Method for reproducible differentiation of clinical-grade retinal pigment epithelium 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 priority to U.S. Provisional Patent Application No. 62 / 215,579, filed on 8 September 2015, the entire contents of which are incorporated herein by reference.

[0002] Parties to the joint research agreement This invention is the result of activities carried out within the scope of a collaborative research agreement that was in effect at the time the invention was made. The parties to the said collaborative research agreement were the U.S. Government, the U.S. Department of Health and Human Services represented by the National Eye Institute, the National Institutes of Health, and Cellular Dynamics International, Inc. [Background technology]

[0003] background 1. Field This disclosure relates in general to the field of stem cell biology. More specifically, it relates to an efficient method for producing stem cell-derived retinal pigment epithelial cell populations for use as cell therapy.

[0004] 2. Explanation of related technologies The retina is a photosensitive tissue layer that covers the inner surface of the eye. Photoreceptor cells (either rods or cones) in the retina are directly sensitive to light, converting chemical photosignals into electrical events that trigger nerve impulses. The retinal pigment epithelium (RPE) is a layer of pigment cells that form the blood-retinal barrier. RPE cells play a crucial role in maintaining visual function, as well as in the transport of ions, water, and metabolic end products from the subretinal space to the blood (Strauss et al., 2005). Furthermore, RPE cells establish the eye's immune privilege 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, as well as 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 the various types of terminally differentiated blood cells. Embryonic stem (ES) cells originate from the embryo and are pluripotent, thus having the ability to develop into any organ or tissue type, including RPE cells.

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

[0007] iPSCs have been shown to give rise to ophthalmic cells, including RPE cells (Hirami et al., 2009). However, all known techniques for producing iPSC or ESC-derived RPE cells rely on the use of embryoid body starting populations. There is no efficient method for large-scale production of iPSC or ESC-derived RPE cells needed for therapeutics, screening assays, retinal disease models, and RPE biology studies. [Overview of the project] [Means for solving the problem]

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

[0009] In some embodiments, methods for differentiating pluripotent stem cells into retinal pigment epithelium (RPE) cells are provided. For example, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). In one embodiment, a method for producing human RPE cells, comprising: a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) that dissociate into substantially 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 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 formation of embryoid bodies. In some aspects, the RPE cells are cryopreserved after production.

[0010] In certain aspects, the 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, the at least one cell adhesion protein is human.

[0011] In certain aspects, the iPSCs are cultured without a feeder layer. In some aspects, the iPSCs are cultured in a fully defined culture medium. In another aspect, the iPSCs are cultured in a xenofree culture medium.

[0012] In a further embodiment, 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]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-fur The following group is selected: [nyl)methylene]hydrazide (PNU74654) 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-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, a WNT pathway inhibitor is CKI-7.In a particular embodiment, BMP pathway inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), 4-[6- These are [4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347). For example, a 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-yl-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 determination factor (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-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712) or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4-yl)-1,5-naphthyridine (RepSox). For example, the 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 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]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-fur The following group is selected: [nyl)methylene]hydrazide (PNU74654) 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-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, a WNT pathway inhibitor is CKI-7.In a particular embodiment, BMP pathway inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), 4-[6- These are [4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347). For example, a BMP pathway inhibitor is LDN193189.In a particular aspect, TGFβ pathway inhibitors include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazole-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-yl-butyl- 3H-imidazole-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-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-imidazole-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benz These include amide (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridinyl-2-yl)-1H-pyrazole-4-yl)-1,5-naphthiridine (RepSox). For example, the TGFβ pathway inhibitor is SB431542.In some aspects, 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]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), and 6-(4-bromo-2-fluoroanilino)-7-f These are ruolo-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, a MEK inhibitor is PD0325901. In certain embodiments, the retinal differentiation medium comprises LDN193189, CKI-7, SB431542, and PD0325901.

[0014] In a further embodiment, RPE cells are dissociated after being cultured in RPE maturation medium. In a further embodiment, dissociated RPE cells are seeded and cultured in RPE maturation medium. In a particular embodiment, the RPE maturation medium contains a MEK inhibitor. 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]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7- These are ruolo-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). In some embodiments, the MEK inhibitor is PD0325901.

[0015] In further embodiments, RPE cells are cultured in RPE medium, then dissociated and re-seeded onto a degradable scaffold in RPE maturation medium to produce mature RPE cells. In certain embodiments, the RPE maturation medium may contain at least one primary ciliary inducer. In some embodiments, the at least one primary ciliary inducer is prostaglandin E2 (PGE2) or aphydicolin. In other embodiments, the RPE maturation medium may contain N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1).

[0016] In a further embodiment, the starting population of iPSCs is preconfluent cells dissociated into single cells. In another embodiment, iPSCs are formed at a density of approximately 5,000 to 40,000 cells / cm². 2 Culture the iPSCs at an initial cell density of 5,000, 10,000, 20,000, 30,000, or 40,000 cells / cm³. In a particular embodiment, iPSCs are cultured at 5,000, 10,000, 20,000, 30,000, or 40,000 cells / cm³. 2 Culture at the initial cell density.

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

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

[0019] [Invention 1001] A method for producing human retinal pigment epithelial (RPE) cells, a) A process to obtain a starting population containing human induced pluripotent stem cells (iPSCs) that essentially dissociate into single cells; b) A step of culturing the iPSC in retinal induction medium to initiate the differentiation of the cells into retinal system cells; c) A step of further culturing the retinal cells in a retinal differentiation medium to further differentiate the retinal cells; d) A step of culturing the cells in retinal culture medium to form differentiated RPE cells; and e) A step of culturing the RPE cells in RPE maturation medium to produce human RPE cells. Includes, Herein, the method is one in which the formation of an embryoid body is not included. [Invention 1002] The method of the present invention 1001, wherein the iPSCs of step (b) are cultured on a matrix. [Invention 1003] The method of the present invention 1002, wherein the matrix comprises at least one recombinant cell adhesion protein. [Invention 1004] The method of the present invention 1003, wherein the at least one cell adhesion protein is laminin, vitronectin, or fibronectin. [Invention 1005] The method of the present invention 1003 or 1004, wherein the at least one cell adhesion protein is human. [Invention 1006] The method according to any one of the present invention 1001 to 1005, wherein the retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, and insulin growth factor 1 (IGF1). [Invention 1007] The method according to any one of the present invention 1001 to 1006, wherein the retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor, and IGF1. [Invention 1008] A method according to any one of the present invention 1001 to 1007, 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. [Invention 1009] The method of the present invention 1008, further comprising dissociating the RPE cells and reseeding the RPE cells onto a degradable scaffold in the RPE maturation medium. [Invention 1010] Any method of the present invention 1001 to 1009, further comprising culturing the RPE cells in the RPE maturation medium containing at least one primary ciliary inducer to produce mature RPE cells. [Invention 1011] The method of the present invention 1010, wherein the at least one primary ciliary inducer is prostaglandin E2 (PGE2) or aphydicolin. [Invention 1012] Any method of the present invention 1001 to 1009, 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]pyrimidine-2-yl)thio]-acetamide (IWP2) and / or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). [Invention 1013] Any method 1001 to 1012 of the present invention further comprises cryopreserving the RPE cells. [Invention 1014] The method according to any one of the invention 1001 to 1012, wherein the starting population of iPSCs in step (a) is preconfluent cells that have been dissociated into single cells. [Invention 1015] The iPSCs in step (b) are processed at a rate of approximately 5,000 to 40,000 cells / cm². 2 A method of culturing at the initial cell density, as described in any of the invention 1001 to 1014. [Invention 1016] A method of culturing iPSCs without using a feeder layer, as described in any of the present invention 1001 to 1015. [Invention 1017] A method of culturing the iPSC in a well-defined culture medium, any one of the methods described in items 1001 to 1015 of the present invention. [Invention 1018] A method according to any of items 1001 to 1015 of the present invention, wherein the iPSCs are cultured in a xenofree culture medium. [Invention 1019] The WNT pathway inhibitors are 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]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), and 2-phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydra The method according to any of the invention 1006 to 1018, wherein the material is zide (PNU74654) 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-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 (SFRP1) 1. [Invention 1020] The TGFβ pathway inhibitors are 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazole-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-yl-butyl-3H-imidazole-4- (Iyl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-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-Imidazole-2-yl]benzamide (D4476), 4-[4-[3-(2-Pyridinyl)-1H-Pyrazole-4-yl]-2-Pyridinyl]-N-(Tetrahydro-2H-Pyran-4-yl)-benzamide (GW788388), 4-[3 Any method according to 1006 to 1019 of the present invention, wherein the compound is -(2-pyridinyl)-1H-pyrazole-4-yl]-quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridine-2-yl)-1H-pyrazole-4-yl)-1,5-naphthyridine (RepSox). [Invention 1021] The aforementioned MEK inhibitors are 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]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethyl Any method according to items 1007 to 1020 of the present invention, wherein the material is xy)-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). [Invention 1022] The BMP pathway inhibitors are 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), and 4-[6-[4-(1-methylethoxy)phenyl] The method according to any of items 1006 to 1021 of the present invention, wherein the material is [nyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), or 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347). [Invention 1023] The method according to any one of the present invention 1006 to 1021, wherein the BMP pathway inhibitor in the retinal induction medium is LDN193189. [Invention 1024] The method according to any of the present invention 1007 to 1023, wherein the BMP pathway inhibitor in the retinal differentiation medium is LDN193189 and the MEK inhibitor is PD0325901. [Invention 1025] Any method 1001 to 1024 of the present invention, wherein the starting population of iPSCs is matched in MHC haplotype to a subject requiring it. [Invention 1026] The method according to any one of the present invention 1001 to 1025, wherein the starting population of iPSCs is homozygous for at least one HLA allele. [Invention 1027] The method of the present invention 1026, wherein the at least one HLA allele is HLA-A, HLA-B, or HLA-DR. [Invention 1028] A method for producing human retinal pigment epithelial (RPE) cells, a) A step of obtaining a starting population containing human induced pluripotent stem cells (iPSCs) that essentially dissociate into single cells in a well-defined culture medium; b) A step of culturing the iPSCs on laminin in a retinal induction medium containing LDN193189, CKI-7, and SB431542 to initiate the differentiation of the cells into retinal system cells; c) A step of further culturing the retinal cells in a retinal differentiation medium containing LDN193189, CKI-7, SB431542, and PD0325901 to further differentiate the retinal cells; d) A step of culturing the cells in a retinal medium containing nicotinamide and activin A to form differentiated RPE cells; and e) A step of culturing the RPE cells in RPE maturation medium to produce human RPE cells. Includes, Herein, the method is one in which the formation of an embryoid body is not included. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are given for illustrative purposes only, but to illustrate preferred embodiments of the present invention, because 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 drawing]

