A method for reproducible differentiation of clinical-grade retinal pigment epithelial cells

Abstract

To provide methods of producing an RPE cell population from a starting cell suspension, such as a single cell suspension, of pluripotent stem cells (PSCs).SOLUTION: Provided is a method of producing human retinal pigment epithelial (RPE) cells, comprising: a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) that are dissociated into essentially single cells; b) culturing the iPSCs in a retinal induction medium to initiate differentiation of the cells into retinal lineage cells; c) further culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells; d) culturing the cells in retinal medium to form differentiating RPE cells; and e) culturing the RPE cells in a RPE maturation medium, thereby producing human RPE cells; the method not comprising the formation of embryoid bodies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 215,579, filed September 8, 2015, the entire contents of which are incorporated herein by reference.

[0002] Parties to the Joint Research Agreement This invention was made as a result of activities carried out within the scope of a Cooperative Research Agreement in effect at the time this invention was made, the parties to which are the United States Government, the U.S. Department of Health and Human Services, as represented by the National Eye Institute, the National Institutes of Health, and Cellular Dynamics International, Inc. [Background technology]

[0003] background 1. Field The present disclosure relates generally to the field of stem cell biology. More specifically, it relates to methods for efficiently producing stem cell-derived retinal pigment epithelial cell populations for use as cell therapy.

[0004] 2. Description of Related Technology The retina is a light-sensitive tissue layer covering the inner surface of the eye. Photoreceptor cells (either rods or cones) in the retina are directly sensitive to light and convert chemical light signals into electrical events, which trigger nerve impulses. The retinal pigment epithelium (RPE) is a layer of pigmented cells that form the blood-retinal barrier. RPE cells play an important role in maintaining visual function and transporting ions, water, and metabolic end products from the subretinal space to the blood (Strauss et al., 2005). Furthermore, RPE cells establish immune privilege in the eye by secreting immunosuppressive factors. Damage or injury to RPE cells can lead to retinal degeneration, loss of visual function, and blindness. Several retinal disorders, including acute and age-related macular degeneration and Best's disease, involve RPE degeneration; therefore, cell replacement therapy is a possible treatment option for preserving vision (Buchholz et al., 2009).

[0005] Generally, stem cells are undifferentiated cells that can give rise to a range of mature functional cells. For example, hematopoietic stem cells can give rise to any of various types of terminally differentiated blood cells. Embryonic stem (ES) cells are derived from embryos and are pluripotent, so they have the ability to develop into any organ or tissue type, including RPE cells.

[0006] The generation of induced pluripotent stem cells (iPSCs) from adult somatic mouse cells in 2006 provided a significant breakthrough in stem cell research, drug development, disease modeling, and cellular therapeutics (Takahashi et al., 2006). Human iPSCs can differentiate into specialized cell types and have the potential for patient-specific, immune-compatible cells for regenerative medicine (Yu et al., 2007).

[0007] iPSCs have been shown to give rise to ocular cells, including RPE cells (Hirami et al., 2009). However, all known techniques for producing iPSC- or ESC-derived RPE cells to date rely on the use of starting populations of embryoid bodies. No efficient method exists for the large-scale production of iPSC- or ESC-derived RPE cells, which are needed for therapeutics, screening assays, retinal disease models, and RPE biology research. Summary of the Invention [Means for solving the problem]

[0008] overview Unfortunately, due to the fact that the production process of embryoid bodies itself is not reproducible, has variable efficiency, and is not scalable (which is necessary for commercial-scale production of RPE), routine and reproducible RPE production from pluripotent cells such as iPSCs or ESCs, that is, from a starting group of embryoid bodies, is difficult.Disclosed is a method for obtaining a retinal pigment epithelial (RPE) cell population, which avoids the need to use embryoid bodies, and instead uses a cell suspension group of pluripotent stem cells, preferably single-cell suspension, rather than using embryoid bodies.In certain embodiments, the starting cell population of pluripotent stem cells can be, for example, embryonic stem cells or induced pluripotent stem cells.

[0009] In some embodiments, provided is a method for differentiating pluripotent stem cells into retinal pigment epithelial (RPE) cells.For example, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).In one embodiment, provided is a method for producing human RPE cells, comprising:a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) that are essentially dissociated into single cells;b) culturing the iPSCs in a retinal induction medium to initiate the differentiation of the cells into retinal lineage cells;c) further culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells;d) culturing the cells in a retinal medium to form differentiated RPE cells;and e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells.In some embodiments, the method does not include the formation of embryoid bodies.In some aspects, RPE cells are cryopreserved after production.

[0010] In certain embodiments, iPSCs are cultured on a matrix. In some embodiments, the matrix comprises at least one recombinant cell adhesion protein, such as laminin, vitronectin or fibronectin. In particular, at least one cell adhesion protein is human.

[0011] In certain embodiments, iPSCs are cultured without a feeder layer. In some embodiments, iPSCs are cultured in a well-defined culture medium. In other embodiments, iPSCs are cultured in a xeno-free culture medium.

[0012] In further embodiments, the retinal induction medium comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and insulin growth factor 1 (IGF1). In some embodiments, the WNT pathway inhibitor is N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-fura The WNT pathway inhibitor is selected from the group consisting of: 2,4-diaminoquinazolinone, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1) and secreted Frizzled-related protein (SFRP1)1. For example, the WNT pathway inhibitor is CKI-7.In certain embodiments, the BMP pathway inhibitor is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6- [4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2) or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML347).For example, BMP pathway inhibitor is LDN193189.In certain embodiments, the TGFβ pathway inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-phenyl-butyl- 3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, Left-Right Determinant (Lefty), 3-(6-methyl-2- pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide Amide (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712) or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox).For example, TGFβ pathway inhibitor is SB431542.

[0013] In some embodiments, the retinal differentiation medium comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, a MEK inhibitor, and IGF 1. In some embodiments, the WNT pathway inhibitor is N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-fura ... The WNT pathway inhibitor is selected from the group consisting of: 2,4-diaminoquinazolinone, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1) and secreted Frizzled-related protein (SFRP1)1. For example, the WNT pathway inhibitor is CKI-7.In certain embodiments, the BMP pathway inhibitor is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6- [4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2) or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML347).For example, BMP pathway inhibitor is LDN193189.In certain embodiments, the TGFβ pathway inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-phenyl-butyl- 3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, Left-Right Determinant (Lefty), 3-(6-methyl-2- pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide Amide (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712) or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox).For example, TGFβ pathway inhibitor is SB431542.In some embodiments, the MEK inhibitor is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro- The MEK inhibitor is fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119) or 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).For example, the MEK inhibitor is PD0325901.In certain embodiments, the retinal differentiation medium comprises LDN193189, CKI-7, SB431542 and PD0325901.

[0014] In further embodiments, the RPE cells are dissociated after culturing in the RPE maturation medium. In further embodiments, the dissociated RPE cells are seeded and cultured in the RPE maturation medium. In certain embodiments, the RPE maturation medium comprises a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-2-methyl-2-propanol (GSK1120213), 2-fluoro-4-iodophenyl-3-methyl-2-propanol (GSK1120214), 2-fluoro-4-iodophenyl-3-methyl-2-propanol (GSK1120215), 2-fluoro-4-iodophenyl-3-methyl-2-propanol (GSK1120216 ... In some embodiments, the MEK inhibitor is PD0325901.

[0015] In yet another embodiment, RPE cells are dissociated after culturing in RPE medium and re-seeded on degradable scaffolds in RPE maturation medium, thereby producing mature RPE cells.In certain embodiments, RPE maturation medium can contain at least one primary cilia inducer.In some embodiments, at least one primary cilia inducer is prostaglandin E2 (PGE2) or aphidicolin.In other embodiments, RPE maturation medium can contain N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1).

[0016] In further embodiments, the starting population of iPSCs is pre-confluent cells dissociated into single cells. In other embodiments, iPSCs are cultured at a density of about 5,000-40,000 cells / cm. 2 In certain embodiments, iPSCs are cultured at an initial cell density of 5,000, 10,000, 20,000, 30,000, or 40,000 cells / cm. 2 The cells are cultured at an initial cell density of 1000 kJ / ml.

[0017] In still further embodiments, the starting population of iPSCs is MHC haplotype-matched to the subject in need thereof.In some embodiments, iPSCs are homozygous for at least one HLA allele.For example, iPSCs are homozygous for HLA-A, HLA-B or HLA-DR.In some embodiments, iPSCs are homozygous for HLA-A and HLA-B.

[0018] In another embodiment, provided is a method for producing human retinal pigment epithelial (RPE) cells, comprising: a) obtaining a starting population comprising the human induced pluripotent stem cells (iPSCs) that are essentially dissociated into single cells in a well-defined medium; b) culturing the iPSCs on laminin in a retinal induction medium that comprises LDN193189, CKI-7 and SB431542, to start the differentiation of the cells into retinal cells; c) further culturing the retinal cells in a retinal differentiation medium that comprises LDN193189, CKI-7, SB431542 and PD0325901, to further differentiate the retinal cells; d) culturing the cells in a retinal medium that comprises nicotinamide and activin A, to form differentiated RPE cells; and e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells.In certain embodiments, the method does not include the formation of embryoid body.

[0019] [The present invention 1001] 1. A method for producing human retinal pigment epithelial (RPE) cells, comprising: a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) dissociated into essentially single cells; b) culturing the iPSCs in a retinal induction medium to initiate differentiation of the cells into retinal lineage cells; c) further culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells; d) culturing the cells in retinal medium to form differentiated RPE cells; and e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells. Including, A method wherein the method does not include the formation of embryoid bodies. [The present invention 1002] 1001. The method of claim 1001, wherein said iPSCs of step (b) are cultured on a matrix. [The present invention 1003] 1002. The method of claim 1002, wherein said matrix comprises at least one recombinant cell adhesion protein. [The present invention 1004] 1004. The method of claim 1003, wherein said at least one cell adhesion protein is laminin, vitronectin or fibronectin. [The present invention 1005] 1005. The method of claim 1003 or 1004, wherein said at least one cell adhesion protein is human. [The present invention 1006] The method according to any one of claims 1001 to 1005, wherein said retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor and insulin growth factor 1 (IGF1). [The present invention 1007] The method of any one of claims 1001 to 1006, wherein said retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor and IGF1. [The present invention 1008] Any of the methods of 1001 to 1007 of the present invention, further comprising dissociating the RPE cells following step (e), reseeding the RPE cells, and culturing the RPE cells in the RPE maturation medium containing a MEK inhibitor. [The present invention 1009] The method of claim 1008, further comprising dissociating said RPE cells and reseeding said RPE cells onto a degradable scaffold in said RPE maturation medium. [The present invention 1010] Any of the methods of 1001 to 1009 of the present invention, further comprising culturing the RPE cells in the RPE maturation medium containing at least one primary cilium inducer, thereby producing mature RPE cells. [The present invention 1011] 10. The method of claim 10, wherein said at least one primary cilium inducer is prostaglandin E2 (PGE2) or aphidicolin. [The present invention 1012] Any of the methods of claims 1001 to 1009, further comprising culturing the RPE cells in the RPE maturation medium containing N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2) and / or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). [The present invention 1013] The method of any one of claims 1001 to 1012, further comprising cryopreserving the RPE cells. [The present invention 1014] 13. The method of any of claims 1001 to 1012, wherein said starting population of iPSCs in step (a) are pre-confluent cells dissociated into single cells. [The present invention 1015] The iPSCs of step (b) are cultured at a density of about 5,000 to 40,000 cells / cm. 2 The method of any one of claims 1001 to 1014, wherein the cells are cultured at an initial cell density of 1001 to 1014. [The present invention 1016] The method of any one of claims 1001 to 1015, wherein the iPSCs are cultured without a feeder layer. [The present invention 1017] The method of any one of claims 1001 to 1015, wherein said iPSCs are cultured in a well-defined culture medium. [The present invention 1018] The method of any one of claims 1001 to 1015, wherein the iPSCs are cultured in a xeno-free culture medium. [The present invention 1019] The WNT pathway inhibitor is N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydrazine Any of the methods of claims 1006 to 1018, wherein the compound is 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1), or secreted Frizzled-related protein 1 (SFRP1). [The present invention 1020] The TGFβ pathway inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-ethyl-3H-imidazol-4-yl)-butyl- yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, Left-Right Determinant (Lefty), 3-(6-methyl-2-pyridinyl)-N-phenyl-4 -(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW788388), 4-[3 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox). [The present invention 1021] The MEK inhibitor is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)- 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), or 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244). [The present invention 1022] The BMP pathway inhibitor is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-[4-(1-methylethoxy)phenyl] 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML347). [The present invention 1023] The method of any one of claims 1006 to 1021, wherein the BMP pathway inhibitor in the retinal induction medium is LDN193189. [The present invention 1024] The method of any one of claims 1007 to 1023, wherein the BMP pathway inhibitor in the retinal differentiation medium is LDN193189 and the MEK inhibitor is PD0325901. [The present invention 1025] The method of any of claims 1001 to 1024, wherein said starting population of iPSCs is MHC haplotype-matched to a subject in need thereof. [The present invention 1026] 1026. The method of any of claims 1001 to 1025, wherein said starting population of iPSCs is homozygous for at least one HLA allele. [The present invention 1027] 1027. The method of claim 1026, wherein said at least one HLA allele is HLA-A, HLA-B or HLA-DR. [The present invention 1028] 1. A method for producing human retinal pigment epithelial (RPE) cells, comprising: a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) dissociated into essentially single cells in a well-defined medium; b) culturing the iPSCs on laminin in a retinal induction medium containing LDN193189, CKI-7, and SB431542 to initiate differentiation of the cells into retinal lineage cells; c) further culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, CKI-7, SB431542, and PD0325901 to further differentiate the retinal lineage cells; d) culturing the cells in a retinal medium containing nicotinamide and activin A to form differentiated RPE cells; and e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells. Including, A method wherein the method does not include the formation of embryoid bodies. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given for purposes of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of certain specific embodiments presented herein.

