 100 Insulin-Transferrin-Selenium, (e) Oncostatin M at a concentration of about 10 ng / ml - 50 ng / ml or about0.1 ng / ml - 100 ng / ml, optionally wherein the concentration of Oncostatin M is about 10 ng / ml,(f) recombinant Hepatocyte Growth Factor at a concentration of about 2.5 ng / ml - 100 ng / ml or about 10 ng / ml - 50 ng / ml, optionally wherein the concentration of recombinant Hepatocyte Grow th Factor is about 50 ng / mL, and(g) Forskolin at a concentration of about 2 pM - 10 pM or about 100 nM - 100 pM, optionally wherein the concentration of Forskolin is about 5 pM.DBl / 162996881.8Ref. No.: 123828-02-5143-WO11. The method of any one of the preceding claims, wherein the culture media comprises Essential 6, RPMI-1640. RPMI-1640 ATCC Modification, Hepatozyme, Dulbecco's Modified Eagle Medium (DMEM) / F12, DMEM / F12-base media, William E, and supplemented Minimum Essential Medium (MEM).

12. The method of any one of the preceding claims, further comprising evaluating the differentiation state of the iPSCs.

13. The method of claim 12, wherein the iPSCs are differentiated into hepatocytes and the evaluation comprises: a. evaluating the expression of one or more of: cytochrome P450 1A2 (CYP1 A2), cytochrome P450, family 3, subfamily A (CYP3A), albumin, alpha fetoprotein (AFP), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), sulfotransferases, carnitine palmitoyltransferase I (CPT1), and alpha-1 antitry psin (A1AT); b. evaluating ammonia detoxification; c. evaluating ureagenesis; or d. a combination of any of the foregoing.

14. The method of any one of the preceding claims, wherein the method further comprises preparing a pharmaceutical composition of differentiated iPSCs.

15. A method comprising administering a therapeutically effective amount of differentiated iPSCs to a subject in need thereof, wherein the differentiated iPSCs are produceable or produced by the method of any one of the preceding claims.

16. A pharmaceutical composition suitable for administration to a subject comprising a therapeutically effective amount of differentiated iPSCs, wherein the differentiated iPSCs are produceable or produced by the method of any one of the preceding claims.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition comprises micro encapsulated cells or macro encapsulated cells.

18. The pharmaceutical composition of claim 16 or 17, wherein the subject is a human.

19. The pharmaceutical composition of any one of claims 16-18, wherein the differentiated iPSCs are hepatocytes.

20. The pharmaceutical composition of any one of claims 16-19, further comprising a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.44DB1 / 162996881.8Ref. No.: 123828-02-5143-WO21. A device comprising a therapeutically effective amount of the pharmaceutical composition of any one of claims 16-20.

22. A dosage form of a therapeutically effective amount of the pharmaceutical composition of any one of claims 16-20.

23. A method of generating differentiated cells for iPSCs substantially as shown in FIG. 1.DBl / 162996881.8

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