[0020] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. This disclosure may be better understood by referring to one or more of these drawings together with a detailed description of a specific embodiment presented herein.

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

[0022] [Figure 2A]Flow cytometry analysis of related 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 related 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 associated 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 associated 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 associated 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 observed in the cytoplasm of untreated cells and in 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 observed in the tight junctions of the plasma membrane of 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. Cells treated with PGE2 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 observed in the cytoplasm of untreated cells and in 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 observed in the tight junctions of the plasma membrane of 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. Cells treated with PGE2 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 observed in the cytoplasm of untreated cells and in 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 observed in the tight junctions of the plasma membrane of 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. Cells treated with PGE2 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. Cells treated with LiCl or untreated cells have weak p27 expression in the nucleus. C) RPE65 and ZO1 tight junctions of cells treated with IWP2+IWR1, IWP2, or LiCl. RPE65 staining is high in the cytoplasm of IWP2+IWR1-treated and IWP2 cells, and low in untreated cells; staining is not observed 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. Cells treated with LiCl or untreated cells have weak p27 expression in the nucleus. C) RPE65 and ZO1 tight junctions of cells treated with IWP2+IWR1, IWP2, or LiCl. RPE65 staining is high in the cytoplasm of IWP2+IWR1-treated and IWP2 cells, and low in untreated cells; staining is not observed 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. The data shown represent RPE differentiation set up by multiple operators using an optimized protocol across three cell lines, as measured by flow cytometry of 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. The data represent 109 differentiations performed by 5 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 purities. [Figure 6B]A) Multi-operator RPE differentiation. The data shown represent RPE differentiation set up by multiple operators using an optimized protocol across three cell lines, as measured by flow cytometry of 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. The data represent 109 differentiations performed by 5 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 purities. [Figure 6C] A) Multi-operator RPE differentiation. The data shown represent RPE differentiation set up by multiple operators using an optimized protocol across three cell lines, as measured by flow cytometry of 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. The data represent 109 differentiations performed by 5 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 purities. [Figure 6D]A) Multi-operator RPE differentiation. The data shown represent RPE differentiation set up by multiple operators using an optimized protocol across three cell lines, as measured by flow cytometry of 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. The data represent 109 differentiations performed by 5 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 purities.

[0027] [Figure 7A]A) The functionality of the barrier function of RPE cells produced using the RPE differentiation protocol is demonstrated by transepithelial potential (TEP) measurement of the ion gradient across the entire monolayer. B) Functionality of RPE cells treated with IWP2 or IWP2+endo-IWR2. C-E) Transepithelial electrical resistance (TER) and TEP (bright line) of untreated, PGE2-treated, and IWP2+endo-IWR1-treated cells. F) Functional response (TER) from cells matured in RPE-MM+PGE2 medium with 50 μM PGE2 versus 100 μM PGE2 from day 54 to day 75 of the iPSC differentiation protocol. Compared to iPSC-derived RPE cultured in RPE-MM+PGE2 medium with 50 μM PGE2 from day 54 to day 75 of the differentiation protocol, the TER scale during the differentiation process progressively increased in the case of 100 μM PGE2. This demonstrates that increasing the concentration of PGE2 promotes the maturation and functional efficiency of iPSC-derived RPE cultures. G) Purity of iPSC-derived RPE by expression rate of mature RPE markers at day 75 in cultures treated with 50 μM vs. 100 μM PGE2, with the iPSC-derived RPE differentiation protocol initiated between day 54 and day 75. Expression of Pmel17, Tryp1, and Cralbp (RPE-specific markers) is comparable to that of iPSC-derived RPE cultured with 50 μM PGE2. This indicates that PGE2 promotes the differentiation of iPSC-derived RPE across a range of concentrations. Expression of the Best1 marker (late-maturation RPE marker) is considerably higher in cells treated with 100 μM PGE2 compared to cells treated with 50 μM PGE2, indicating that increasing the concentration of PGE2 enhances 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 [Modes for carrying out the invention]

[0028] Description of Exemplary Embodiments Certain aspects of this disclosure overcome several major problems with the current art by providing a method for producing RPE cell populations from pluripotent stem cell starting cell suspensions, preferably essentially single-cell suspensions 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 embryoid body starting populations. In some embodiments, this disclosure provides a highly efficient and reproducible method for differentiating pluripotent stem cells (PSCs) into functionally 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. Therefore, a purified cell population is approximately 90% or more, 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 not” with respect to a particular component is used herein to mean that no particular component is intentionally included in the composition and / or is present only as an impurity or in trace amounts. Therefore, the total amount of a particular component resulting from any unintended contamination of the composition is considerably less than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which no amount of the particular component can be detected using standard analytical methods.

[0031] As used herein, “a” or “an” may mean one or more. As used herein, as used in a claim (may include) with the word “comprising,” the word “a” or “an” may mean one or more.

[0032] The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to substitutes, or that such substitutes are not mutually exclusive; however, this disclosure supports the definition of substitutes and “and / or” only. Where used herein, “another” may mean at least a second or more.

[0033] Throughout this application, the term "about" is used to indicate that a value includes inherent error variations of the device or method used to determine the value, or variations that exist between the subjects of study.

[0034] The term “cell” is used herein to mean a structural and functional unit of an organism that is capable of independently replicating, is enclosed by a membrane, and contains biomolecules and genetic material. As used herein, cells may be naturally occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0035] The term “cell population” is used herein to typically refer to a group of cells of a common type. A cell population may originate from a common precursor or may contain one or more cell types. A “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 that proportion of that cell type in the starting population. A cell population may be enriched in one or more cell types and depleted in one or more cell types.

[0036] The term “stem cell” as used herein refers to a cell that, under appropriate conditions, can differentiate into a variety of specialized cell types, and under other appropriate conditions, can self-replicate and remain essentially undifferentiated and pluripotent. The term “stem cell” also encompasses pluripotent cells, compound pluripotent cells, precursor cells, and progenitor cells. Exemplary human stem cells may 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 may also be produced from somatic cells by reprogramming them to a pluripotent state by expressing 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 characteristic of cells to differentiate into all other cell types in an organism, with the exception of extraembryonic or placental cells. Pluripotent stem cells can differentiate into all three germ layer cell types (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 other embodiments, pluripotent stem cells are induced pluripotent stem cells, which are produced by reprogramming somatic cells.

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

[0039] As used herein, “undifferentiated” refers to cells that exhibit 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 endoderm, mesoderm, and ectoderm cells. When pluripotent stem cells aggregate and non-adherent culture of EBs in suspension becomes possible, a spheroid structure is formed.

[0041] "Isolated" cells are substantially separated or purified from other cells in an organism or culture. Isolated cells may be, for example, at least 99%, at least 98%, at least 95%, or at least 90% pure.

[0042] An "embryo" refers to a mass of cells obtained by one or more divisions of an activated oocyte that has a zygote or artificially reprogrammed nucleus.

[0043] Embryonic stem (ES) cells are undifferentiated pluripotent cells obtained from the inner cell mass of an early-stage embryo, such as the blastocyst, or produced by artificial means (e.g., nuclear transfer), and are capable of giving 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 (hereinafter referred to as reprogramming factors). iPSCs may be produced using fetal somatic cells, postnatal somatic cells, neonatal somatic cells, immature somatic cells, or adult somatic cells. Factors that may be used to reprogram somatic cells into pluripotent stem cells in certain embodiments 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 somatic cells into pluripotent stem cells.