[0021] [Figure 1A] A) Image of pre-confluent iPSCs. B) Example image of day 25 of RPE differentiation. C) Example image of day 40 of RPE differentiation. D) Example image at 100x bright field at day 60 after reseeding the culture in RPE-MM at day 40. [Figure 1B] A) Image of pre-confluent iPSCs. B) Example image of day 25 of RPE differentiation. C) Example image of day 40 of RPE differentiation. D) Example image at 100x bright field at day 60 after reseeding the culture in RPE-MM at day 40. [Figure 1C] A) Image of pre-confluent iPSCs. B) Example image of day 25 of RPE differentiation. C) Example image of day 40 of RPE differentiation. D) Example image at 100x bright field at day 60 after reseeding the culture in RPE-MM at day 40. [Figure 1D] A) Image of pre-confluent iPSCs. B) Example image of day 25 of RPE differentiation. C) Example image of day 40 of RPE differentiation. D) Example image at 100x bright field at day 60 after reseeding the culture in RPE-MM at day 40.

[0022] [Figure 2A]Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) before cell sorting of iPSC-derived RPE cell populations. [Figure 2B] Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) before cell sorting of iPSC-derived RPE cell populations.

[0023] [Figure 3A] Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24-positive and CD56-positive cells, CD24-positive and CD90-positive cells, and CD24-positive, CD56-positive, and CD90-positive cells. [Figure 3B] Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24-positive and CD56-positive cells, CD24-positive and CD90-positive cells, and CD24-positive, CD56-positive, and CD90-positive cells. [Figure 3C] Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24-positive and CD56-positive cells, CD24-positive and CD90-positive cells, and CD24-positive, CD56-positive, and CD90-positive cells.

[0024] [Figure 4A]A) β-catenin and F-actin staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. β-Catenin staining is seen in the cytoplasm of untreated cells and the membrane of treated cells. B) pERM (ezrin) and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. ERM staining is low in the cytoplasm of untreated cells and high in the cytoplasm of treated cells, while ZO1 staining is seen in the tight junctions of the plasma membrane in both untreated and treated cells. C) RPE65 and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. RPE65 staining is low in the cytoplasm of untreated cells and high in the cytoplasm of treated cells. D) Transmission electron micrographs of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. PGE2-treated cells have more extensive apical processes. [Figure 4BC] A) β-catenin and F-actin staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. β-Catenin staining is seen in the cytoplasm of untreated cells and the membrane of treated cells. B) pERM (ezrin) and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. ERM staining is low in the cytoplasm of untreated cells and high in the cytoplasm of treated cells, while ZO1 staining is seen in the tight junctions of the plasma membrane in both untreated and treated cells. C) RPE65 and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. RPE65 staining is low in the cytoplasm of untreated cells and high in the cytoplasm of treated cells. D) Transmission electron micrographs of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. PGE2-treated cells have more extensive apical processes. [Figure 4D]A) β-catenin and F-actin staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. β-Catenin staining is seen in the cytoplasm of untreated cells and the membrane of treated cells. B) pERM (ezrin) and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. ERM staining is low in the cytoplasm of untreated cells and high in the cytoplasm of treated cells, while ZO1 staining is seen in the tight junctions of the plasma membrane in both untreated and treated cells. C) RPE65 and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. RPE65 staining is low in the cytoplasm of untreated cells and high in the cytoplasm of treated cells. D) Transmission electron micrographs of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. PGE2-treated cells have more extensive apical processes.

[0025] [Figure 5AB] A) β-catenin staining of cells treated with IWP2 + endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2 + endo-IWR1 have β-catenin on the cell membrane. Cells treated with LiCl have β-catenin in the nucleus, while untreated cells have β-catenin in the cytoplasm. B) p27 staining of cells treated with IWP2 + endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2 + endo-IWR1 have higher p27 expression in the nucleus, suggesting that the cells have exited the cell cycle. LiCl-treated or untreated cells have weak p27 expression in the nucleus. C) RPE65 and ZO1 tight junctions in cells treated with IWP2 + IWR1, IWP2, or LiCl. RPE65 is high in the cytoplasm of IWP2 + IWR1-treated and IWP2-treated cells, but low in untreated cells. No staining is seen in LiCl-treated cells. D) Electron microscopy images of functional tight junctions in cells treated with IWP2+IWR1, IWP2, or LiCl. [Figure 5CD]A) β-catenin staining of cells treated with IWP2 + endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2 + endo-IWR1 have β-catenin on the cell membrane. Cells treated with LiCl have β-catenin in the nucleus, while untreated cells have β-catenin in the cytoplasm. B) p27 staining of cells treated with IWP2 + endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2 + endo-IWR1 have higher p27 expression in the nucleus, suggesting that the cells have exited the cell cycle. LiCl-treated or untreated cells have weak p27 expression in the nucleus. C) RPE65 and ZO1 tight junctions in cells treated with IWP2 + IWR1, IWP2, or LiCl. RPE65 is high in the cytoplasm of IWP2 + IWR1-treated and IWP2-treated cells, but low in untreated cells. No staining is seen in LiCl-treated cells. D) Electron microscopy images of functional tight junctions in cells treated with IWP2+IWR1, IWP2, or LiCl.

[0026] [Figure 6A] A) Multi-operator RPE differentiation. Data shown represent RPE differentiations set up by multiple operators using an optimized protocol across three lines, as measured by flow cytometry for the RPE marker retinaldehyde-binding protein 1 (Cralbp). B) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, AMD Donor3C, and HLA LineA. C-D) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations. Data represent 10 differentiations performed by five operators on 28 iPSC lines derived from 13 donors. The percentage of Cralbp-positive cells increased to 90-100% compared to pre-purified cell populations of different purity. [Figure 6B]A) Multi-operator RPE differentiation. Data shown represent RPE differentiations set up by multiple operators using an optimized protocol across three lines, as measured by flow cytometry for the RPE marker retinaldehyde-binding protein 1 (Cralbp). B) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, AMD Donor3C, and HLA LineA. C-D) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations. Data represent 10 differentiations performed by five operators on 28 iPSC lines derived from 13 donors. The percentage of Cralbp-positive cells increased to 90-100% compared to pre-purified cell populations of different purity. [Figure 6C] A) Multi-operator RPE differentiation. Data shown represent RPE differentiations set up by multiple operators using an optimized protocol across three lines, as measured by flow cytometry for the RPE marker retinaldehyde-binding protein 1 (Cralbp). B) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, AMD Donor3C, and HLA LineA. C-D) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations. Data represent 10 differentiations performed by five operators on 28 iPSC lines derived from 13 donors. The percentage of Cralbp-positive cells increased to 90-100% compared to pre-purified cell populations of different purity. [Figure 6D]A) Multi-operator RPE differentiation. Data shown represent RPE differentiations set up by multiple operators using an optimized protocol across three lines, as measured by flow cytometry for the RPE marker retinaldehyde-binding protein 1 (Cralbp). B) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, AMD Donor3C, and HLA LineA. C-D) Reproducibility of the RPE differentiation protocol is shown across different starting cell line populations. Data represent 10 differentiations performed by five operators on 28 iPSC lines derived from 13 donors. The percentage of Cralbp-positive cells increased to 90-100% compared to pre-purified cell populations of different purity.

[0027] [Figure 7A]A) Functionality of barrier function in RPE cells generated using the RPE differentiation protocol is demonstrated by transepithelial potential (TEP) measurements of the ion gradient across the monolayer. B) Functionality of RPE cells treated with IWP2 or IWP2 + endo-IWR2. C-E) Transepithelial electrical resistance (TER) and TEP (light lines) for untreated, PGE2-treated, and IWP2 + endo-IWR1-treated cells. F) Functional response (TER) from cells matured with 50 μM PGE2 versus 100 μM PGE2 in RPE-MM + PGE2 medium from days 54 to 75 of the iPSC-derived differentiation protocol. Measures of TER progressively increased over the course of differentiation in iPSC-derived RPE cells cultured with 50 μM PGE2 in RPE-MM + PGE2 medium compared with 100 μM PGE2 from days 54 to 75 of the differentiation protocol. This demonstrates that increasing concentrations of PGE2 promote the maturation and functional efficiency of iPSC-derived RPE cultures. G) Purity of iPSC-derived RPE as measured by the expression of mature RPE markers at day 75 in cultures using 50 μM versus 100 μM PGE2, with the iPSC-derived RPE differentiation protocol initiated between days 54 and 75. Expression of Pmel17, Tryp1, and Cralbp (RPE-specific markers) is comparable to that of iPSC-derived RPE cultured using 50 μM PGE2, demonstrating that PGE2 promotes differentiation of iPSC-derived RPE across a range of concentrations. Expression of Best1 (a late mature RPE marker) is significantly higher in cells treated with 100 μM PGE2 compared to cells treated with 50 μM PGE2, demonstrating that increasing concentrations of PGE2 enhance the purity and maturation of iPSC-derived RPE. [Figure 7B] Same as above [Figure 7C] Same as above [Figure 7D] Same as above [Figure 7E] Same as above [Figure 7F] Same as above [Figure 7G] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0028] Description of exemplary embodiments Certain aspects of the present disclosure overcome several major problems associated with the current state of the art by providing a method for producing RPE cell populations from a starting cell suspension of pluripotent stem cells, preferably an essentially single-cell suspension of pluripotent stem cells. RPE cells can be derived from pluripotent stem cells, such as ES cells and iPSC cells; however, current methods rely on a starting population of embryoid bodies. In some embodiments, the present disclosure provides a highly efficient and reproducible method for differentiating pluripotent stem cells (PSCs) into functional, mature RPE cells without the use of embryoid bodies. Further embodiments and advantages are described below. I. Definition

[0029] The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, a purified cell population is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, or most preferably essentially free of other cell types.

[0030] As used herein, "essentially" or "essentially free" with respect to a particular component means that the particular component is not intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintentional incorporation of the composition is significantly less than 0.05%, preferably less than 0.01%. Most preferred is a composition in which the amount of the particular component cannot be detected using standard analytical methods.

[0031] As used herein, "a" or "an" may mean one or more. As used herein, in a claim(s), when used in conjunction with the word "comprising," the word "a" or "an" may mean one, or two or more.

[0032] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0033] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among study subjects.

[0034] The term "cell" is used herein to refer to a structural and functional unit of an organism that is capable of independent replication, is surrounded by a membrane, and contains biomolecules and genetic material. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fusion cell, a genetically modified cell, etc.).

[0035] The term "cell population" is used herein to refer to a group of cells, typically of a common type. A cell population may be derived from a common precursor or may contain more than one cell type. An "enriched" cell population refers to a cell population derived from a starting cell population (e.g., an unfractionated heterogeneous cell population) that contains a greater proportion of a particular cell type than the proportion of that cell type in the starting population. A cell population may be enriched for one or more cell types and depleted for one or more cell types.

[0036] The term "stem cell" as used herein refers to a cell that can differentiate into a variety of specialized cell types under appropriate conditions, and that can self-renew and remain essentially in an undifferentiated, pluripotent state under other appropriate conditions. The term "stem cell" also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state through the expression of specific transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs."

[0037] The term "pluripotency" refers to the property of cells that can differentiate into all other cell types in an organism, except for extraembryonic or placental cells. Pluripotent stem cells can differentiate into all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types) even after long-term culture. Pluripotent stem cells are embryonic stem cells derived from the inner cell mass of a blastocyst. In another embodiment, pluripotent stem cells are induced pluripotent stem cells that are derived by reprogramming somatic cells.

[0038] The term "differentiation" refers to the process by which unspecialized cells become more specialized, with changes in structural and / or functional properties. Mature cells typically have altered cell structure and tissue-specific proteins. More specifically, in the context of this method, it refers to the process of human stem cells acquiring the cell type of retinal pigment epithelium (RPE) cells, which have characteristics that indicate that said RPE cells are mature terminally differentiated cells.

[0039] As used herein, "undifferentiated" refers to cells that display characteristic markers and morphological features of undifferentiated cells that are clearly distinguishable from terminally differentiated cells of embryonic or adult origin.

[0040] "Embryoid bodies (EBs)" are aggregates of pluripotent stem cells that can differentiate into cells of the endoderm, mesoderm, and ectoderm. Spheroid structures are formed when pluripotent stem cells aggregate and allow non-adherent culture of EBs in suspension.

[0041] An "isolated" cell is one that has been substantially separated or purified from other cells in an organism or culture. An isolated cell can be, for example, at least 99%, at least 98% pure, at least 95% pure, or at least 90% pure.

[0042] "Embryo" refers to a mass of cells resulting from one or more divisions of a zygote or an activated oocyte having an artificially reprogrammed nucleus.

[0043] An "embryonic stem (ES) cell" is an undifferentiated pluripotent cell obtained from the inner cell mass of an early stage embryo, e.g., the blastocyst stage, or produced by artificial means (e.g., nuclear transfer), that can give rise to any differentiated cell type in the embryo or adult, including germ cells (e.g., sperm and eggs).

[0044] "Induced pluripotent stem cells (iPSCs)" are cells produced by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be produced using fetal somatic cells, postnatal somatic cells, neonatal somatic cells, juvenile somatic cells, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram the somatic cells into pluripotent stem cells.

[0045] "Allele" refers to one of two or more forms of a gene. Diploid organisms, such as humans, contain two copies of each chromosome, and therefore have one allele for each.

[0046] The term "homozygous" is defined as containing two identical alleles at a particular locus. The term "heterozygous" is defined as containing two different alleles at a particular locus.

[0047] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. A haplotype can be based on a set of single nucleotide polymorphisms (SNPs) on a single chromosome and / or alleles in the major histocompatibility complex.

[0048] As used herein, the term "haploidentical" is defined as a cell (e.g., iPSC cell) and a subject being treated sharing one or more major histocompatibility locus haplotypes. A subject's haplotype can be easily determined using assays well known in the art. Haploidentical iPSC cells can be autologous or allogeneic. Autologous cells grown in tissue culture and essentially differentiated into RPE cells are haploidentical to the subject.

[0049] "Substantially the same HLA type" indicates that the donor's HLA type matches that of the patient to such an extent that transplant cells obtained by inducing differentiation of iPSCs derived from the donor's somatic cells can be engrafted when transplanted into the patient.

[0050] "Super donor" as used herein refers to an individual homozygous for specific MHC class I and II genes. These homozygous individuals can function as super donors, and their cells (including tissues and other materials containing those cells) can be transplanted into individuals who are either homozygous or heterozygous for that haplotype. Super donors can be homozygous for the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus(s) allele(s), respectively.

[0051] "Feeder-free" or "feeder-independent" is used herein to refer to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as a substitute for a feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. In some cases, feeder-free cultures utilize an animal-based matrix (e.g., MATRIGEL™) or are grown on substrates such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to remain essentially undifferentiated without the need for a mouse fibroblast "feeder layer."

[0052] A "feeder layer" is defined herein as a coating layer of cells, such as on the bottom of a culture dish. Feeder cells may release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, may attach.

[0053] When used in reference to a medium, extracellular matrix, or culture condition, the term "defined" or "well-defined" refers to a medium, extracellular matrix, or culture condition in which the chemical composition and amount of almost all components are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Generally, a defined medium includes a basal medium (e.g., Dulbecco's Modified Eagle's Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640 containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and an energy source) supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin. An exemplary well-defined medium is Essential 8™ medium.

[0054] The term "xeno-free (XF)" when used with respect to a medium, extracellular matrix, or culture conditions refers to a medium, extracellular matrix, or culture conditions that are essentially free of xenogeneic animal-derived components. In the culture of human cells, any proteins from non-human animals, such as mice, are xenogeneic components. In certain embodiments, a xeno-free matrix may be essentially free of any non-human animal-derived components, thus excluding mouse feeder cells or MATRIGEL™. MATRIGEL™ is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (a tumor rich in extracellular matrix proteins) to contain laminin (a major component), collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0055] "KNOCKOUT™ Serum Replacement" refers herein to a serum-free formulation optimized for the growth and maintenance of undifferentiated cells, such as stem cells, in culture.

[0056] "Preconfluent" refers to a cell culture in which the percentage of the culture surface covered by cells is approximately 60-80%. Typically, preconfluent refers to a culture in which approximately 70% of the culture surface is covered by cells.

[0057] "Retina" refers to the light-sensitive layer of tissue that lines the inside of the eye.

[0058] "Retinal pigment epithelium" refers to the single layer of pigmented cells between the retina and the blood vessel-filled layer, the choroid.

[0059] "Retinal lineage cells" as used herein refers to cells that can give rise to or differentiate into RPE cells.

[0060] "Retinal induction medium (RIM)" herein refers to a growth medium containing a WNT pathway inhibitor and a BMP pathway inhibitor, which can result in the differentiation of PSCs into retinal lineage cells. RIM also contains a TGFβ pathway inhibitor.

[0061] "Retinal differentiation medium (RDM)" is defined herein as a medium containing a WNT pathway inhibitor, a BMP pathway inhibitor, and a MEK inhibitor, which allows retinal cells to differentiate. RDM also contains a TGFβ pathway inhibitor.

[0062] "Retinal medium (RM)" is defined as a growth medium for the culture of retinal cells, which growth medium contains activin A and nicotinamide.

[0063] "RPE maturation medium (RPE-MM)" refers herein to a medium for maturation of RPE cells, which contains taurine and hydrocortisone. RPE-MM also contains triiodothyronine. RPE-MM may also contain PD0325901 or PGE2.

[0064] "Mature" RPE cells herein refer to RPE cells in which the expression of immature RPE markers, such as Pax6, is downregulated and the expression of mature RPE markers, such as RPE65, is upregulated.

[0065] RPE cell "maturation" herein refers to the process of modulating the RPE developmental pathway to generate mature RPE cells. For example, modulation of ciliary function can result in RPE maturation.

[0066] As used herein, a "therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease or condition, is sufficient to effect such treatment.

[0067] An "inducer" is defined herein as a molecule that controls gene expression (e.g., activates a gene in a cell). Inducers can bind to repressors or activators. Inducers function by disabling repressors. II. Pluripotent stem cells A. Embryonic stem cells

[0068] ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiation capacity. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner cell mass cells on a feeder layer of non-growing cells. The replated cells continue to proliferate and can generate new colonies of ES cells, which can be removed, dissociated, replated, and grown. This process of "passaging" undifferentiated ES cells can be repeated multiple times to produce cell lines containing undifferentiated ES cells (U.S. Patent No. 5,843,780; U.S. Patent No. 6,200,806; U.S. Patent No. 7,029,913). ES cells have the potential to proliferate while maintaining their pluripotency. For example, ES cells are useful in research on cells and the genes that control cell differentiation. The pluripotency of ES cells, combined with genetic manipulation and selection, can be used for in vivo genetic analysis studies by generating transgenic, chimeric, and knockout mice.

[0069] Methods for generating mouse ES cells are well known. In one method, preimplantation blastocysts from 129 mouse strains are treated with mouse antisera to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse fetal fibroblasts in medium containing fetal bovine serum. The resulting colonies of undifferentiated ES cells are passaged on a feeder layer of mouse fetal fibroblasts in the presence of fetal bovine serum to generate a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000). In another method, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0070] Human ES cells can be produced or derived from zygotes or blastocyst-stage mammalian embryos produced by sperm-egg fusion, nuclear transfer, pathogenic mechanisms, or by reprogramming chromatin using previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000), followed by integration of the reprogrammed chromatin into the plasma membrane to produce embryonic cells. In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass cultured on a feeder layer of mouse fetal fibroblasts. Furthermore, cell clusters derived from the inner cell mass are chemically or mechanically dissociated and replated, and colonies with undifferentiated morphology are selected with a micropipette, dissociated, and replated (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown in the absence of serum by culturing the cells on a protein matrix (e.g., MATRIGEL) in the presence of a "conditioned" medium containing basic fibroblast growth factor. TM They can be grown without a feeder cell layer by culturing them on a layer of cellulose or laminin (Xu et al., 2001).

[0071] ES cells can also be derived from other organisms, including rhesus monkeys and marmosets, by previously described methods (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000), as well as from established mouse and human cell lines.For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14 and ACT30.As a further example, established mouse ES cell lines include the CGR8 cell line established from the inner cell mass of mouse strain 129 embryos, and the culture of CGR8 cells can be grown in the presence of LIF without the use of a feeder layer.

[0072] ES stem cells can be detected by protein markers, including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REX1. B. Induced pluripotent stem cells

[0073] Induction of pluripotency was achieved by reprogramming somatic cells with the introduction of pluripotency-associated transcription factors, initially using mouse cells in 2006 (Yamanaka et al., 2006) and human cells in 2007 (Yu et al., 2007; Takahashi et al., 2007). Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into almost any cell type. The use of iPSCs circumvents most of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients with iPSC-derived autologous transplants may not require lifelong immunosuppressive treatment to prevent graft rejection.

[0074] Any cell can be used as the starting point for iPSCs, except for germ cells. For example, cell types can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or gastric cells. T cells can also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648). There is no limit to the degree of cell differentiation or the age of the animal from which the cells are collected; undifferentiated progenitor cells (including somatic stem cells) and even terminally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cells themselves are RPE cells, such as human RPE cells. The RPE cells can be adult or fetal RPE cells. iPSCs can be grown under conditions known to differentiate human ES cells into specific cell types and express human ES cell markers, including SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0075] Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art.Those skilled in the art can easily produce induced pluripotent stem cells.For example, see US Patent Application Publication No. 20090246875, US Patent Application Publication No. 2010 / 0210014; US Patent Application Publication No. 20120276636; US Patent No. 8,058,065; US Patent No. 8,129,187; US Patent No. 8,278,620; PCT Publication No. WO2007 / 069666 and US Patent No. 8,268,620 (which are incorporated herein by reference).Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from somatic cells.In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc and Klf4 are utilized.

[0076] Cells are treated with nuclear reprogramming material (which is generally one or more factors that can induce iPSCs from somatic cells) or the nucleic acid encoding these materials (including in the form of vector).Nuclear reprogramming material generally includes at least Oct3 / 4, Klf4 and Sox2, or the nucleic acid encoding these molecules.P53 function inhibitor, L-myc, or the nucleic acid encoding L-myc, and Lin28 or Lin28b, or the nucleic acid encoding Lin28 or Lin28b, can be used as additional nuclear reprogramming material.Nanog can also be used for nuclear reprogramming.As disclosed in published U.S. Patent Application No. 20120196360, exemplary reprogramming factors for the production of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18; Klf4 can be replaced with Klf1, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil;(7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28;(8)Oct3 / 4 , Klf4, Sox2, L-Myc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oct (11) Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT; (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HPVI (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmil; (18) Oct3 / 4, Klf4, Sox2, Lin28 (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced with Esrrg).In one non-limiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are utilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized. See, for example, U.S. Patent No. 7,682,828, which is incorporated herein by reference. These factors include, but are not limited to, Oct3 / 4, Klf4, and Sox2. In other examples, factors include, but are not limited to, Oct3 / 4, Klf4, and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3 / 4, Klf4, Sox2, and Sal4 are utilized. Factors such as Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency and can be expressed from several different expression vectors. For example, integrative vectors such as EBV element-based systems can be used (U.S. Patent No. 8,546,140). In further embodiments, reprogramming proteins can be directly introduced into somatic cells by protein transduction. Reprogramming can further include contacting cells with one or more signaling receptors, including glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signal transduction inhibitors, leukemia inhibitory factor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These regulatory factors can include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that virtually any iPS cell or cell line can be used.

[0077] The mouse and human cDNA sequences of these nuclear reprogramming agents are available by reference to the NCBI accession numbers referenced in PCT Publication No. WO2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming agents or nucleic acids encoding these reprogramming agents are known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference.

[0078] Once induced, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with various media and techniques developed for culturing pluripotent stem cells, more specifically embryonic stem cells, as described in U.S. Patent No. 7,442,548 and U.S. Patent Publication No. 2003 / 0211603. In the case of mouse cells, culturing is carried out by adding leukemia inhibitory factor (LIF) as a differentiation inhibitor to a regular culture medium. In the case of human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. As known to those skilled in the art, other methods for culturing and maintaining iPSCs can be used.

[0079] In certain embodiments, undefined conditions may be used; for example, to maintain stem cells in an undifferentiated state, pluripotent cells may be cultured on fibroblast feeder cells or in a medium exposed to fibroblast feeder cells. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts treated with radiation or antibiotics to terminate cell division as feeder cells. Alternatively, pluripotent cells may be cultured using a defined feeder-independent culture system, such as TESR™ medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8™ medium (Chen et al., 2011), and maintained in an essentially undifferentiated state.

[0080] In some embodiments, iPSCs can be modified to express an exogenous nucleic acid (e.g., to include a tyrosinase enhancer operably linked to a promoter and a nucleic acid sequence encoding a first marker). The tyrosinase gene is disclosed, for example, in GENBANK® Accession No. 22173, available as of January 1, 2013. This sequence aligns to positions 5286971-5291691 (reverse orientation) on chromosome 7 of the C57BL / 6 mouse strain. A 4721 base pair sequence is sufficient for expression in RPE cells. See Murisier et al., Dev. Biol. 303:838-847, 2007, which is incorporated herein by reference. This construct is expressed in retinal pigment epithelial cells. Other enhancers can also be utilized. Other RPE-specific enhancers include D-MITF, DCT, TYRP1, RPE65, VMD2, MERTK, MYRIP and RAB27A. Suitable promoters include, but are not limited to, any promoter that is expressed in retinal pigment epithelial cells, including the tyrosinase promoter. The construct may also contain other elements, such as a ribosome binding site (internal ribosome binding sequence) for translation initiation and a transcription / translation terminator. Generally, it is advantageous to transfect cells with the construct. Suitable vectors for stable transfection include, but are not limited to, retroviral vectors, lentiviral vectors and Sendai virus.