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

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

[0047] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. Haplotypes 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 “haplotype matching” is defined as the sharing of one or more major histocompatibility locus haplotypes between cells (e.g., iPSC cells) and the subject being treated. The haplotype of a subject can be readily determined using assays well known in the Art. Haplotype-matched iPSC cells can be autologous or allogeneic. Autologous cells grown in tissue culture and essentially differentiated into RPE cells are haplotype-matched to a subject.

[0049] "Substantially identical HLA type" indicates that, when transplanted cells obtained by inducing differentiation of iPSCs derived from donor somatic cells are transplanted into a patient, the donor's HLA type matches that of the patient to a degree that allows the transplanted cells to engraft.

[0050] "Superdonor" as used herein refers to an individual homozygous for specific MHC class I and II genes. These homozygous individuals can function as superdonors, and their cells (including tissues and other materials containing their cells) can be transplanted into individuals that are homozygous or heterozygous for that haplotype. Superdonors may be homozygous for the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus alleles, respectively.

[0051] In this specification, “feeder-free” or “feeder-independent” refers to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as a substitute for the 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 proliferating state. In some cases, feeder-free cultures utilize an animal-based matrix (e.g., MATRIGEL®) or are grown on a substrate such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to remain essentially undifferentiated without requiring a mouse fibroblast “feeder layer.”

[0052] In this specification, the “feeder layer” is defined as a coating layer of cells, such as on the bottom surface of a culture dish. Feeder cells release nutrients into the culture medium and may provide a surface to which other cells, such as pluripotent stem cells, can adhere.

[0053] When used in relation to culture media, extracellular matrices, or culture conditions, the terms “defined” or “well-defined” refer to culture media, extracellular matrices, or culture conditions in which the chemical composition and quantities of virtually 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 supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin (e.g., Dulbecco’s Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, which contains amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources). An exemplary well-defined medium is Essential 8® Medium.

[0054] When used in relation to culture media, extracellular matrix, or culture conditions, the term "xeno-free (XF)" refers to culture media, extracellular matrix, or culture conditions that are essentially free of heterologous animal-derived components. In human cell culture, any protein from a non-human animal, such as a mouse, is a heterologous component. In certain embodiments, a xeno-free matrix cannot essentially contain any heterologous 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 (the main component), collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0055] "KNOCKOUT® Serum Substitute" as used herein refers to serum-free formulations optimized for growing and maintaining undifferentiated cells, such as stem cells, in culture.

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

[0057] The term "retina" refers to the photosensitive layer of tissue that covers the inner surface of the eye.

[0058] The "retinal pigment epithelium" refers to the single layer of pigment cells between the choroid, a layer filled with blood vessels, and the retina.

[0059] In this specification, "retinal cells" refers to cells that can give rise to or differentiate into RPE cells.

[0060] "Retinal Induction Medium (RIM)" as used herein refers to a growth medium comprising a WNT pathway inhibitor and a BMP pathway inhibitor that can induce differentiation of PSCs into retinal system cells. RIM also comprises a TGFβ pathway inhibitor.

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

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

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

[0064] In this specification, "mature" RPE cells 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] In this specification, RPE cell "maturation" refers to the process of modulating the RPE development pathway to produce mature RPE cells. For example, modification of ciliary function can lead to RPE maturation.

[0066] As used herein, “therapeutic dose” refers to an amount of a compound sufficient to achieve a treatment when administered to a subject for the treatment of a disease or condition.

[0067] In this specification, "inducer" is defined as a molecule that regulates gene expression (e.g., activates a gene within a cell). Inducers may bind to repressors or activators. Inducers function by neutralizing repressors. II. Pluripotent stem cells A. Embryonic stem cells

[0068] ES cells are derived from the inner cell mass of a blastocyst and possess high in vitro differentiation potential. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner mass cells on a non-growing cell feeder layer. Resowed cells can continue to proliferate, generating new colonies of ES cells, which can be isolated, dissociated, reseeded, and grown. This process of "passaging" undifferentiated ES cells can be repeated many 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 genes related to cells and genes that control cell differentiation. The pluripotency of ES cells, combined with genetic manipulation and selection, can be used in in vivo genetic analysis studies by creating transgenic mice, chimeric mice, 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 antiserum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse fetal fibroblasts in a medium containing bovine fetal serum. The resulting undifferentiated ES cell colonies are subcultured on the feeder layer of mouse fetal fibroblasts in the presence of bovine fetal serum to generate a population of ES cells. In some methods, mouse ES cells can grow in the absence of a feeder layer by adding the cytokine leukemia suppressor (LIF) to a serum-containing culture medium (Smith, 2000). In other methods, mouse ES cells can grow 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 induced from zygote-egg fusion, nuclear transfer, or zygote- or blastocyst-stage mammalian embryos produced by pathogenesis, or by reprogramming chromatin using previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000), followed by incorporating the reprogrammed chromatin into the plasma membrane to produce embryonic cells. In one method, human blastocysts are exposed to anti-human serum, trophectoderm cells are lysed, and the inner cell mass is isolated from the inner cell mass cultured on a feeder layer of mouse embryonic fibroblasts. Furthermore, the cell clusters derived from the inner cell mass are chemically or mechanically dissociated and reseeded, and colonies with undifferentiated morphology are selected using a micropipette, dissociated, and reseeded (U.S. Patent No. 6,833,269). In some methods, human ES cells can grow without serum by culturing them on a fibroblast feeder layer in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can grow in a protein matrix (e.g., MATRIGEL) in the presence of a "conditioned" medium containing basic fibroblast growth factor. TM Alternatively, by culturing on laminin, they can grow without a feeder cell layer (Xu et al., 2001).

[0071] ES cells can also be derived from other organisms, including rhesus monkeys and marmosets, as well as from established mouse and human cell lines, by previously described methods (Thomson, and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000). For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As a further example, an established mouse ES cell line is the CGR8 cell line, established from the inner cell mass of mouse strain 129 embryos, and cultures 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 the 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] Pluripotency induction was initially achieved in 2006 using mouse cells (Yamanaka et al., 2006) and in 2007 using human cells (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells through the introduction of pluripotency-related transcription factors. Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into virtually any cell type. The use of iPSCs avoids many of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients with iPSC-derived autologous grafts may not require lifelong immunosuppressive treatment to prevent graft rejection.

[0074] Any cell type other than germ cells can be used as a starting point for iPSCs. For example, cell types may be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, 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 are no restrictions on 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 manner disclosed herein. In one embodiment, the somatic cells are themselves RPE cells, such as human RPE cells. RPE cells may be adult or fetal RPE cells. iPSCs may 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. See, for example, U.S. Patent Application Publication 20090246875, U.S. Patent Application Publication 2010 / 0210014; U.S. Patent Application Publication 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,278,620; PCT Publication WO2007 / 069666 and U.S. Patent No. 8,268,620 (these 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 used.

[0076] Cells are treated with nuclear reprogramming agents (which are generally one or more factors that can induce iPSCs from somatic cells) or nucleic acids encoding these agents (including in vector-integrated forms). Nuclear reprogramming agents generally include at least Oct3 / 4, Klf4, and Sox2, or nucleic acids encoding these molecules. P53 functional inhibitors, L-myc, or nucleic acids encoding L-myc, and Lin28 or Lin28b, or nucleic acids encoding Lin28 or Lin28b, may be used as further nuclear reprogramming agents. Nanog may also be used for nuclear reprogramming.As disclosed in the published U.S. Patent Application No. 20120196360, exemplary reprogramming factors for the production of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 is replaceable with Soxl, Sox3, Soxl5, Soxl7 or Soxl8; Klf4 is replaceable with Klfl, 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 3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced with Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HPVI 6 E6;(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 used. In other embodiments, Oct4, Nanog, and Sox2 are used. 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 used. In other non-limiting examples, Oct3 / 4, Klf4, Sox2, and Sal4 are used. Factors such as Nanog, Lin28, Klf4, or c-Myc may increase reprogramming efficiency and can be expressed from several different expression vectors. For example, embedded vectors such as EBV element-based systems may be used (U.S. Patent No. 8,546,140). In a further embodiment, the reprogramming protein may be directly introduced into somatic cells by protein transduction. Reprogramming may further involve contacting the cell with one or more signaling receptors, including glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor β (TGF-β) receptor inhibitors or signaling inhibitors, leukemia inhibitor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These regulators may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that virtually any iPS cell or cell line may be used.

[0077] The mouse and human cDNA sequences of these nuclear reprogramming materials are available by reference to the NCBI accession numbers mentioned in PCT Publication WO2007 / 069666 (which is incorporated herein by reference). Methods for introducing one or more reprogramming materials or nucleic acids encoding these reprogramming materials are known in the art and are disclosed, for example, in U.S. Patent Application Publication 2012 / 0196360 and U.S. Patent No. 8,071,369 (both 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, culture is carried out by adding leukemia inhibitor (LIF) as a differentiation inhibitor to the standard medium. In the case of human cells, it is preferable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs can be used, as are known to those skilled in the art.