[0081] Plasmids have been designed with several goals in mind, including achieving controlled high copy number, avoiding potential sources of plasmid instability in bacteria, and providing a means for selecting plasmids suitable for use in mammalian cells, including human cells. Particular attention has been paid to two requirements for plasmids for use in human cells. First, they must be suitable for maintenance and fermentation in E. coli so that large amounts of DNA can be produced and purified. Second, they must be safe and suitable for use in human patients and animals. The first requirement requires high-copy-number plasmids that are selectable for bacterial fermentation and can be relatively easily stably maintained during bacterial fermentation. The second requirement requires attention to elements such as selectable markers and other coding sequences. In some embodiments, the marker-encoding plasmid is composed of (1) a high-copy number origin of replication, (2) a selectable marker (e.g., but not limited to, the neo gene for antibiotic selection with kanamycin), (3) a transcription termination sequence containing a tyrosinase enhancer, and (4) a multiple cloning site for incorporating various nucleic acid cassettes; and (5) a nucleic acid sequence encoding the marker operably linked to a tyrosinase promoter. Numerous plasmid vectors are known in the art for introducing nucleic acids encoding proteins. These include, but are not limited to, the vectors disclosed in U.S. Pat. Nos. 6,103,470; 7,598,364; 7,989,425; and 6,416,998, which are incorporated herein by reference.

[0082] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Episomal gene delivery systems can be plasmids, Epstein-Barr virus (EBV)-based episomal vectors, yeast-based vectors, adenovirus-based vectors, simian virus 40 (SV40)-based episomal vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0083] Markers include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, firefly / renilla luciferase, or nanoluc), or other proteins. Markers can be proteins (including secreted proteins, cell surface proteins, or internal proteins; synthesized or taken up by the cell); nucleic acids (e.g., mRNA or enzymatically active nucleic acid molecules), or polysaccharides. Included are determinants of any such cellular components that are detectable by antibodies, lectins, probes, or nucleic acid amplification reactions and are specific for the marker of the cell type of interest. Markers can also be identified by biochemical or enzymatic assays, or biological responses that depend on the function of the gene product. Nucleic acid sequences encoding these markers can be operably linked to a tyrosinase enhancer. In addition, other genes, such as genes that can affect stem cells for RPE differentiation or RPE function or physiology or pathology, can be included. Thus, in some embodiments, nucleic acids encoding one or more of MITF, PAX6, TFEC, OTX2, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOLV4, ADAM10, CD55, CD59, and ARMS2 are included. 1.MHC haplotype match

[0084] The major histocompatibility complex (HLA) is the primary cause of immune rejection of allogeneic organ transplants. There are three major class I MHC haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA locus is highly polymorphic and distributed across 4 Mb on chromosome 6. This region is associated with autoimmune and infectious diseases, and HLA haplotype compatibility between donors and recipients can affect the clinical outcome of transplants. Therefore, the ability to haplotype HLA genes within this region is clinically important. HLA corresponding to MHC class I presents peptides from the inside of the cell, while HLA corresponding to MHC class II presents antigens from the outside of the cell to T lymphocytes. MHC haplotype incompatibility between the graft and the host triggers an immune response against the graft, leading to its rejection. Therefore, to prevent rejection, patients can be treated with immunosuppressants. HLA-matched stem cell lines can overcome the risk of immune rejection.

[0085] Given the importance of HLA in transplantation, HLA loci are typically typed by serology and PCR to identify favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be achieved using a complement-mediated lymphocytotoxicity test with purified T or B lymphocytes. This procedure is primarily used for matching HLA-A and HLA-B loci. Molecular-based tissue typing can often be more accurate than serological testing. Low-resolution molecular methods, such as SSOP (sequence-specific oligonucleotide probe) methods, which test PCR products against a series of oligonucleotide probes, can be used to identify HLA antigens; these methods are currently the most common methods used for class II HLA typing. High-resolution techniques, such as SSP (sequence-specific primer) methods, which utilize allele-specific primers for PCR amplification, can identify specific MHC alleles.

[0086] If donor cells are HLA homozygous (i.e., contain identical alleles for each antigen-presenting protein), the MHC compatibility between the donor and recipient is significantly increased. Most individuals are heterozygous for MHC class I and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can function as super donors, and grafts made from their cells can be transplanted into all individuals who are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype that is frequently found in the population, these cells can be applied to transplantation therapy in a large number of individuals.

[0087] Therefore, iPSCs can be produced from the somatic cells of the subject to be treated or another subject with the same or substantially the same HLA type as that of the patient. In some cases, the donor's major HLA (for example, the three major loci of HLA-A, HLA-B, and HLA-DR) is identical to the recipient's major HLA. In some cases, the somatic cell donor can be a super donor; thus, iPSCs derived from an MHC homozygous super donor can be used to generate RPE cells. Thus, iPSCs derived from a super donor can be transplanted into a subject that is either homozygous or heterozygous for its haplotype. For example, iPSCs can be homozygous for two HLA alleles, such as HLA-A and HLA-B. In this way, iPSCs produced from a super donor can be used in the methods disclosed herein to produce RPE cells that can potentially "match" with a large number of potential recipients. 2. Episomal Vectors

[0088] In certain embodiments, reprogramming factors are expressed from expression cassettes contained in one or more exogenous episomal genetic elements (see U.S. Patent Publication No. 2010 / 0003757, which is incorporated herein by reference). Thus, iPSCs can essentially be free of exogenous genetic elements, such as retroviral or lentiviral vector elements. These iPSCs are prepared by using extrachromosomally replicating vectors (i.e., episomal vectors), which are vectors that can replicate episomally, to create iPSCs that are essentially free of exogenous vectors or viral elements (see U.S. Patent No. 8,546,140, ​​which is incorporated herein by reference; Yu et al., 2009). Some DNA viruses, such as adenovirus, simian vacuolar virus 40 (SV40) or bovine papillomavirus (BPV), or budding yeast ARS (autonomously replicating sequence)-containing plasmids, replicate extrachromosomally or episomally in mammalian cells. These episomal plasmids essentially avoid all of the drawbacks associated with integrative vectors (Bode et al., 2001). For example, lymphotrophic herpes virus-based vectors (including those defined above) or Epstein-Barr virus (EBV) can replicate extrachromosomally and support the delivery of reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1 or their mutants or functional equivalents. An additional advantage of episomal vectors is that after introduction into cells, exogenous elements disappear over time, resulting in self-sustaining iPSCs essentially free of these elements.

[0089] Other extrachromosomal vectors include other lymphotropic herpesvirus-based vectors. Lymphotropic herpesviruses are herpesviruses that replicate in lymphoblasts (e.g., human B lymphoblasts) and become plasmids during part of their natural life cycle. Herpes simplex virus (HSV) is not a "lymphotropic" herpesvirus. Exemplary lymphotropic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); herpesvirus saimiri (HS) and Marek's disease virus (MDV). Other sources of episome-based vectors are also contemplated (e.g., yeast ARS, adenovirus, SV40 or BPV). C. Somatic cell nuclear transfer

[0090] Pluripotent stem cells can be prepared by somatic cell nuclear transfer (SCNT). SCNT involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, donor fibroblast nuclei from rhesus monkey skin fibroblasts are introduced into the cytoplasm of spindle-free, mature metaphase II rhesus monkey oocytes by electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to ionomycin and then incubated until the blastocyst stage. Embryonic stem cell lines are then obtained by culturing the inner cell mass of selected blastocysts. These embryonic stem cell lines exhibit normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo. III.Retinal pigment epithelial cells

[0091] RPE cells are produced in the method disclosed herein. The cells of the retina that are directly sensitive to light are photoreceptors. Photoreceptors are light-sensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, photoreceptors convert incident light energy focused by the lens into electrical signals, which are then sent to the brain via the optic nerve. Vertebrates have two types of photoreceptors, including cones and rods. Cones are adapted to detect detailed central vision and color vision and function well in bright light. Rods are responsible for peripheral vision and scotopic vision. The neural signals from rods and cones are processed by other neurons in the retina.

[0092] The retinal pigment epithelium (RPE) acts as a barrier between the bloodstream and the retina and interacts closely with photoreceptors to maintain visual function. It is composed of a single layer of hexagonal cells densely packed with melanin granules, which absorb light energy reaching the retina. The primary functions of specialized RPE cells include transporting nutrients such as glucose, retinol, and fatty acids from the blood to photoreceptors; transporting water, metabolic end products, and ions from the subretinal space to the blood; absorbing light and protecting against photooxidation; re-isomerizing all-trans-retinol to 11-cis-retinal; phagocytosis of shed photoreceptor membranes; and secreting various factors essential for the structural integrity of the retina.

[0093] The retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, Best vitelliform macular dystrophy gene (VMD2), and pigment epithelium-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with many vision-altering conditions, including retinal pigment epithelial detachment, dysplasia, atrophy, retinopathies, retinitis pigmentosa, macular dystrophies, or degeneration.

[0094] Retinal pigment epithelial (RPE) cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually recognized by their cobblestone morphology and initial appearance of pigment. In addition, differentiated RPE cells possess transepithelial resistance (TER) and transepithelial potential (TEP) across the monolayer (TER > 100 ohms.cm2; TEP > 2 mV), transport fluid and CO2 from the apical to the basal side, and regulate polarized cytokine secretion.

[0095] RPE cells express several proteins that can serve as markers for detection using methodologies such as immunocytochemistry, Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). For example, RPE-specific markers include cellular retinaldehyde-binding protein (CRALBP), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP-1), retinal pigment epithelium-specific 65 kDa protein (RPE65), premelanosome protein (PMEL17), bestrophin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-2. Specifically, expression of these genes is approximately 100-1000-fold lower in RPE cells than in ES or iPSC cells, as assessed by quantitative RT-PCR.

[0096] RPE cell markers can be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis using sequence-specific primers in standard amplification methods using publicly available sequence data (GENBANK®). Expression of a tissue-specific marker, when detected at the protein or mRNA level, is considered positive if its level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, more specifically, more than 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more than that of control cells, such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0097] Dysfunction, injury, and loss of RPE cells are factors in many ocular diseases and disorders, including age-related macular degeneration (AMD), hereditary macular degeneration (including Best's disease), and retinitis pigmentosa. A possible treatment for such diseases is the transplantation of RPE cells into the retina of individuals in need of such treatment. It is speculated that the replacement of RPE cells by such transplantation may slow, halt, or reverse deterioration, improve retinal function, and prevent blindness resulting from such conditions. However, obtaining RPE cells directly from human donors and embryos is difficult. A. Induction of RPE cells from embryoid bodies of PSCs

[0098] iPSCs reprogrammed using known reprogramming factors can give rise to neuronal lineage ocular cells, including RPE cells (Hirami et al., 2009). PCT Publication No. 2014 / 121077 (incorporated herein by reference in its entirety) discloses a method in which embryoid bodies (EBs) produced from iPSCs are treated with Wnt and Nodal antagonists in suspension culture to induce the expression of retinal progenitor cell markers. This publication also discloses a method for inducing RPE cells from iPSCs by the differentiation process of iPSC EBs into RPE cell-enriched cultures. For example, embryoid bodies are produced from iPSCs by adding a rho-associated coiled-coil kinase (ROCK) inhibitor and cultured in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor. The EBs are plated onto MATRIGEL™-coated tissue culture media in a second medium containing a basic fibroblast growth factor (bFGF), a Nodal pathway inhibitor, about 20 ng to about 90 ng of Noggin, and about 1% to about 5% knockout serum replacement to form differentiated RPE cells. The differentiated RPE cells are cultured in a third medium containing activin and WNT3a. The RPE cells are then cultured in RPE medium containing about 5% fetal serum, a canonical WNT inhibitor, a non-canonical WNT inhibitor, and an inhibitor of the sonic hedgehog and FGF pathways to produce human RPE cells.

[0099] There are some drawbacks to using EBs for the production of differentiated cell types. For example, EB production is an inconsistent and unreproducible process due to variable efficiency. The size and shape of EBs produced from iPSCs or ES cells are not uniform, and the production of EBs also involves a rate-limiting centrifugation process. The present disclosure provides a method that enables the large-scale production of iPSCs or ES-derived cells required for clinical, research, or therapeutic applications, and is EB-independent. B. Derivation of RPE Cells from Virtually Single-Cell PSCs

[0100] In some embodiments, a method for producing RPE cells from an essentially single-cell suspension of pluripotent stem cells (PSCs), such as human iPSCs, is provided. In some embodiments, PSCs are cultured to preconfluence to prevent any cell aggregation. In certain embodiments, PSCs are dissociated by incubation with a cell dissociation enzyme, such as TRYPSIN™ or TRYPLE™. PSCs can also be dissociated into an essentially single-cell suspension by pipetting. In addition, blebbistatin (e.g., about 2.5 μM) can be added to the culture medium while the cells are not attached to the culture vessel to increase PSC survival after dissociation into single cells. Alternatively, a ROCK inhibitor can be used instead of blebbistatin to increase PSC survival after dissociation into single cells.

[0101] To efficiently differentiate RPE cells from single-cell PSCs, an accurate injection density can increase RPE differentiation efficiency. Therefore, single-cell suspensions of PSCs are generally counted before seeding. For example, single-cell suspensions of PSCs are counted using a hemocytometer or an automated cell counter, such as a VICELL® or TC20. Cells can be diluted to a cell density of approximately 10,000 to 500,000 cells / mL, approximately 50,000 to 200,000 cells / mL, or approximately 75,000 to 150,000 cells / mL. In a non-limiting example, a single-cell suspension of PSCs is diluted to a density of approximately 100,000 cells / mL in a well-defined culture medium, such as ESSENTIAL 8™ (E8™) medium.

[0102] Once a single-cell suspension of PSCs is obtained at a known cell density, the cells are generally seeded into an appropriate culture vessel, such as a tissue culture plate, such as a flask, a 6-well plate, a 24-well plate, or a 96-well plate. The culture vessel used to culture the cell(s) can include, but is not limited to, a flask, a flask for tissue culture, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, a CELLSTACK® chamber, a culture bag, and a roller bottle, as long as stem cells can be cultured therein. Cells can be cultured in volumes of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel can be a bioreactor, which can refer to any ex vivo device or system that supports a biologically active environment in which cells can grow. The bioreactor can have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0103] In certain embodiments, PSCs, such as iPSCs, are plated at a cell density appropriate for efficient differentiation. Generally, cells are plated at a density of about 1,000 to about 75,000 cells / cm. 2 For example, about 5,000 to about 40,000 cells / cm 2In a 6-well plate, cells can be seeded at a cell density of about 50,000 to about 400,000 cells per well. In an exemplary method, cells are seeded at a cell density of about 100,000, about 150,000, about 200,000, about 250,000, about 300,000, or about 350,000 cells per well, e.g., about 200,000 cells per well.

[0104] In order to promote cell adhesion while maintaining cell viability, PSCs, such as iPSCs, are generally cultured on culture plates coated with one or more cell adhesion proteins. For example, preferred cell adhesion proteins include extracellular matrix proteins, such as vitronectin, laminin, collagen, and / or fibronectin, which can be used to coat culture surfaces as a means of providing solid support for the growth of pluripotent cells. The term "extracellular matrix" is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and cullin. In an exemplary method, PSCs are grown on culture plates coated with vitronectin or fibronectin. In some embodiments, the cell adhesion protein is a human protein.

[0105] Extracellular matrix (ECM) proteins can be of natural origin, purified from human or animal tissue, or alternatively, they can be genetically engineered recombinant proteins or essentially synthetic. ECM proteins can be in the form of whole proteins or peptide fragments, native or engineered. Examples of ECM proteins that may be useful in matrices for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition comprises synthetically produced peptide fragments of fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is xeno-free. For example, xeno-free matrices for culturing human cells may use matrix components of human origin, which may exclude any non-human animal components.

[0106] In some embodiments, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0107] Cells, such as RPE cells or PSCs, can be cultured with the nutrients necessary to support the growth of each specific cell population. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidants, pyruvate, buffering agents, and inorganic salts. To enhance stem cell growth, an exemplary growth medium contains a minimal essential medium, such as Dulbecco's Modified Eagle's Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins. Examples of minimal essential medium include, but are not limited to, Minimal Essential Medium Eagle's (MEM) Alpha Medium, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. Additionally, the minimal essential medium may be supplemented with additives such as horse serum, calf serum, or fetal bovine serum. Alternatively, the medium may be serum-free. In other cases, the growth medium may contain a "knockout serum replacement," referred to herein as a serum-free formulation optimized for growing and maintaining undifferentiated cells, such as stem cells, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a well-defined, feeder-free medium.

[0108] Thus, after plating, single-cell PSCs are generally cultured in a well-defined culture medium. In certain embodiments, about 18-24 hours after plating, the medium is aspirated and fresh medium, such as E8™ medium, is added to the culture. In certain embodiments, after plating, single-cell PSCs are cultured in a well-defined culture medium for about 1, 2, or 3 days. Preferably, single-cell PSCs are cultured in a well-defined culture medium for about 2 days before proceeding with the differentiation process.

[0109] In some embodiments, the medium may or may not contain a serum replacement. Serum replacements may include materials that suitably contain albumin (e.g., lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Serum replacements may be prepared, for example, by the methods disclosed in International Publication No. WO 98 / 30679. Alternatively, for greater convenience, any commercially available material may be used. Commercially available materials include KNOCKOUT™ Serum Replacement (KSR), Chemically Defined Lipid Concentrate (Gibco), and GLUTAMAX™ (Gibco).

[0110] Other culture conditions can be defined as appropriate. For example, the culture temperature may be about 30 to 40°C, e.g., at least or about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration may be about 1 to 10%, e.g., about 2 to 5%, or any derivable range therein. The oxygen tension may be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any derivable range therein. a. Differentiation medium Retinal induction medium

[0111] After the single-cell PSCs are attached to the culture plate, the cells are preferably cultured in a retinal induction medium to initiate the differentiation process into retinal lineage cells. The retinal induction medium (RIM) contains a WNT pathway inhibitor, which can lead to the differentiation of PSCs into retinal lineage cells. The RIM further contains a TGFβ pathway inhibitor and a BMP pathway inhibitor. An exemplary RIM medium is shown in Table 3.

[0112] The RIM may comprise DMEM and F12 in a ratio of about 1:1. In an exemplary method, the RIM may include a WNT pathway inhibitor such as CKI-7, a BMP pathway inhibitor such as LDN193189, and a TGFβ pathway inhibitor such as SB431542. For example, the RIM may contain about 5 nM to about 50 nM, e.g., about 10 nM, LDN193189, about 0.1 μM to about 5 μM, e.g., about 0.5 μM, CKI-7, and about 0.5 μM to about 10 μM, e.g., about 1 μM, SB431542. In addition, the RIM may include a knockout serum replacement, e.g., about 1% to about 5% MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin growth factor 1 (IGF1). Preferably, the IGF1 is animal-free IGF1 (AF-IGF1), and is contained in the RIM at about 0.1 ng / mL to about 10 ng / mL, e.g., about 1 ng / mL. For example, the medium is aspirated daily and replaced with fresh RIM. Generally, cells are cultured in the RIM for about 1 to about 5 days, e.g., about 1, 2, 3, 4, or 5 days, e.g., about 2 days, to produce retinal cells. Retinal differentiation medium

[0113] Retinal cells can then be cultured in retinal differentiation medium (RDM) for further differentiation. RDM contains a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. In one embodiment, RDM contains a WNT pathway inhibitor such as CKI-7, a BMP pathway inhibitor such as LDN193189, a TGFβ pathway inhibitor such as SB431542, and a MEK inhibitor such as PD0325901. Alternatively, RDM can contain a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a bFGF inhibitor. Generally, the concentrations of the Wnt pathway inhibitor, BMP pathway inhibitor, and TGFβ pathway inhibitor in RDM are, for example, about 9 to about 11 times higher, e.g., about 10 times higher, than in RIM. In an exemplary method, the RDM comprises about 50 nM to about 200 nM, e.g., about 100 nM, LDN193189, about 1 μM to about 10 μM, e.g., about 5 μM, CKI-7, about 1 μM to about 50 μM, e.g., about 10 μM, SB431542, and about 0.1 μM to about 10 μM, e.g., about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM, PD0325901. One exemplary RDM medium is shown in Table 3.

[0114] Typically, RDM contains approximately 1:1 DMEM and F12, knockout serum replacement (e.g., about 1% to about 5%, e.g., about 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., about 1 ng / mL to about 50 ng / mL, e.g., about 10 ng / mL). In certain methods, cells are fed daily with fresh RDM after aspirating the medium from the previous day. Typically, cells are cultured in RDM for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, e.g., about 7 days, to induce differentiation of retinal cells. Retinal culture medium

[0115] The differentiated retinal cells can then be further differentiated by culturing them in retinal medium (RM). The retinal medium contains activin A and may further contain nicotinamide. The RM may contain about 50 to about 200 ng / mL, e.g., about 100 ng / mL, of activin A and about 1 mM to about 50 mM, e.g., about 10 mM, of nicotinamide. Alternatively, the RM may contain other TGF-β pathway activators, such as GDF1 and / or WNT pathway activators, e.g., WAY-316606, IQ1, QS11, SB-216763, BIO(6-bromoindirubin-3'-oxime), or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. Alternatively, the RM may further contain WNT3a. An exemplary RM medium is shown in Table 3.

[0116] The RM may contain DMEM and F12 in a ratio of about 1:1, about 1% to about 5%, e.g., about 1.5%, of knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. The medium may be changed daily at room temperature in the RM. Typically, cells are cultured in the RM for about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days, e.g., about 10 days, to induce differentiation of RPE cells. RPE maturation medium

[0117] For further differentiation of RPE cells, the cells are preferably cultured in RPE maturation medium (RPE-MM). An exemplary RPE-MM medium is shown in Table 3. RPE maturation medium may contain about 100 μg / mL to about 300 μg / mL, e.g., about 250 μg / mL, taurine, about 10 μg / L to about 30 μg / L, e.g., about 20 μg / L, hydrocortisone, and about 0.001 μg / L to about 0.1 μg / L, e.g., about 0.013 μg / L, triiodothyronine. In addition, RPE-MM may contain MEM alpha, N-2 supplement, MEM non-essential amino acids (NEAA), and sodium pyruvate, as well as fetal bovine serum (e.g., about 0.5% to about 10%, e.g., about 1% to about 5%). The medium may be changed every other day in room temperature RPE-MM. Generally, cells are cultured in RPE-MM for about 5 to about 10 days, e.g., about 5 days. The cells can then be dissociated using a cell dissociation enzyme or the like, replated, and cultured for an additional period of time, e.g., about 5 to about 30 days, e.g., about 15 to 20 days, for further differentiation into RPE cells. In a further embodiment, the RPE-MM does not contain a WNT pathway inhibitor. At this stage, the RPE cells can be cryopreserved. b. RPE cell maturation

[0118] RPE cells can then be cultured in RPE-MM for a continued period of time for maturation. In some embodiments, RPE cells are grown in wells, such as 6-well, 12-well, or 24-well plates, or in 10-cm plates. RPE cells can be maintained in RPE medium for about 4 to about 10 weeks, for example, about 6 to 8 weeks, such as 6, 7, or 8 weeks. In an exemplary method for continued maturation of RPE cells, cells can be dissociated with a cell dissociation enzyme, such as TRYPLE™, and replated onto a degradable scaffold assembly, such as in a specialized SNAPWELL™ design, in RPE-MM containing an MEK inhibitor, such as PD0325901, for about 1 to 2 weeks. Alternatively, the RPE-MM can contain a bFGF inhibitor instead of the MEK inhibitor. Methods for culturing RPE cells on degradable scaffolds are taught and described in PCT Publication No. WO 2014 / 121077, which is incorporated herein by reference in its entirety. Briefly, the key elements of the method are the CORNING® COSTAR® SNAPWELL™ plate, a bioinert O-ring, and a biodegradable scaffold. The SNAPWELL™ plate provides the structure and platform for the biodegradable scaffold. The microporous membrane creating the apical and basal sides not only provides support for the scaffold but is ideal for separating distinct sides of the polarized layer of cells. The ability of the SNAPWELL™ insert to detach the membrane allows the support ring of the insert to be used as an anchor for the scaffold. The resulting monolayer of differentiated, polarized, and confluent functional RPE cells can be cryopreserved at this stage (e.g., in xeno-free CS10 medium).

[0119] In some embodiments, mature RPE cells can be further developed into a functional RPE cell monolayer (which functions as intact RPE tissue) by continued culture in RPE-MM containing additional chemicals or small molecules that promote RPE maturation. For example, these small molecules are primary cilia inducers, such as prostaglandin E2 (PGE2) or aphidicolin. PGE2 can be added to the culture medium at a concentration of about 25 μM to about 250 μM, e.g., about 50 μM to about 100 μM. Alternatively, the RPE-MM can contain a canonical WNT pathway inhibitor. Exemplary canonical WNT pathway inhibitors are N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). Cells can be cultured in this medium for an additional period, e.g., about 1 to about 5 weeks, e.g., about 2 to 4 weeks, to obtain a mature, functional RPE cell monolayer. Thus, the disclosed method provides mature RPE cells derived from a single-cell suspension of pluripotent cells that can be consistently reproduced on a large scale for clinical use. c. Cryopreservation of RPE cells

[0120] Retinal pigment epithelial cells produced by the methods disclosed herein can be cryopreserved. See, for example, PCT Publication No. 2012 / 149484A2, which is incorporated herein by reference. Cells can be cryopreserved with or without a substrate. In some embodiments, storage temperatures range from about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and overlapping ranges. In some embodiments, lower temperatures are used for storage (e.g., maintenance) of cryopreserved cells. In some embodiments, liquid nitrogen (or other similar coolant) is used to store the cells. In further embodiments, the cells are stored for more than about 6 hours. In further embodiments, the cells are stored for about 72 hours. In some embodiments, the cells are stored for 48 hours to about 1 week. In still other embodiments, the cells are stored for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In further embodiments, the cells are stored for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. Cells can also be stored for longer periods. Cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.

[0121] In some embodiments, an additional cryoprotectant may be used. For example, cells may be cryopreserved in a cryopreservation solution containing one or more cryoprotectants, such as DM80, serum albumin, such as human or bovine serum albumin. In certain embodiments, the solution contains about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution contains about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In certain embodiments, the solution contains 2.5% DMSO. In another specific embodiment, the solution contains 10% DMSO.