[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 on a medium exposed to fibroblast feeder cells. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts, which have been treated with radiation or antibiotics to terminate cell division, as feeder cells. Alternatively, pluripotent cells may be cultured using defined feeder-independent culture systems, 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 may be modified to express exogenous nucleic acids (e.g., to include an operaby-linked tyrosinase enhancer to nucleic acid sequences encoding a promoter and a first marker). The tyrosinase gene is disclosed, for example, in GENBANK® accession number 22173, available as of January 1, 2013. This sequence is aligned at positions 5286971–5291691 (reverse orientation) on chromosome 7 of the C57BL / 6 mouse strain. The 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 may also be available. 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 expressed in retinal pigment epithelial cells (including tyrosinase promoters). The construct may also include other elements, such as a ribosome binding site (internal ribosome binding sequence) for translation initiation and a transcription / translation terminator. In general, 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 viruses.

[0081] Plasmids are designed with several goals in mind, such as achieving controlled high copy numbers, avoiding potential causes of plasmid instability in bacteria, and providing 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 are suitable for maintenance and fermentation in E. coli so that large amounts of DNA can be produced and purified. Second, they are safe and suitable for use in human patients and animals. The first requirement demands high copy number plasmids that are selectable for bacterial fermentation and can be maintained relatively easily and stably during bacterial fermentation. The second requirement demands attention to elements such as selection markers and other coding sequences. In some embodiments, a plasmid encoding a marker comprises (1) a high copy number origin of replication, (2) a selection marker (e.g., a neogene for antibiotic selection by kanamycin, but not limited to these), (3) a transcription termination sequence containing a tyrosinase enhancer, and (4) a multicloning site for incorporating various nucleic acid cassettes; and (5) a nucleic acid sequence encoding the marker operably linked to a tyrosinase promoter. There are numerous plasmid vectors known in the art for inducing protein-encoding nucleic acids. These include, but are not limited to, vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No. 7,989,425; and U.S. Patent No. 6,416,998 (these are incorporated herein by reference).

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

[0083] Markers may include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), enzymes (e.g., horseradish peroxidase or alkaline phosphatase or firefly / sea mushroom luciferase or nanoluc) or other proteins. Markers may be proteins (including secreted proteins, cell surface proteins or endoproteins; synthesized or taken up by cells); nucleic acids (e.g., mRNA or enzymatically active nucleic acid molecules) or polysaccharides. Any such cellular component detectable by antibodies, lectins, probes or nucleic acid amplification reactions may include determinants of cellular components specific to the marker of the cell type of interest. Markers may also be identified by biochemical or enzymatic assays or by biological responses dependent on the function of gene products. Nucleic acid sequences encoding these markers may be operably ligated to tyrosinase enhancers. In addition, other genes may be included, such as genes that can influence stem cells regarding RPE differentiation or RPE function or physiological function or pathology. Therefore, in some embodiments, nucleic acids encoding one or more of the following are included: 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. 1. MHC haplotype matching

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

[0085] Due to the importance of HLA in transplantation, HLA loci are typically classified by serology and PCR to identify preferred donor-recipient pairs. Serological detection of HLA class I and II antigens can be achieved using complement-mediated lymphocyte toxicity tests 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, and 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] When donor cells are HLA homozygous (i.e., contain identical alleles for each antigen-presenting protein), MHC compatibility between donor and recipient increases significantly. 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 superdonors, and grafts made from their cells can be transplanted into any individual that is either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype that is frequently observed in the population, these cells can be used in transplant therapy in a large number of individuals.

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

[0088] In certain embodiments, the reprogramming factor is expressed from an expression cassette contained within one or more exogenous episomal gene elements (see U.S. Patent Publication 2010 / 0003757, which is incorporated herein by reference). Thus, iPSCs may not essentially contain exogenous gene elements such as retroviral vector elements or lentiviral vector elements. These iPSCs are prepared by creating iPSCs that are essentially free of exogenous vectors or viral elements using an extrachromosomal replication vector (i.e., an episomal vector) (which is a vector that can replicate as an episome) (see U.S. Patent No. 8,546,140, ​​which is incorporated herein by reference; Yu et al., 2009). Some DNA viruses, such as adenoviruses, monkey vacuolated virus 40 (SV40), or bovine papillomavirus (BPV), or budding yeast ARS (autonomous replication sequence)-containing plasmids replicate extrachromosomally or as episomes in mammalian cells. These episomal plasmids are essentially free from all the drawbacks associated with embedded vectors (Bode et al., 2001). For example, lymphocyte-targeted herpes virus-based (including) or Epstein-Barr virus (EBV), as defined above, can replicate extrachromosomally and assist in the delivery of reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1 or their variants or functional equivalents. A further advantage of episomal vectors is that, after introduction into cells, the exogenous elements disappear over time, resulting in self-persistent iPSCs that essentially do not contain these elements.

[0089] Other extrachromosomal vectors include other lymphocytropic herpesvirus-based vectors. Lymphotropic herpesviruses are herpesviruses that replicate in lymphoblasts (e.g., human B lymphoblasts) and become plasmids for part of their natural life cycle. Herpes simplex virus (HSV) is not a “lymphotropic” herpesvirus. Exemplary lymphocytropic herpesviruses include, but are not limited to, EBV, Kaposi’s sarcoma herpesvirus (KSHV); squirrel monkey herpesvirus (HS); and Marek’s disease virus (MDV). Other sources for episome-based vectors are also considered (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. Somatic cell nuclear transfer involves the transfer of donor nuclei into spindle-free oocytes. In one method, donor fibroblast nuclei from rhesus monkey dermal fibroblasts are introduced into the cytoplasm of spindle-free mature metaphase II rhesus monkey oocytes (ooctye) by electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to ionomycin and then incubated to 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 manner disclosed herein. Retinal cells that are directly sensitive to light are photoreceptor cells. Photoreceptors are photosensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, photoreceptor cells convert the energy of incident light focused by a lens into an electrical signal, which is then transmitted to the brain via the optic nerve. Vertebrates have two types of photoreceptor cells, 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. Nerve signals from rods and cones are processed by other neurons in the retina.

[0092] The retinal pigment epithelium acts as a barrier between blood flow and the retina and interacts closely with photoreceptors in maintaining visual function. It consists of a single layer of hexagonal cells densely packed with melanin granules that absorb light energy reaching the retina. The main functions of specialized RPE cells include the transport of nutrients such as glucose, retinol, and fatty acids from the blood to photoreceptors; the transport of water, metabolic end products, and ions from the subretinal space to the blood; protection against light absorption and photo-oxidation; re-isomerization of all-trans-retinol to 11-cis-retinal; phagocytosis of detached photoreceptor membranes; and the secretion of various essential factors for the structural integrity of the retina.

[0093] The retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, the best vitiligo macular dystrophy gene (VMD2), and pigment epithelial-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with many visual impairments, including retinal pigment epithelial detachment, malformations, atrophy, retinopathy, retinitis pigmentosa, macular dystrophy, 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 pigment appearance. In addition, differentiated RPE cells have transepithelial resistance / TER and transepithelial potential / TEP throughout the monolayer (TER > 100 ohms.cm2; TEP > 2 mV), transport fluids and CO2 from the apical to the basal side, and regulate the polarized secretion of cytokines.

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

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

[0097] Dysfunction, damage, and loss of RPE cells are contributing factors to many eye 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 those who require such treatment. It is hypothesized that supplementing RPE cells through such transplantation can delay, 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 PSC embryoid bodies

[0098] iPSCs reprogrammed using known reprogramming factors can give rise to ophthalmic cells of the neuronal lineage, including RPE cells (Hirami et al., 2009). PCT Publication 2014 / 121077 (which is incorporated herein by whole reference) discloses a method for inducing the expression of retinal progenitor cell markers by treating embryoid bodies (EBs) produced from iPSCs with Wnt and Nodal antagonists in suspension culture. This publication discloses a method for inducing RPE cells from iPSCs by the differentiation process of iPSC EBs into an RPE cell-enriched culture. For example, embryoid bodies are produced from iPSCs by adding a rho-related coiled-coil kinase (ROCK) inhibitor and cultured in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor. Furthermore, differentiated RPE cells are formed by plating EB onto MATRIGEL® coated tissue cultures in a second medium that does not contain basic fibroblast growth factor (bFGF), contains a Nodal pathway inhibitor, contains approximately 20 ng to 90 ng of noggin, and contains approximately 1 to 5% of a knock-out serum substitute. Differentiated RPE cells are cultured in a third medium containing activin and WNT3a. Then, human RPE cells are produced by culturing RPE cells in an RPE medium containing approximately 5% fetal serum, a canonical WNT inhibitor, a non-canonical WNT inhibitor, and inhibitors of the sonic hedgehog and FGF pathways.