[0122] During cryopreservation, cells can be cooled, for example, at about 1°C / min. In some embodiments, the cryopreservation temperature is about -80°C to about -180°C or about -125°C to about -140°C. In some embodiments, cells are cooled to 4°C before being cooled at about 1°C / min. Cryopreserved cells can be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, once the cells reach about -80°C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be performed using a controlled-rate freezer. Cryopreserved cells can be thawed, for example, at a temperature of about 25°C to about 40°C, typically about 37°C. d. inhibitor WNT pathway inhibitors

[0123] WNTs are a family of highly conserved secreted signaling molecules that regulate cell-cell interactions and are related to the Drosophila segment polarity gene wingless. In humans, WNT family genes encode 38-43 kDa cysteine-rich glycoproteins. WNT proteins have a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence (see, e.g., Shimizu et al., Cell Growth Differ 8:1349-1358 (1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote the stabilization of cytoplasmic β-catenin, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain WNT proteins has been shown to be associated with certain cancers.

[0124] WNT inhibitor in this specification generally refers to WNT inhibitor.Therefore, WNT inhibitor refers to any inhibitor of WNT family protein member, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11 and Wnt16.A certain embodiment of this method relates to the WNT inhibitor in differentiation medium. Examples of suitable WNT inhibitors already known in the art include N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl Examples of WNT inhibitors include: 2,4-diaminoquinazolinone (2-furanyl)methylenehydrazide (PNU74654), quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1) and secreted Frizzled-related protein (SFRP1) 1. In addition, WNT inhibitors may include antibodies against WNT, dominant-negative variants of WNT, and siRNA and antisense nucleic acids that suppress WNT expression. Inhibition of WNT can also be achieved using RNA-mediated interference (RNAi). BMP pathway inhibitors

[0125] Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β (TGFβ) superfamily. BMPs are thought to constitute a group of important morphogenetic signals that organize structures throughout the body. The important function of BMP signals in physiology is highlighted by the numerous roles for dysregulated BMP signaling in pathological processes.

[0126] BMP pathway inhibitors can include inhibitors of BMP signaling generally, or inhibitors specific for BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Exemplary BMP inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-[4-(1 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML347). TGFβ pathway inhibitors

[0127] Transforming growth factor-β (TGF-β) is a secreted protein that regulates proliferation, cell differentiation, and other functions in most cells. It is a type of cytokine that plays a role in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. TGF-β exists in at least three isoforms, designated TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor-β superfamily, which also includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplegic, and Vg-1.

[0128] TGFβ pathway inhibitors can generally include any inhibitor of TGFβ signal transduction. For example, TGFβ pathway inhibitors include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-ylie-butyl-3H-imidazol-4-yl)quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl) ... 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, Left-Right Determinant (Lefty), 3-(6-methyl-2-pyridinyl) 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712) or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox). MEK inhibitors

[0129] MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 or MEK2. They can be used to affect the MAPK / ERK pathway. For example, MEK inhibitors include N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N -(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), and 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244). bFGF inhibitors

[0130] Basic fibroblast growth factor (bFGF, also known as FGF2 or FGF-β) is a member of the fibroblast growth factor family. bFGF is present in the basement membrane and in the subendothelial extracellular matrix of blood vessels. In addition, bFGF is a common component of human ESC culture medium, which is necessary for cells to remain in an undifferentiated state.

[0131] The bFGF inhibitor in this specification generally refers to the bFGF inhibitor.For example, bFGF inhibitor includes but is not limited to N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea (PD173074), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1-tert-butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyridin [2,3-d]pyrimidin-7-yl]urea (PD161570), 6-(2,6-dichlorophenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one dihydrochloride hydrate (PD166285), N-[2-amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)-urea (PD166866), and MK-2206. IV. Use of Retinal Pigment Epithelial Cells

[0132] Certain embodiments provide methods for producing RPE or RPE-enriched cell populations that can be used for many important research, development and commercial purposes.

[0133] In some embodiments, the methods disclosed herein involve the production of at least or about 10 RPE cells that comprise at least or about 90% (e.g., at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivable therein). 6 , 10 7 , 10 8 , 5x10 8 , 10 9 , 10 10 This results in a cell population of cells (or any derivable range therein).

[0134] In certain embodiments, the starting cells for the method are at least or about 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The starting cells may include at least or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The seeding density may be 100 cells / ml, or any range derivable therein.

[0135] The RPE cell produced by the method disclosed herein can be used in any method and application currently known in the art for RPE cell.For example, the method for evaluating a compound can be provided, comprising measuring the pharmacological or toxicological properties of the compound on RPE cell.The method for evaluating a compound for the effect on RPE cell can also be provided, comprising: a) contacting the RPE cell provided herein with the compound; and b) measuring the effect of the compound on the RPE cell. A. Screening of Test Compounds

[0136] RPE cells can be commercially used to screen factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (e.g., culture conditions or manipulations) that affect the characteristics of such cells and their various progeny. For example, test compounds can be chemical compounds, small molecules, polypeptides, growth factors, cytokines, or other biological agents.

[0137] In one embodiment, the method includes contacting RPE cells with a test agent and determining whether the test agent modulates the activity or function of RPE cells in the population. In some applications, the screening assay is used to identify agents that modulate the proliferation of RPE cells or change the differentiation of RPE cells. The screening assay can be performed in vitro or in vivo. Methods for screening and identifying ophthalmic or RPE agents include those suitable for high-throughput screening. For example, for the identification of potential therapeutic molecules, RPE cells can be positioned or placed on culture dishes, flasks, roller bottles, or plates (e.g., single multi-well dishes or dishes, such as 8-, 16-, 32-, 64-, 96-, 384-, and 1536-well multi-well plates or dishes), optionally in a defined location. Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0138] Other screening applications involve testing pharmaceutical compounds for their effect on the maintenance or repair of retinal tissue. Screening can be done because compounds are designed to have a pharmacological effect on cells, or because compounds designed to have an effect elsewhere may have unintended side effects on cells of this tissue type. B. Treatment and Transplantation

[0139] Other embodiments may also provide for the use of RPE cells to enhance the maintenance of ocular tissue as well as repair for any condition requiring repair, including retinal degeneration or severe injury.

[0140] To determine the suitability of cell composition for therapeutic administration, cells can first be tested in suitable animal models.In one embodiment, RPE cells are evaluated for their ability to survive and maintain their phenotype in vivo.Cell composition is administered to immunodeficient animals (for example, nude mice or animals that are immunodeficient chemically or by irradiation).After a period of growth, tissue is harvested and evaluated for whether pluripotent stem cell-derived cells still exist.

[0141] Several animals are available for testing the suitability of RPE cell compositions. For example, the Royal College of Surgeons (RCS) rat is a well-known model of retinal dystrophy (Lund et al., 2006). In addition, the suitability and survival of RPE cells can be determined by Matrigel transplantation (e.g., subcutaneous or subretinal) in immunodeficient animals such as NOG mice (Kanemura et al., 2014).

[0142] The human RPE cells described herein or pharmaceutical compositions comprising these cells can be used to manufacture medicaments for treating conditions in patients in need thereof. RPE cells can be cryopreserved in advance. In certain embodiments, the disclosed RPE cells are derived from iPSCs and can be used to provide "personalized medicine" to patients with ocular diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct disease-causing mutations, differentiate into RPE, and form RPE tissue. This RPE tissue can be used to replace the patient's endogenous degenerated RPE. Alternatively, iPSCs generated from healthy donors or HLA-homozygous "super donors" can be used. To create an anti-inflammatory and immunosuppressive environment in vivo, RPE cells can be treated in vitro with specific factors, such as pigment epithelium-derived factor (PEDF), transforming growth factor (TGF)-β, and / or retinoic acid.

[0143] Various ocular conditions can be treated or prevented by introducing RPE cells obtained using the methods disclosed herein. Conditions include retinal diseases or disorders that are generally associated with retinal dysfunction or deterioration, retinal damage, and / or loss of retinal pigment epithelium. Conditions that can be treated include, but are not limited to, retinal degenerative diseases, such as Stargardt's macular dystrophy, retinitis pigmentosa, macular degeneration (e.g., age-related macular degeneration), glaucoma, and diabetic retinopathy. Additional conditions include Leber's congenital amaurosis, hereditary or acquired macular degeneration, Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, other dystrophies of the RPE, and RPE and retinal damage caused by any one of light injury, laser injury, inflammatory injury, infectious injury, radiation injury, neovascular injury, or traumatic injury. In certain embodiments, methods for treating or preventing conditions characterized by retinal degeneration are provided, comprising administering an effective amount of a composition comprising RPE cells to a subject in need of treatment or prevention of a condition characterized by retinal degeneration. These methods may include selecting a subject with one or more of these conditions and administering a therapeutically effective amount of RPE cells sufficient to treat the condition and / or ameliorate the symptoms of the condition. RPE cells can be transplanted in various formats. For example, RPE cells can be introduced to the target site in the form of a cell suspension, or can be attached as a monolayer on a matrix, extracellular matrix, or substrate, such as a biodegradable polymer, or a combination thereof. RPE cells can also be transplanted (co-transplanted) with other retinal cells, such as photoreceptors. In some embodiments, RPE cells are autologous because they are produced from iPSCs derived from the subject to be treated. In other embodiments, RPE cells are produced from an MHC-matched donor.

[0144] In some embodiments, RPE cells can be used for autologous RPE transplantation to subjects suitable for regenerative medicine. RPE cells can be transplanted in combination with other retinal cells, such as photoreceptor-bearing cells. Transplantation of RPE cells produced by the disclosed method can be performed by various techniques known in the art. For example, methods for performing RPE transplantation are described in U.S. Patent Nos. 5,962,027 and 6,045,791 (each of which is incorporated herein by reference in its entirety). According to one embodiment, transplantation is performed by pars plana vitrectomy (PPV). The procedure involves performing a pana vitrectomy (pana vitrectomy) followed by delivery of the cells into the subretinal space through a small retinal opening or by direct injection. RPE cells can be introduced to the target site in the form of a cell suspension, adhered to a matrix, such as an extracellular matrix, or provided on a substrate such as a biodegradable polymer. RPE cells can also be transplanted with other cells, such as photoreceptor-bearing retinal cells (co-transplantation). Thus, compositions containing RPE cells obtained by the methods disclosed herein are provided. In some embodiments, these RPE cells contain a tyrosinase enhancer operably linked to a promoter and a nucleic acid encoding a marker. In other embodiments, the RPE cells also contain a second constitutive promoter operably linked to a nucleic acid encoding a second marker.

[0145] Pharmaceutical compositions of RPE cells are produced by the methods disclosed herein. These compositions contain at least about 1 x 10 3 RPE cells, approximately 1x10 4 RPE cells, approximately 1x10 5 RPE cells, approximately 1x10 6 RPE cells, approximately 1x10 7 RPE cells, approximately 1x10 8 RPE cells, or approximately 1x10 9The composition may comprise RPE cells. In certain embodiments, the composition is a substantially purified preparation (relative to non-RPE cells) comprising differentiated RPE cells produced by the methods disclosed herein. Also provided are compositions comprising a scaffold, e.g., a polymeric carrier and / or extracellular matrix, and an effective amount of RPE cells produced by the methods disclosed herein. For example, the cells are provided as a monolayer of cells. The matrix material is generally physiologically acceptable and suitable for use in vivo. For example, physiologically acceptable materials include, but are not limited to, absorbable and / or non-absorbable solid matrix materials, such as small intestinal submucosa (SIS), cross-linked or non-cross-linked alginate, hydrocolloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, fleeces, and bioadhesives.

[0146] Suitable polymer carriers also include porous meshes or sponges formed from synthetic or natural polymers and polymer solutions. For example, the matrix is ​​a polymer mesh or sponge or a polymer hydrogel. Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; and polysaccharides such as alginate and hyaluronic acid polymers. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include hydroxy acid polymers such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-glycolic acid (PGLA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof. Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohol.

[0147] Polymers capable of forming malleable ionically or covalently crosslinked hydrogels can be used. Hydrogels are materials formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. Examples of materials that can be used to form hydrogels include polysaccharides such as alginate, polyphosphazine, and polyacrylate (which are ionically crosslinked), or block copolymers such as PLURON1CS™ or TETRON1CS™, polyethylene oxide-polypropylene glycol block copolymers (which are crosslinked by temperature or H, respectively). Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen.

[0148] The pharmaceutical composition may optionally be packaged in a suitable container with written instructions for the desired purpose (e.g., reconstitution of RPE cell function to ameliorate a disease or abnormality of retinal tissue). In some embodiments, the RPE cells produced by the disclosed methods may be engineered to form RPE, which may be used to replace degenerated RPE in a subject in need thereof. C. Distribution for Commercial, Therapeutic, and Research Purposes

[0149] In some embodiments, a reagent system is provided that includes a set or combination of cells, including an RPE-enriched cell population, present at any time during manufacture, distribution, or use. The cell set includes any combination of the cell populations disclosed herein, in combination with undifferentiated pluripotent stem cells or other differentiated cell types, often sharing the same genome. Each cell type may be packaged together or in separate containers, in the same facility or at different locations, at the same or different times, under the control of the same entity or different entities that share a business relationship.