[0099] Using EBs for the production of differentiated cell types has several drawbacks. For example, EB production is an inconsistent and non-reproducible process due to its variability in efficiency. The size and shape of EBs produced from iPSCs or ES cells are not uniform, and EBS production also involves rate-limiting centrifugation. This disclosure provides an EB-independent method for enabling the large-scale production of iPSCs or ES-derived cells required for clinical, research, or therapeutic applications. B. Induction of RPE cells from substantially single-cell PSCs

[0100] In some embodiments, methods are provided for producing RPE cells from essentially single-cell suspensions of pluripotent stem cells (PSCs), such as human iPSCs. In some embodiments, the PSCs are cultured to preconfluent to prevent any cell aggregation. In certain embodiments, the PSCs are dissociated by incubation with a cell dissociation enzyme, such as TRYPSIN® or TRYPLE®. The PSCs may also be dissociated into essentially single-cell suspensions by pipetting. In addition, while the cells are not adhered to the culture vessel, brevistatin (e.g., about 2.5 μM) may be added to the culture medium to increase the survival of PSCs after dissociation into single cells. Alternatively, a ROCK inhibitor may be used instead of brevistatin to increase the survival of PSCs after dissociation into single cells.

[0101] To efficiently differentiate RPE cells from single-cell PSCs, accurate counting and 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 by a hemocytometer or automated cell counter, e.g., VICELL® or TC20. Cells may be diluted to cell densities 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 non-limiting examples, single-cell suspensions of PSCs are diluted to a density of approximately 100,000 cells / mL in a well-defined culture medium, e.g., ESSENTIAL 8® (E8®) medium.

[0102] Once single-cell suspensions of PSCs are obtained at a known cell density, the cells are generally seeded into appropriate culture vessels, such as tissue culture plates, flasks, 6-well plates, 24-well plates, or 96-well plates. Culture vessels used to culture cells(s) may include, but are not limited to, flasks, tissue culture flasks, dishes, petri dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, insofar 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 within that range, depending on the needs of the culture. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment in which cells can grow. The bioreactor may have a capacity of at least 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 within that which can be derived.

[0103] In certain configurations, PSCs, such as iPSCs, are plated at a cell density appropriate for efficient differentiation. Generally, cells are plated at approximately 1,000 to 75,000 cells / cm³. 2 For example, approximately 5,000 to 40,000 cells / cm² 2Cells are plated at the following cell densities. In a 6-well plate, cells can be seeded at a cell density of approximately 50,000 to 400,000 cells per well. In an exemplary method, cells are seeded at a cell density of approximately 100,000, 150,000, 200,000, 250,000, 300,000, or 350,000 cells per well, for example, at a cell density of approximately 200,000 cells per well.

[0104] 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 the culture surface as a means of providing a 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, enterin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosine, ancholine, chondronectin, link proteins, bone sialoproteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and karin. 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 may be of natural origin, may be purified from human or animal tissue, or ECM proteins may be genetically modified recombinant proteins or essentially synthetic products. ECM proteins may 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 includes synthetically produced peptide fragments of fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is xeno-free. For example, in a xeno-free matrix for culturing human cells, human-derived matrix components may be used, from which any non-human animal components may be excluded.

[0106] In some embodiments, the total protein concentration in the matrix composition may 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 a more preferred embodiment, 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 may be cultured with 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 buffers to maintain pH. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, and inorganic salts. To enhance stem cell growth, exemplary growth media contain minimally essential media supplemented with various nutrients such as non-essential amino acids and vitamins, such as Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8® (E8®) medium. Examples of minimally essential media include, but are not limited to, Minimum Essential Medium Eagle® Alpha Medium, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. In addition, minimally essential media 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 serum-free formulation optimized for growing and maintaining undifferentiated cells, such as stem cells, in the culture medium, and referred to herein as a “Knockout serum substitute.” KNOCKOUT® serum substitutes are 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] Therefore, after plating, single-cell PSCs are generally cultured in a well-defined culture medium. In a particular embodiment, the medium is aspirated approximately 18–24 hours after seeding, and fresh medium such as E8® medium is added to the culture. In a particular embodiment, after plating, single-cell PSCs are cultured in a well-defined culture medium for approximately 1, 2, or 3 days. Preferably, before progression in the differentiation process, single-cell PSCs are cultured in a well-defined culture medium for approximately 2 days.

[0109] In some embodiments, the culture medium may or may not contain a serum substitute. Examples of serum substitutes include materials appropriately containing albumin (e.g., lipid-rich albumin, albumin substitutes, e.g., recombinant albumin, vegetable 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 substitutes may be prepared, for example, by the method disclosed in International Publication No. WO98 / 30679. Alternatively, for greater convenience, any commercially available material may be used. Examples of commercially available materials include KNOCKOUT® serum substitute (KSR), chemically defined lipid concentrates (Gibco), and GLUTAMAX® (Gibco).

[0110] Other culture conditions can be defined as appropriate. For example, the culture temperature may be about 30-40°C, for example, 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 1-10%, for example, about 2-5%, or any range that can be derived therefrom. The oxygen tension may be at least, at most, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range that can be derived therefrom. a. Differentiation medium Retinal induction medium

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

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

[0113] Next, retinal cells can 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 may contain a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a bFGF inhibitor. Generally, in RDM, the concentrations of Wnt pathway inhibitors, BMP pathway inhibitors, and TGFβ pathway inhibitors are, for example, about 9 to 11 times higher, and for example, about 10 times higher, compared to RIM. In an exemplary method, RDM contains approximately 50 nM to 200 nM of LDN193189, e.g., approximately 100 nM; approximately 1 μM to 10 μM of CKI-7, e.g., approximately 5 μM; approximately 1 μM to 50 μM of SB431542, e.g., approximately 10 μM; and approximately 0.1 μM to 10 μM of PD0325901, e.g., approximately 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM. One exemplary RDM medium is shown in Table 3.

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

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

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

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

[0118] RPE cells can then be cultured in RPE-MM for a continuous period 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, e.g., about 6 to 8 weeks, e.g., 6, 7, or 8 weeks. An exemplary method for continuous maturation of RPE cells involves dissociating the cells with a cell-dissociating enzyme such as TRYPLE® and reseeding them for about 1 to 2 weeks on a degradable scaffold assembly such as in a specialized SNAPWELL® design in RPE-MM containing a MEK inhibitor such as PD0325901. Alternatively, RPE-MM may contain a bFGF inhibitor instead of a MEK inhibitor. A method for culturing RPE cells on a degradable scaffold is taught and described in PCT Publication WO2014 / 121077 (which is incorporated herein by reference in its entirety). In short, the main elements of the method are CORNING® COSTAR® SNAPWELL® plates, bioinactive O-rings, and a biodegradable scaffold. The SNAPWELL® plates provide structure and platform for the biodegradable scaffold. The microporous membrane, which creates apical and basal sides, is ideal not only for providing support to the scaffold but also for separating distinct sides of the cellular polarization layer. The ability of the SNAPWELL® insert to delaminate 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 further chemicals or small molecules that promote RPE maturation). For example, these small molecules are primary ciliary inducers, such as prostaglandin E2 (PGE2) or aphydicolin. PGE2 may be added to the culture medium at concentrations of about 25 μM to about 250 μM, for example, about 50 μM to about 100 μM. Alternatively, RPE-MM may 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]pyrimidine-2-yl)thio]acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). Cells can be cultured in this medium for a further period, e.g., about 1 to 5 weeks, or e.g., about 2 to 4 weeks, to obtain a monolayer of mature, functional RPE cells. Thus, the method of this disclosure provides mature RPE cells derived from single-cell suspensions of pluripotent cells that can be reproduced on a large scale and consistently for clinical use. c. Cryopreservation of RPE cells

[0120] Retinal pigment epithelial cells produced by the methods disclosed herein may be cryopreserved. See, for example, PCT Publication 2012 / 149484A2 (which is incorporated herein by reference). Cells may 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 their overlapping ranges. In some embodiments, lower temperatures are used for storage (e.g., maintenance) of cryopreserved cells. In several embodiments, liquid nitrogen (or other similar coolant) is used to store the cells. In further embodiments, cells are stored for more than about 6 hours. In further embodiments, cells are stored for about 72 hours. In several embodiments, cells are stored for 48 hours to about 1 week. In yet another embodiment, the cells are stored for about one, two, three, four, five, six, seven, or eight weeks. In yet another embodiment, the cells are stored for one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve months. The cells may also be stored for longer periods. The cells may be cryopreserved separately or on a substrate such as one of the substrates disclosed herein.

[0121] In some embodiments, additional cryoprotectants may be used. For example, cells can 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 another embodiment, 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 a specific embodiment, the solution contains 2.5% DMSO. In another specific embodiment, the solution contains 10% DMSO.

[0122] Cells can be cooled, for example, at about 1 °C / min during cryopreservation. 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 gas phase of liquid nitrogen before thawing for use. In some embodiments, for example, when the cells reach about -80 °C, they are transferred to the liquid nitrogen storage area. Cryopreservation can also be performed using a rate-controlled freezer. Cryopreserved cells can be thawed at a temperature of, for example, about 25 °C to about 40 °C, typically about 37 °C. d. Inhibitor WNT pathway inhibitor

[0123] WNTs are a family of highly conserved secretory signaling molecules associated with the wingless segment polarity gene in Drosophila, regulating intercellular interactions. In humans, WNT family genes encode 38-43 kDa cysteine-rich glycoproteins. WNT proteins possess a hydrophobic signaling sequence, a conserved asparagine-binding 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 cytoplasmic β-catenin stabilization, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of specific WNT proteins has been shown to be associated with certain cancers.