[0150] The pharmaceutical composition may optionally be packaged in a suitable container with written instructions for a desired purpose (eg, reconstitution of RPE cell function to ameliorate disease or injury of ocular tissue). V. Kit

[0151] In some embodiments, kits are provided that may include one or more media and components for, for example, the production of RPE cells. The reagent system may be packaged in either aqueous or lyophilized form, as appropriate. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed (preferably, appropriately aliquoted). If there is more than one component in the kit, the kit also generally contains a second, third, or additional container into which additional components may be separately placed. However, various combinations of components may be included in vials. The components of the kit may be provided as dry powder(s). If the reagents and / or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in a separate container means. The kit also typically includes a means for containing the kit component(s) in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. The kit may also include instructions for use in printed or electronic, eg, digital, format. [Example]

[0152] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be recognized by those of skill in the art that the techniques disclosed in the examples which follow are techniques discovered by the inventors to function well in the practice of the invention and, therefore, can be considered to constitute preferred modes for practicing the same. However, those of skill in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and which can still obtain like or similar results without departing from the spirit and scope of the invention. Example 1 - Preparation of starting pluripotent stem cell populations

[0153] The starting population of RPE cells can be derived from pluripotent stem cells such as ES cells and iPSCs.In an exemplary method, RPE cells are derived from the human iPSCs that are reprogrammed from somatic cells by methods known in the art, such as United States Patent No. 8,546,140, ​​United States Patent No. 8,741,648, United States Patent No. 8,691,574, United States Patent Application Publication No. 20090246875, United States Patent No. 8,278,104, United States Patent No. 9,005,967, United States Patent No. 8,058,065, United States Patent No. 8,129,187, PCT Publication No. WO2007 / 069666A1, United States Patent No. 8,183,038 and United States Patent No. 8,268,620 (which are incorporated herein by reference). In one exemplary method, nuclear programming factors Oct4, Sox2, c-Myc, and Klf4 were used to generate pluripotent stem cells from somatic cells. In another exemplary method, nuclear programming factors Oct4, Sox2, Nanog, Lin28, L-Myc, and SV40 large T antigen were used to generate pluripotent stem cells from somatic cells.

[0154] iPSCs were grown on vitronectin-coated plates in a well-defined culture medium, such as ESSENTIAL 8™ (E8™) medium, without mouse or human feeder layers. Vitronectin was diluted 1:200 in DPBS without calcium or magnesium, and the culture plates were coated with vitronectin and incubated at room temperature for approximately 1 hour. iPSCs were split when preconfluent and prevented from overgrowth to prevent unhealthy and / or differentiated cells (Figure 1A).

[0155] To induce RPE cells, iPSCs were dissociated into a single-cell suspension to remove any aggregates or embryoid bodies. To obtain a single-cell suspension, cells were washed with DPBS and incubated in a cell dissociation enzyme, such as TRYPLE™, at 37°C for approximately 10 minutes. Cells were then detached by pipetting using a serological pipette, and the cell suspension was collected in a conical tube. If the cells did not detach with gentle pipetting, the culture was incubated for a longer period, e.g., 2–3 minutes. To collect all cells, the culture vessel was washed with room temperature E8™ medium, and the medium was then added to the tube containing the cell suspension. Additionally, blebbistatin (e.g., 2.5 μM) was added to the E8™ medium while the cells were not attached to the culture vessel to increase PSC survival after dissociation into single cells. To collect the cells, they were centrifuged at 400 × g for approximately 5 minutes, the supernatant was aspirated, and the cells were resuspended in an appropriate volume of E8™ medium.

[0156] To efficiently differentiate RPE cells from single-cell iPSCs, accurately count the injection density of single-cell iPSCs by an automated cell counter such as VICELL™ and incubate at approximately 1 x 10 in room temperature E8™ medium. 5The cells were diluted to a cell suspension of 100,000 cells / mL. Once a single-cell suspension of iPSCs was obtained at a known cell density, the cells were placed into an appropriate culture vessel, such as a vitronectin-coated 6-well plate. The cells were seeded at a cell density of approximately 200,000 cells / well and placed in a humidified incubator at 37°C. After approximately 18–24 hours, the medium was aspirated and fresh E8™ medium was added to the culture. After seeding, the cells were cultured in E8™ medium for approximately 2 days to allow for proper adhesion to the plate. Example 2 - Differentiation of iPSCs into RPE cells

[0157] As in Example 1, single-cell iPSCs seeded at the appropriate cell density were cultured for approximately two days and then cultured in various differentiation media to induce RPE cells. On day 3, the E8™ medium was aspirated and room-temperature retinal induction medium (RIM) (e.g., Table 3) was added. Briefly, RIM contained approximately a 1:1 ratio of DMEM and F12, knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, RIM contained a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and insulin growth factor 1 (IGF1). The medium was aspirated daily, and fresh RIM was added to the cells. Cells were cultured in RIM for approximately two to four days.

[0158] Next, cells were cultured in retinal differentiation medium (RDM) for approximately 7–14 days. Briefly, RDM (Table 2) contained approximately 1:1 DMEM and F12, knockout serum replacement, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, RDM contained a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFβ pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD325901). The concentrations of the Wnt pathway inhibitor, BMP pathway inhibitor, and TGFβ pathway inhibitor were 10-fold higher in RDM compared to RIM. The medium was aspirated daily, and room-temperature RDM was added to the cells to produce differentiated retinal cells.

[0159] To induce RPE cells, the cells were then cultured in retinal medium (RM) for 7–10 days. RM contained approximately a 1:1 ratio of DMEM and F12, knockout serum replacement, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, RM contained nicotinamide and activin A. The medium was replaced daily with room-temperature RM to obtain RPE cells.

[0160] For RPE cell maturation, cells were cultured in RPE maturation medium (RPE-MM) for 5–10 days. RPE-MM (Table 2) contained MEM alpha, fetal bovine serum, N-2 supplement, MEM NEAA, and sodium pyruvate. In addition, RPE-MM contained taurine, hydrocortisone, and 3,3',5-triiodo-L-thyronine (Figure 1C). The medium was replaced with room-temperature RPE-MM every other day. Cells were then dissociated with cell dissociation enzymes and replated on vitronectin-coated plates. At this stage, induced PRE cells can be cryopreserved in xeno-free CS10 medium. Plated cells are cultured for approximately 15 more days to continue RPE maturation. Example 3 - RPE Cell Maturation

[0161] For continued maturation of the RPE cells produced in Example 2, the cells were dissociated with cell dissociation enzymes such as TRYPLE™ and replated for 1-2 weeks on specialized SNAPWELL™-designed degradable scaffold assemblies in RPE-MM containing a MEK inhibitor such as PD0325901. This resulted in a differentiated, polarized, and confluent monolayer of functional RPE cells (Figure 1D), which could be cryopreserved at this stage in xeno-free CS10 medium.

[0162] By continuing to culture in RPE-MM containing additional small molecules, such as primary cilia inducers like PGE2 or aphidicolin, mature RPE cells were further developed into functional RPE cell monolayers that function as intact RPE tissue. Without being bound by theory, these primary cilia inducers suppress the canonical WNT pathway, inducing cell cycle exit in RPE cells and inducing apical-basal polarization in the RPE monolayer. Alternatively, RPE maturation can be induced by canonical WNT pathway inhibitors, such as IWP2 and endo-IWR1, which also induce cell cycle exit in RPE cells and promote RPE maturation. Cells were further cultured in this medium for 2–3 weeks to obtain functional mature RPE cell monolayers. Thus, the disclosed method provides mature RPE cells derived from pluripotent cells that can be consistently reproduced on a large scale for clinical use. Example 4 - Cryopreservation of RPE cells

[0163] For cryopreservation of differentiated RPE cells in Example 2, the medium was aspirated and the cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS). The cells were then incubated with cell dissociation enzymes, and the cell suspension was pipetted into a conical tube. The cells were centrifuged, the supernatant was aspirated, and the cells were resuspended in room temperature RPE-MM. The cell suspension was then filtered through a STERIFLIP® cell strainer, and the cells were counted. The cells were then centrifuged and collected at an appropriate density (e.g., 1 × 10 7 The cells were resuspended in cold CryoStor® CS10 at 1000x the cell count (1000x the cells / mL). The cell suspension was aliquoted into pre-labeled cryovials, which were placed in a freezing container and transferred to a -80°C freezer for 12-24 hours. The vials were then transferred to liquid nitrogen for storage. Example 5 - Enrichment of a starting population of RPE cells by MACS depletion of contaminating non-RPE cells and CD24, CD56 and / or CD90 depletion

[0164] The population of RPE cells obtained in Examples 2 or 3 may contain residual contaminating non-RPE cells and immature RPE cells (collectively referred to as "contaminating cells"), both of which can be separated and removed to obtain a mature RPE-enriched cell population. Contaminating cells can be removed from the culture by various methods, such as magnetic-activated cell sorting (MACS®), fluorescence-activated cell sorting (FACS), or single-cell sorting. The MACS® method, which is known in the art to separate various cell populations according to surface antigens, was used to separate the contaminating cells from the desired, more mature RPE cells.

[0165] Contaminating cells in the starting population of RPE cells have specific cell surface markers that can be used to separate them from the desired mature RPE cells. For example, CD24, CD56, and / or CD90 are cell surface antigens expressed on (but not limited to) pluripotent stem cells and other neural cell types. CD24 is a glycoprotein expressed on the surface of pluripotent stem cells, some B lymphocytes, and differentiating neuroblasts. CD56, or neural cell adhesion molecule (NCAM), is a glycoprotein expressed on the surface of neurons and natural killer cells. CD90, or Thy-1, is a marker expressed on the surface of various stem cells and neurons. Expression of CD24, CD56, and / or CD90 is lost during differentiation of stem cells into many mature cell types, including RPE cells. Therefore, removal of cells positive for CD24, CD56, and / or CD90 results in the depletion of remaining contaminating cells.

[0166] To perform the isolation technique, it is desirable to dissociate the starting population of RPE cells into a single-cell suspension for sorting (e.g., MACS) to be performed. For previously cryopreserved cells, the cells must be thawed and reseeded. To obtain a single-cell suspension from cells in adherent culture, the cells were washed (e.g., with DPBS) and a cell dissociation enzyme (e.g., TRYPLE™) was added. After incubating the cells at 37°C for approximately 5 minutes, the vessel was gently tapped to detach neuronal clusters. The cells were washed twice with DPBS and a cell dissociation enzyme (e.g., TRYPLE™) was added. After incubating the cells at 37°C for approximately 30 minutes, the cell suspension was collected in RPE-MM plating medium and centrifuged at 400 × g for 5 minutes. The cell pellet was resuspended in RPE-MM plating medium, and the cell suspension was filtered through a cell strainer (e.g., a 20 μM SteriFlip cell strainer) to dissociate any remaining cell clusters. The cell suspension was counted for viable cells (e.g., using a ViCell counter) to obtain a cell concentration. From the counted cell suspension, a single cell suspension was obtained that could be used for sorting or flow cytometry purity assays.

[0167] To remove contaminating cells from the starting population of RPE cells, MACS was used to deplete CD24-, CD56-, and / or CD90-positive cells. After dissociating cells from the starting population of RPE cells into a single cell suspension, the cells were diluted to, for example, 1 x 10 7 The cells were resuspended in MACS buffer at 1.11 x 10 cells / mL. An example of a MACS buffer is included in Table 3. Next, the cells were stained with anti-CD24, anti-CD56, and / or anti-CD90 antibodies (each diluted 1:500) and incubated at 4°C for 20 minutes to allow the antibodies to bind to the antigens on the cells. The antibodies used should be tagged with a label (e.g., FITC) that binds to the secondary antibody. After incubation, 20 mL of MACS buffer was added, and the cells were centrifuged at 400 x g for 5 minutes. The cell pellet was resuspended in 20 mL of MACS buffer, mixed vigorously, and centrifuged at 400 x g for 5 minutes to remove unbound antibody. The cell pellet was then resuspended (e.g., 1.11 x 10 cells / mL) in MACS buffer.8 The cells were resuspended in MACS buffer (cells / mL) and diluted (1:10) secondary antibody (e.g., anti-FITC) coated microbeads were added and the cells were incubated for 20 min at 4 °C. After incubation, the cells were washed with MACS buffer to remove unbound microbeads, up to 1.25 × 10 8 The cells were resuspended in 500 μL of MACS buffer. The cell suspension was transferred to an LD column placed in a strong magnetic field. Cells expressing the antigens CD24, CD56, and / or CD90 attached to the microbeads remained on the column. The LD column was washed twice with MACS buffer. Unlabeled cells that did not express the antigens CD24, CD56, and / or CD90 were allowed to flow through and collected. For further characterization and culture, the collected unlabeled cell suspension was centrifuged (400 × g for 5 min) and reseeded in RPE-MM plating medium. An aliquot of the cell suspension was used for flow cytometry purity assays. Thus, MACS cell sorting yielded an enriched RPE cell population depleted of cells positive for CD24, CD56, and / or CD90. It should be noted that the use of this method is not limited to the starting population obtained from the method detailed in Example 2, but can be used to remove contaminating cells from RPE populations produced by other methods, such as, but not limited to, the methods described in U.S. Patent Application No. 12 / 523,444 and U.S. Patent Application No. 14 / 405,730. [Table 1] The pre-sort percentage of cells positive for RPE markers is that present in the starting population of RPE cells in Example 2. Combined depletion of CD24-positive cells and CD56-positive cells resulted in greater enrichment of RPE cells than depletion of CD24-positive cells alone. Depletion of CD24-positive cells, CD56-positive cells, and CD90-positive cells resulted in greater than 99% RPE cells in the cell population. Example 6 - Flow cytometry purity assay for characterization of RPE-enriched cell populations

[0168] Before and after MACS sorting, RPE cells were characterized (e.g., pre- and post-sorting) by a panel of relevant markers, including BEST1, CRALBP, TYRP1, PMEL17, MAP2, NES, and MITF. Flow cytometry purity assays were performed before and after MACS depletion of CD24-, CD56-, and / or CD90-positive cells to obtain measurements of the percentage of cells positive for each marker (Table 1) (Figures 2 and 3).