[0124] As used herein, WNT inhibitors generally refer to WNT inhibitors. Therefore, WNT inhibitors refer to any inhibitor of a member of the WNT family of proteins, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11, and Wnt16. Certain embodiments of this method relate to WNT inhibitors in differentiation media. 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]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), and 2-phenoxybenzoic acid-[(5-methyl Examples of WNT inhibitors include 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-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 siRNAs and antisense nucleic acids that suppress WNT expression. WNT inhibition can also be achieved using RNA-mediated interference (RNAi). BMP pathway inhibitors

[0125] Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging 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 crucial physiological functions of BMP signaling are highlighted by their numerous roles in uncontrolled BMP signaling during pathological processes.

[0126] BMP pathway inhibitors generally include inhibitors of BMP signaling, or inhibitors specific to BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Examples of BMP inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), and 4-[6-[4-(1 Examples include -methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347). TGFβ pathway inhibitors

[0127] Transforming growth factor β (TGFβ) 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. TGFβ 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, decapentaplesic, and Vg-1.

[0128] TGFβ pathway inhibitors can generally include any inhibitor of TGFβ signaling. For example, TGFβ pathway inhibitors include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazole-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-yl-butyl-3H-imidazole Zole-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide hydrate, left-right determinant (Lefty), 3-(6-methyl-2-pyridinyl (A83-01)-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-imidazole-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide These are (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridinyl-2-yl)-1H-pyrazole-4-yl)-1,5-naphthiridine (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. Examples of 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]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), and 6-(4-bromo-2-fluoroanilino)-7-fluoro-N Examples include -(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 media, which is necessary for cells to remain in an undifferentiated state.

[0131] The bFGF inhibitor in this specification generally refers to a bFGF inhibitor. For example, bFGF inhibitors include, but are 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]pyrido[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 a method for producing an RPE or RPE-enriched cell population that can be used for many important research, development, and commercial purposes.

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

[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 This may include the use of individual cells or any range that can be derived from them. The starting cells are at least or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 It may have a seeding density of a certain number of cells / ml, or any range within that that can be derived.

[0135] RPE cells produced by the methods disclosed herein can be used in any method and application currently known in the Art for RPE cells. For example, a method for evaluating a compound may be provided, comprising assaying the pharmacological or toxicological properties of the compound against RPE cells. A method for evaluating a compound for its effect on RPE cells may also be provided, comprising a) contacting the RPE cells provided herein with the compound; and b) assaying the effect of the compound on the RPE cells. A. Screening of test compounds

[0136] RPE cells can be commercially used to screen for factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (e.g., culture conditions or procedures) that affect the characteristics of such cells and their various offspring. For example, the test compound may be a chemical compound, small molecule, polypeptide, growth factor, cytokine, or other biological agent.

[0137] In one embodiment, the method includes contacting RPE cells with a test drug and determining whether the test drug modulates the activity or function of RPE cells in a population. In some applications, screening assays are used to identify drugs that modulate the proliferation of RPE cells or alter the differentiation of RPE cells. Screening assays may be performed in vitro or in vivo. Methods for screening and identifying ophthalmic or RPE drugs include those suitable for high-throughput screening. For example, for the identification of potential therapeutic molecules, RPE cells may be positioned or placed on a culture dish, flask, roller bottle, or plate (e.g., single multi-well dish or dish, e.g., 8, 16, 32, 64, 96, 384, and 1536 multi-well plate or dish) at a predetermined position of any choice. Libraries that can be screened may include, for example, small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0138] Other screening applications relate to testing pharmaceutical compounds for their effects on maintaining or repairing retinal tissue. Since compounds are designed to have pharmacological effects on cells, or because compounds designed to have effects elsewhere may have unintended side effects on cells of this tissue type, screening may be performed. B. Treatment and transplantation

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

[0140] To determine the suitability of a cell composition for therapeutic administration, the cells may first be tested in a suitable animal model. In one embodiment, RPE cells are evaluated for their ability to survive in vivo and maintain their phenotype. The cell composition is administered to immunodeficient animals (e.g., nude mice or animals immunodeficient by chemical or radiation). After a period of growth, tissue is collected and evaluated for the continued presence of pluripotent stem cell-derived cells.

[0141] Several animals are available to test 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 viability 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] Human RPE cells or pharmaceutical compositions containing these cells described herein may be used in the manufacture of pharmaceuticals for treating conditions in patients requiring treatment of the condition. RPE cells may be cryopreserved in advance. In certain embodiments, the disclosed RPE cells are derived from iPSCs and may be used to provide “personalized medicine” to patients with eye diseases. In some embodiments, somatic cells obtained from a patient may be genetically engineered to correct disease-causing mutations, differentiate into RPE, and form RPE tissue. This RPE tissue may be used to replace endogenous degenerated RPE from the same patient. Alternatively, iPSCs produced from healthy donors or HLA homozygous “superdonors” may be used. To create an anti-inflammatory and immunosuppressive environment in vivo, RPE cells may be treated in vitro with specific factors, such as pigment epithelial-derived factor (PEDF), transforming growth factor (TGF)-β, and / or retinoic acid.

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

[0144] In some embodiments, RPE cells may be used for autologous RPE transplantation into subjects suitable for regenerative medicine. RPE cells may be transplanted in combination with other retinal cells, such as those possessing photoreceptors. Transplantation of RPE cells produced by the disclosed method may be carried out by various techniques known in the art. For example, methods for carrying out RPE transplantation are described in U.S. Patent No. 5,962,027 and U.S. Patent No. 6,045,791 (which are each incorporated herein by reference in their entirety). According to one embodiment, transplantation is performed by transciliary vitrectomy (pars This is performed by pana vitrectomy, followed by delivery of the cells into the subretinal space via a retinal opening or by direct injection. The 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. The RPE cells can also be transplanted together with other cells, such as retinal cells having photoreceptors (co-transplantation). Thus, compositions comprising RPE cells obtained by the methods disclosed herein are provided. In some embodiments, these RPE cells include a tyrosinase enhancer operably linked to a nucleic acid encoding a promoter and a marker. In other embodiments, the RPE cells also include 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 1 x 10⁶ 4 RPE cells, approximately 1 x 10⁶ 5 RPE cells, approximately 1 x 10⁶ 6 RPE cells, approximately 1 x 10⁶ 7 RPE cells, approximately 1 x 10⁶ 8 A single RPE cell, or approximately 1 x 10⁶ 9It may contain 100 RPE cells. In certain embodiments, the composition is a substantially purified preparation (with respect to non-RPE cells) containing differentiated RPE cells produced by the method disclosed herein. Compositions are also provided that include a scaffold, e.g., a polymer carrier and / or an extracellular matrix, and an effective amount of RPE cells produced by the method 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 in vivo applications. Examples of physiologically acceptable materials include, but are not limited to, absorbable and / or non-absorbable solid matrix materials, e.g., small intestinal submucosa (SIS), cross-linked or uncross-linked alginates, hydrophilic colloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, fleece, and bioadhesives.

[0146] Suitable polymer carriers include porous meshes or sponges formed from synthetic or natural polymers and polymer solutions. For example, the matrix may be a polymer mesh or sponge or a polymer hydrogel. Natural polymers that can be used include proteins, such as collagen, albumin, and fibrin; as well as polysaccharides, such as alginates and hyaluronic acid polymers. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include polymers of hydroxy acids, such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PGLA), polyo-esters, polyacid anhydrides, 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 may be used. A hydrogel is a substance formed when an organic polymer (natural or synthetic) is crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules and forms a gel. Examples of materials that can be used to form hydrogels include polysaccharides, e.g., alginates, polyphosphatidines, and polyacrylates (which are ionically crosslinked), or block copolymers, e.g., PLURON1CS® or TETRON1CS®, polyethylene oxide-polypropylene glycol block copolymers (which are crosslinked by temperature or H, respectively). Other materials include proteins, e.g., fibrin, and polymers, e.g., 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 improve disease or abnormality of retinal tissue). In some embodiments, RPE cells produced by the disclosed method may be manipulated to form RPEs, which may be used to replace degenerated RPEs in subjects requiring replacement of degenerated RPEs. C. Distribution for commercial, therapeutic, and research purposes.

[0149] In some embodiments, reagent systems are provided that include a set or combination of cells containing RPE-enriched cell populations present at any time during manufacturing, distribution, or use. The cell sets include any combination of the cell populations disclosed herein, often in combination with undifferentiated pluripotent stem cells or other differentiated cell types that share the same genome. Each cell type may be packaged together or in separate containers at the same facility or different locations, at the same or different time periods, under the control of the same or different entities sharing a trading relationship.