[0169] A flow cytometry purity assay was performed to determine the percentage of RPE cells obtained by the disclosed sorting method. Aliquots of the cell suspension collected from the MACS assay (2 x 10 per sample in 5 mL FACS tubes) were collected. 6 The cells (cells) were centrifuged at 400 x g for 3 minutes. The cell pellet was resuspended in 1 mL of staining solution (e.g., Live-Dead Red staining solution) and incubated in the dark at room temperature for 15 minutes. After incubation, 2 mL of wash buffer was added, and the cells were centrifuged at 400 x g for 3 minutes to remove unbound staining solution. The cell pellet was resuspended in fixation buffer and incubated in the dark at room temperature for 15 minutes. After incubation, 2 mL of wash buffer was added, and the cells were centrifuged at 400 x g for 3 minutes, and the supernatant was decanted. The cell pellet was resuspended in 2 mL of wash buffer to obtain 1 x 10 cells per mL of suspension. 6The cells were collected and 200 μL of the cell suspension was transferred to a FACS tube. 2 mL of Perm buffer was added to each tube, and the cells were centrifuged at 400 × g for 3 minutes. Primary antibodies against RPE-specific markers were diluted in Perm buffer, and 100 μL of the diluted antibody solution was added to each tube. After overnight incubation at 4°C in the dark, the cells were washed twice with 2 mL of Perm buffer. Secondary antibody solution was added to each tube, and the cells were incubated at room temperature in the dark for 1–2 hours. After incubation, the cells were washed twice with Perm buffer, centrifuged (at 400 × g for 3 minutes), and resuspended in 100 μL of wash buffer for flow cytometry analysis. Flow cytometry analysis was performed by methods known to those skilled in the art, such as U.S. Patent No. 8,682,810 and Herzenberg et al., 2006 (both of which are incorporated herein by reference), to obtain the percentage of cells positive for each marker tested (Table 1). Flow cytometry purity assays showed that MACS sorting, which depletes contaminating cells positive for CD24, CD56, and / or CD90, resulted in an RPE cell-enriched population (95-99%) compared to the starting cell population (78.6%), as determined by the BEST1 marker. Example 7 - Alternative methods for differentiation of RPE cells

[0170] For the methods described in Examples 2 and 3, including PD0325901 at a concentration of 1 μM in the culture medium over a specific time frame, starting from day 2 after iPSC plating and continuing through the end of the differentiation process (including post-MACS culture), can improve both the purity of the RPE population (meaning a reduction in contaminating cells) and the maturation of the resulting RPE population. The inclusion of 1 μM PD0325901 was shown to improve both the purity and maturation of the RPE population not only in the RDM but also when included in the RPE-MM (approximately days 42-50) of the RPE process described herein. Example 8 - Alternative methods for differentiation of RPE cells

[0171] With respect to the methods described in Examples 2 and 3, reducing the percentage of fetal bovine serum in the RPE-MM and RPE-MM plating medium from 5% to 0.5-1% can improve both the purity of the RPE population (which means a reduction in contaminating cells) and the maturation of the resulting RPE population. Example 9 - Functionality of mature RPE cells

[0172] To analyze the mature RPE cells generated from PGE2 treatment, immunostaining of the RPE monolayer was performed to confirm ZO1 staining in iPSC-RPE cells and the hexagonal structure of tight junctions by transmission electron microscopy (Figure 4A-4C) (Figure 4D). Staining demonstrated that PGE2-treated RPE cells had decreased β-catenin and increased RPE65. In addition, treatment with IWP2 + endo-IWR1 or IWP2 also resulted in decreased β-catenin (Figure 5A) and increased RPE65 (Figure 5C). The combination of IWP2 + endo-IWR1 was found to be more effective than IWP2 or endo-IWR1 alone. Thus, treatment with PGE2, IWP2, or IWP2 + endo-IWR1 resulted in mature RPE cells.

[0173] To assess the barrier function of RPE cells generated by the methods of the present invention, transepithelial potential (TEP) measured the ion gradient across the monolayer generated by energy-driven ion pumps that control cross-cellular passage, and transepithelial electrical resistance (TER) measured the resistance of substances passing through the paracellular space, primarily via the fine structure of tight junctions (Figure 7A).

[0174] The functionality of mature RPE cells treated with IWP2 or endo-IWR1 was also characterized. TEP and TER measurements of untreated RPE cells versus RPE cells treated with PGE2 or IWP2 + endo-IWR1 demonstrate increased functionality of treated mature RPE cells (Figures 7C-7E). Next, we tested whether increasing the concentration of PGE2 in the RPE-MM + PGE2 medium from 50 μM to 100 μM would improve both the purity of the RPE population (i.e., reduction of contaminating cells) and the maturation of the resulting RPE population. To determine the maturation and functionality of 50 μM vs. 100 μM PGE2-treated cultures, we measured barrier function in terms of transepithelial electrical resistance (TER) (Figure 7F) to compare the resistance of substances through the paracellular space as described in Example 9. To determine the percentage of pure RPE cells obtained after treatment of iPSC-derived RPE cultures with 50 μM or 100 μM PGE2 in RPE-MM+PGE2 medium, a flow cytometry purity assay was performed for RPE-specific markers as described in Example 6 (Figure 7G). The results showed that higher concentrations of the primary cilium inducer PGE2 promoted both the purity and maturation of the RPE population during iPSC-derived RPE differentiation. Example 10 - Reproducibility of the RPE differentiation method

[0175] To test the reproducibility of the RPE differentiation process, multiple operators differentiated three iPSC lines into RPE cells (Figure 6A). The average purity of the resulting RPE cells was characterized by measuring the RPE marker retinaldehyde-binding protein 1 (Craplbp) by flow cytometry (Table 2). The RPE differentiation process was found to be highly reproducible across different starting cell populations and different operators. Additionally, reproducibility was confirmed by RPE differentiation from different starting cell lines, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, and HLA LineA (Figure 6B). HLA LineA (21525.102) is an iPSC line generated from a donor homozygous for HLA-A*01 and HLA-B*08, which may provide a beneficial match for 11.38% of the US population. Furthermore, we have successfully produced RPE using this process using iPSC lines generated from donors homozygous for HLA-A*03 and HLA-B*07, designated HLA Line C (21526.101), which potentially provides a beneficial match to 7.63% of the US population. The HLA Line A (21525.102) and HLA Line C (21526.101) homozygous for HLA-A and HLA-B are the property of Cellular Dynamics International, Inc. Additionally, we further confirmed reproducibility in 10 RPE differentiations performed on 28 iPSC lines from 13 donors by measuring the percentage of Cralbp-positive cells before and after purification (Figure 6C-D). Although the percentage of Cralbp-positive cells after RPE differentiation varied, MACS purification consistently yielded purity above 95%, and in most cases nearly 100%. Thus, the present RPE differentiation method has a distinct advantage over any method of producing RPE cells from embryoid bodies, as it provides more consistent and reproducible results across donor genotypes when performed by different operators. [Table 2] Example 11 - Materials and Methods

[0176] The materials used in Examples 1 to 10 are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10]

[0177] Flow cytometry wash buffer was prepared by adding 20 mL of FBS to 1000 mL of DPBS (i.e., calcium and magnesium free). The buffer was filter sterilized and can be stored at 4°C for up to 4 weeks.

[0178] Flow cytometry Perm buffer was prepared by adding 20 mL of FBS to 1000 mL of DPBS (i.e., calcium and magnesium free). 1 gram of saponin was added and mixed thoroughly. The buffer was filter sterilized and can be stored at 4°C for up to 4 weeks.

[0179] Flow cytometry Live-Dead Red staining solution was prepared by diluting Live-Dead staining solution 1:1000 in DPBS (i.e., calcium and magnesium free). Assay 1 x 10 cells. 6 1 mL of staining solution was prepared per specimen. The staining solution was freshly prepared before use.

[0180] Flow cytometry fixation buffer was prepared by adding 1 mL of 36.5% formaldehyde to 8.1 mL of DPBS (i.e., calcium and magnesium free). 6 1 mL of staining solution was prepared per sample. The buffer solution was freshly prepared before use.

[0181] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and steps or sequence of steps of those methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein while the same or similar results are achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. 1. A method for producing human retinal pigment epithelial (RPE) cells, comprising: a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) dissociated into single cells; b) Incubate the iPSCs in retinal induction medium at 1,000-40,000 cells / cm ２ to initiate differentiation of the cells into retinal lineage cells, wherein the retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, and insulin growth factor 1 (IGF1); c) further culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells, wherein the retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor, and IGF1; d) culturing the cells obtained in step c) in retinal culture medium to form differentiated RPE cells; and e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells. Including, A method wherein the method does not include the formation of embryoid bodies.

2. 10. The method of claim 1, wherein the iPSCs of step (b) are cultured on a matrix.

3. The method of claim 2 , wherein the matrix comprises at least one recombinant cell adhesion protein.

4. The method of claim 3, wherein the at least one cell adhesion protein is laminin, vitronectin, or fibronectin.

5. The method of claim 3 or 4, wherein the at least one cell adhesion protein is human.

6. The method of any one of claims 1 to 5, further comprising, following step (e), dissociating the RPE cells obtained in step e), reseeding the RPE cells, and culturing the RPE cells in the RPE maturation medium.

7. The method according to claim 6, further comprising dissociating the RPE cells obtained by the culture process described in claim 6, and cryopreserving the RPE cells.

8. 8. The method of claim 7, further comprising reseeding the RPE cells onto the degradable scaffold in the RPE maturation medium.

9. The method of claim 7 or 8, wherein the RPE maturation medium comprises a MEK inhibitor.

10. The method of any one of claims 1 to 9, further comprising culturing the RPE cells in the RPE maturation medium comprising at least one primary cilium inducer, thereby producing mature RPE cells.

11. 11. The method of claim 10, wherein the at least one primary cilium inducer is prostaglandin E2 (PGE2) or aphidicolin.

12. 10. The method of any one of claims 1 to 9, further comprising culturing the RPE cells in the RPE maturation medium comprising N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2) and / or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1).

13. The method of any one of claims 1 to 12, further comprising cryopreserving the RPE cells.

14. The method of any one of claims 1 to 10, wherein the starting population of iPSCs in step (a) are pre-confluent cells dissociated into single cells.

15. The iPSCs of step (b) are cultured at a density of 5,000 to 40,000 cells / cm ２ The method according to any one of claims 1 to 14, wherein the cells are cultured at an initial cell density of

16. The method of any one of claims 1 to 15, wherein the iPSCs are cultured without a feeder layer.

17. The method of any one of claims 1 to 15, wherein the iPSCs are cultured in a well-defined culture medium.

18. The method of any one of claims 1 to 15, wherein the iPSCs are cultured in a xeno-free culture medium.

19. The WNT pathway inhibitor is selected from the group consisting of N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydrazine, and the like.

19. The method of any one of claims 6 to 18, wherein the compound is selected from the group consisting of 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1), and secreted Frizzled-related protein (SFRP1).

20. The TGFβ pathway inhibitor is selected from the group consisting of 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-yely-butyl-3H-imidazol-4-yl)quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-yely-butyl-3H-imidazol-4-yl)quinoxaline (SB525334). yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, left-right determinant (Lefty), 3-(6-methyl-2-pyridinyl)-N-phenyl-4 -(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW788388), 4-[3 20. The method according to any one of claims 6 to 19, wherein the compound is 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox).

21. The MEK inhibitor is selected from the group consisting of N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)- 21. The method of any one of claims 7 to 20, wherein the compound is N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119) or 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).

22. The BMP pathway inhibitor is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-[4-(1-methylethoxy)phenyl] 22. The method of any one of claims 6 to 21, wherein the compound is 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML347).

23. The method of any one of claims 6 to 21, wherein the BMP pathway inhibitor in the retinal induction medium is LDN193189.

24. The method of any one of claims 7 to 23, wherein the BMP pathway inhibitor is LDN193189 and the MEK inhibitor is PD0325901 in the retinal differentiation medium.

25. 25. The method of any one of claims 1 to 24, wherein the starting population of iPSCs is MHC haplotype-matched to a subject in need thereof.

26. 26. The method of any one of claims 1 to 25, wherein the starting population of iPSCs is homozygous for at least one HLA allele.

27. 27. The method of claim 26, wherein the at least one HLA allele is HLA-A, HLA-B, or HLA-DR.

28. 1. A method for producing human retinal pigment epithelial (RPE) cells, comprising: a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) dissociated into single cells in a well-defined medium; b) culturing the iPSCs on laminin, vitronectin, or a combination thereof in retinal induction medium containing LDN193189, CKI-7, IGF1, and SB431542 at a density of 1,000 to 40,000 cells / cm ２ to initiate differentiation of the cells into retinal lineage cells; c) further culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, CKI-7, SB431542, IGF1 and PD0325901 to further differentiate the retinal lineage cells; d) culturing the cells obtained in step c) in a retinal medium containing nicotinamide and activin A to form differentiated RPE cells; and e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells. Including, A method wherein the method does not include the formation of embryoid bodies.

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