[0150] 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 improve disease or injury of eye tissue). V. Kit

[0151] In some embodiments, kits are provided that may include, for example, one or more media and components for the production of RPE cells. The reagent system may be packaged in either an aqueous medium or a lyophilized form, as required. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the components may be placed (preferably appropriately ali-coated). If there are more than one component in the kit, the kit also generally includes a second, third or further container in which further components may be placed separately. However, various combinations of components may be included in the vial. The kit's components may be supplied as dry powder(s). If the reagents and / or components are supplied as dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is assumed that the solvent may also be supplied in a separate container means. The kit also typically includes means for sealing and containing the kit's components(s) for commercial sale. Such containers may include injection or blow-molded plastic containers in which the desired vials are held. The kit may also include user instructions in printed or electronic format, such as digital. [Examples]

[0152] VI. Examples The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art should recognize that the techniques disclosed in the following embodiments are techniques found by the inventors to function well in carrying out the present invention and therefore may constitute a preferred form for carrying it out. However, those skilled in the art should recognize that, in view of this disclosure, many modifications may be made to the specific embodiments disclosed, and that such modifications may still yield similar or similar results without departing from the spirit and scope of the present invention. Example 1 - Preparation of a starting pluripotent stem cell population

[0153] Starting populations of RPE cells can be induced from pluripotent stem cells such as ES cells and iPSCs. Exemplary methods include induced RPE cells from human iPSCs reprogrammed from somatic cells by methods known in the art, such as U.S. Patent Nos. 8,546,140, ​​8,741,648, 8,691,574, U.S. Patent Application Publication 20090246875, Published U.S. Patent Nos. 8,278,104, Published U.S. Patent Nos. 9,005,967, 8,058,065, 8,129,187, PCT Publication WO2007 / 069666A1, 8,183,038, and 8,268,620 (these are incorporated herein by reference). In one exemplary method, pluripotent stem cells were produced from somatic cells using nuclear programming factors Oct4, Sox2, c-Myc, and Klf4. In another exemplary method, pluripotent stem cells were produced from somatic cells using nuclear programming factors Oct4, Sox2, Nanog, Lin28, L-Myc, and the SV40 large T antigen.

[0154] iPSCs were grown on plates coated with vitronectin in well-defined culture media such as ESSENTIAL 8® (E8®) medium, without the use of mouse or human feeder layers. Vitronectin was diluted 1:200 with calcium or magnesium-free DPBS, coated with the mixture, and incubated at room temperature for approximately 1 hour. Once preconfluent, the iPSCs were divided to prevent overgrowth and thus prevent the development of non-healthy cells and / or differentiated cells (Figure 1A).

[0155] To induce RPE cells, iPSCs were dissociated into single-cell suspensions, removing any aggregates or embryoid bodies. To obtain single-cell suspensions, the cells were washed with DPBS and incubated in a cell dissociation enzyme such as TRYPLE® at 37°C for approximately 10 minutes. The cells were then detached by pipetting using a serological pipette, and the cell suspensions were collected in a conical tube. If the cells did not detach with gentle pipetting, the culture was incubated for a longer period, e.g., an additional 2-3 minutes. To collect all cells, the culture vessel was washed with E8® medium at room temperature, and then the medium was added to the tube containing the cell suspensions. In addition, while the cells were not adhering to the culture vessel, brevistatin (e.g., 2.5 μM) was added to the E8® medium to increase PSC viability 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 single-cell iPSCs into RPE cells, an automated cell counter such as VICELL® is used to accurately count the injection density of single-cell iPSCs, and approximately 1 × 10⁶ cells are injected into room temperature E8® medium. 5The cells were diluted to a cell suspension of 200,000 cells / mL. Once single-cell suspensions of iPSCs were obtained at a known cell density, the cells were placed in a suitable culture vessel, such as a 6-well plate coated with vitronectin. 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 for proper adhesion to the plate. Example 2 - Differentiation of iPSCs into RPE cells

[0157] As in Example 1, single-cell iPSCs seeded at an appropriate cell density were cultured for approximately 2 days, and then cultured in various differentiation media to induce RPE cells. On day 3, E8® medium was aspirated and room temperature retinal induction medium (RIM) (e.g., Table 3) was added. Briefly, RIM contained DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, RIM contained WNT pathway inhibitors, BMP pathway inhibitors, TGFβ pathway inhibitors, 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 2–4 days.

[0158] Next, cells were cultured in retinal differentiation medium (RDM) for approximately 7–14 days. Briefly, RDM (Table 2) contained DMEM and F12 in a ratio of approximately 1:1, a knockout serum substitute, 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 times 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. The RM contained DMEM and F12 in a ratio of approximately 1:1, a knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RM contained nicotinamide and activin A. The medium was changed 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 changed every other day with room temperature RPE-MM. The cells were then dissociated with cell dissociation enzymes and re-seeded on a vitronectin-coated plate. At this stage, induced PRE cells can be cryopreserved in xenofree CS10 medium. To continue RPE maturation, the plated cells were cultured for a further 15 days. Example 3 - Maturation of RPE cells

[0161] For the continued maturation of the RPE cells produced in Example 2, the cells were dissociated with a cell-dissociating enzyme such as TRYPLE™ and re-seeded for 1-2 weeks on a specialized SNAPWELL™ design degradable scaffold assembly in RPE-MM containing a MEK inhibitor such as PD0325901. This yielded a monolayer of differentiated, polarized, and confluent functional RPE cells (Figure 1D), which could then be cryopreserved in xenofree CS10 medium.

[0162] Mature RPE cells were further developed into a functional RPE cell monolayer that functions as intact RPE tissue by continued culture in RPE-MM containing additional small molecules such as primary ciliary inducers like PGE2 or aphydicolin. While not bound by theory, these primary ciliary inducers suppress the canonical WNT pathway, induce exit from the cell cycle in cells, and induce 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 exit from the cell cycle in RPE cells and promote RPE maturation). Cells were cultured in this medium for a further 2-3 weeks to obtain a functional mature RPE cell monolayer. Thus, the disclosed method provides pluripotent cell-derived mature RPE cells that can be reproduced on a large scale and consistently for clinical use. Example 4 - Cryopreservation of RPE cells

[0163] For cryopreservation of differentiated RPE cells in Example 2, the culture 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 pipetteed 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 to an appropriate density (e.g., 1 × 10⁶). 7 The cells were resuspended in cold CryoStor® CS10 at a concentration of 10 cells / mL. The cell suspension was aliquoted into pre-labeled cryovials, placed in a freezer 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 the starting population of RPE cells by MACS depletion and CD24, CD56 and / or CD90 depletion of contaminated non-RPE cells

[0164] The RPE cell populations obtained in Example 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 isolated and removed to obtain a mature RPE-enriched cell population). Contaminating cells can be removed from the culture by various methods, such as magnetically activated cell sorting (MACS®), fluorescence-activated cell sorting (FACS), or single-cell sorting. Contaminating cells were isolated from the more mature, desired RPE cells using the MACS® method (which is known in the art to separate various cell populations depending on surface antigens).

[0165] Contaminated cells in a starting population of RPE cells possess specific cell surface markers that can be used to isolate contaminated cells from 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 neuronal cell types. CD24 is a glycoprotein expressed on the surface of pluripotent stem cells, some B lymphocytes, and differentiating neuroblasts. CD56, or neuronal 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. The expression of CD24, CD56, and / or CD90 is lost during the 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 contaminated cells.

[0166] To perform the separation technique, it is desirable to dissociate the starting population of RPE cells into single-cell suspensions for the sorting to be performed (e.g., MACS). In the case of pre-frozen cells, the cells must be thawed and reseeded. To obtain single-cell suspensions from cells in adherent cultures, the cells were washed (e.g., with DPBS) and a cell dissociation enzyme was added (e.g., TRYPLE®). After incubating the cells at 37°C for about 5 minutes, the container was gently tapped to detach the 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 about 30 minutes, the cell suspensions were 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 suspensions were filtered through a cell strainer (e.g., a 20 μM SteriFlip cell strainer) to dissociate any remaining cell clusters. Cell suspensions were counted for viable cells (for example, using a ViCell counter) to obtain cell concentrations. Single-cell suspensions were obtained from the counted cell suspensions, which could be used for sorting or flow cytometry purity assays.

[0167] To remove contaminating cells from the RPE cell starting population, MACS was used to deplete CD24-positive, CD56-positive, and / or CD90-positive cells. After dissociating cells from the RPE cell starting population into single-cell suspensions, the cells were then separated, for example, into 1 × 10⁶ cells. 7 The cells were resuspended in MACS buffer at a concentration of 10 cells / mL. Examples of MACS buffers are included in Table 3. Next, the cells were stained with anti-CD24 antibody, anti-CD56 antibody, and / or anti-CD90 antibody (each at a 1:500 dilution), 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 that binds to the secondary antibody (e.g., FITC). After incubation, 20 mL of MACS buffer was added, and the cells were centrifuged at 400 × g for 5 minutes. The cell pellet was resuspended in 20 mL of MACS buffer, mixed vigorously, and centrifuged at 400 × g for 5 minutes to remove unbound antibodies. The cell pellet was then centrifuged (e.g., 1.11 × 10⁶).8 The cells were resuspended in MACS buffer (at 10⁴ cells / mL), and microbeads coated with a diluted (1:10) secondary antibody (e.g., anti-FITC) were added. The cells were incubated at 4°C for 20 minutes. After incubation, the cells were washed with MACS buffer to remove unbound microbeads, up to a maximum of 1.25 × 10⁶. 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, and 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 do not express the antigens CD24, CD56, and / or CD90 were passed through and collected. For further characterization and culture, the collected unlabeled cell suspension was centrifuged (at 400 × g for 5 minutes), re-seeded in RPE-MM plating medium, and aliquots of the cell suspension were used for flow cytometry purity assays. Thus, MACS cell sorting yielded an RPE-enriched 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, but can be used to remove contaminating cells from RPE populations produced by other methods, such as those described in U.S. Patent Application No. 12 / 523,444 and U.S. Patent Application No. 14 / 405,730. [Table 1] The pre-sorting percentage of RPE marker-positive cells is that present in the starting population of RPE cells in Example 2. Depletion of a combination of CD24-positive and CD56-positive cells resulted in a higher RPE cell enrichment than depletion of CD24-positive cells alone. Depletion of CD24-positive, CD56-positive, and CD90-positive cells resulted in over 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., before and after sorting) using a panel of relevant markers including BEST1, CRALBP, TYRP1, PMEL17, MAP2, NES, and MITF. Flow cytometry purity assays were performed before and after MACS removal of CD24-positive, CD56-positive, and / or CD90-positive cells to obtain measurements of the percentage of cells positive for each marker (Table 1) (Figures 2 and 3).

[0169] Flow cytometry purity assays were performed to determine the proportion of RPE cells obtained by the selection method described herein. Aliquots of cell suspensions collected from MACS assays (2 × 10⁶ cells per sample in 5 mL FACS tubes) were used. 6 The cells were centrifuged at 400 × 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 × 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 × g for 3 minutes, and the supernatant was decanted. The cell pellet was resuspended in 2 mL of wash buffer, and 1 × 10⁶ cells per mL of suspension were added. 6Cells were separated into individual cells, and 200 μL of 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. The primary antibody against the RPE-specific marker was diluted in Perm buffer, and 100 μL of the diluted antibody solution was added to each tube. After incubation overnight in the dark at 4°C, the cells were washed twice with 2 mL of Perm buffer. The secondary antibody solution was added to each tube, and the cells were incubated in the dark at room temperature 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 washing 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 (these 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 method for differentiation of RPE cells

[0170] With respect to the methods described in Examples 2 and 3, the inclusion of 1 μM PD0325901 in the culture medium over a specific timeframe, starting from day 2 after iPSC plating and continuing until the end of the differentiation process (including after MACS culture), can improve both the purity of the RPE population (which means a reduction in contaminating cells) and the maturation of the resulting RPE population. The inclusion of 1 μM PD0325901 has been shown to improve both the purity and maturation of the RPE population not only in RDM but also when included in RPE-MM (approximately 42-50 days) of the RPE process described herein. Example 8 - Alternative method for differentiation of RPE cells

[0171] With respect to the methods described in Examples 2 and 3, reducing the proportion of fetal bovine serum in 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 mature RPE cells produced from PGE2 treatment, immunostaining of the RPE monolayer was performed, and ZO1 staining and transmission electron microscopy of iPSC-RPE cells confirmed the presence of tight junction hexagonal structures (Figures 4A-4C) (Figure 4D). Staining showed that PGE2-treated RPE cells had reduced β-catenin and increased RPE65. In addition, treatment with IWP2+endo-IWR1 or IWP2 also resulted in reduced β-catenin (Figure 5A) and increased RPE65 (Figure 5C). The IWP2+endo-IWR1 combination was found to be more effective than IWP2 alone or endo-IWR1 alone. Therefore, treatment with PGE2, IWP2, or IWP2+endo-IWR1 results in mature RPE cells.

[0173] To measure the barrier function of RPE cells prepared by the method of the present invention, transepithelial potential (TEP) was used to measure the ion gradient across the entire monolayer generated by energy-driven ion pumps that control the passage across the cell. Transepithelial electrical resistance (TER) was used to measure the resistance of substances passing through the paracellular space, mainly through 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 showed increased functionality of treated mature RPE cells (Figures 7C-7E). Next, we tested whether increasing the concentration of PGE2 in RPE-MM+PGE2 medium from 50 μM to 100 μM improved both the purity of the RPE population (i.e., reduction in contaminating cells) and the maturation of the resulting RPE population. To determine the maturation and functionality of 50 μM PGE2-treated cultures versus 100 μM PGE2-treated cultures, barrier function with respect to transepithelial electrical resistance (TER) was measured (Figure 7F) to compare the resistance of substances passing through the paracellular space as described in Example 9. To determine the percentage of pure RPE cells obtained after treating iPSC-derived RPE cultures with 50 μM or 100 μM in RPE-MM+PGE2 medium, flow cytometry purity assays were performed on RPE-specific markers as described in Example 6 (Figure 7G). The results showed that higher concentrations of the primary ciliary inducer PGE2 promoted both the purity and maturation of the RPE population in the process of 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 mean purity of the resulting RPE cells was characterized by flow cytometry measurement of the RPE marker retinaldehyde-binding protein 1 (Craplbp) (Table 2). The RPE differentiation process was found to be highly reproducible across different starting cell populations and different operators. In addition, 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 produced from a homozygous donor for HLA-A*01 and HLA-B*08, which may offer a beneficial match for 11.38% of the US population. Furthermore, we have also successfully produced RPEs using this process with iPSC strains produced from homozygous donors for HLA-A*03 and HLA-B*07, referred to as HLA Line C (21526.101) (which may offer a potentially beneficial match for 7.63% of the US population). The above-mentioned homozygous HLA-A and HLA-B HLA Line A (21525.102) and HLA Line C (21526.101) are the property of Cellular Dynamics International, Inc. In addition, we further confirmed reproducibility in 109 RPE differentiations performed on 28 iPSC strains 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% purity. Therefore, this RPE differentiation method has a clear advantage over any other method for 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 thoroughly mixed. 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 the Live-Dead staining solution 1:1000 with DPBS (i.e., calcium and magnesium-free). Cells to be assayed: 1 × 10⁶ 6 One mL of staining solution was prepared for each sample. A fresh staining solution was prepared before each 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). Cells to be assayed: 1 × 10⁶ 6 One mL of staining solution was prepared for each sample. A fresh buffer solution was prepared before use.

[0181] All methods disclosed herein and claimed can be made and performed without excessive experimentation in view of this disclosure. While the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and steps or sets of steps 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 active substances can be used in place of the active substances described herein, but the same or similar results can be achieved. All such similar substitutes and modifications, which will be apparent to those skilled in the art, are considered to be within the spirit, scope and concept of the invention as defined by the appended claims. References The following references are incorporated herein by reference, in particular, to the extent that they provide details of the exemplary methods or other details that supplement those described herein.

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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 in a well-defined medium; b) culturing the iPSCs on laminin, vitronectin, or a combination thereof in a retinal induction medium containing a BMP pathway inhibitor, a WNT pathway inhibitor, an IGF1 and a TGFβ pathway inhibitor at a density of 1,000-40,000 cells / cm ２ to initiate differentiation of the cells into retinal lineage cells, the BMP pathway inhibitor, the WNT pathway inhibitor, and the TGFβ pathway inhibitor are 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), respectively; c) further culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, XAV939, SB431542, IGF1 and a MEK inhibitor to further differentiate the retinal lineage cells; the MEK inhibitor is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901); and d) culturing the cells obtained in step c) in a retinal medium containing nicotinamide and activin A to produce RPE cells. Including, A method wherein the method does not include the formation of embryoid bodies.

2. The method of claim 1 , further comprising the step of cryopreserving the RPE cells.

3. The method of claim 1, further comprising culturing the RPE cells in an RPE maturation medium, thereby producing mature human RPE cells.

4. 1. A method for producing mature 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, wherein said starting population of human iPSCs is pre-confluent cells dissociated into single cells; b) culturing the iPSCs on laminin, vitronectin, or a combination thereof in a retinal induction medium containing a BMP pathway inhibitor, a WNT pathway inhibitor, and a TGFβ pathway inhibitor to initiate differentiation of the cells into retinal lineage cells; the BMP pathway inhibitor, the WNT pathway inhibitor, and the TGFβ pathway inhibitor are 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), respectively; c) further culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, XAV939, SB431542 and a MEK inhibitor to further differentiate the retinal lineage cells; the MEK inhibitor is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901); d) culturing the cells obtained in step c) in a retinal medium containing nicotinamide and activin A to form differentiated RPE cells; e) culturing the RPE cells in an RPE maturation medium, thereby producing human RPE cells; and f) culturing said human RPE cells in an RPE maturation medium comprising at least one primary cilium inducer, thereby producing mature human RPE cells, wherein the at least one primary cilium inducer is prostaglandin E2 (PGE2); Including, A method wherein the method does not include the formation of embryoid bodies.

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