Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells

Abstract

Provided herein are methods of producing an RPE cell population from a starting cell suspension, such as a single cell suspension, of pluripotent stem cells (PSCs). Such a method may comprise culturing the starting single cell suspension of PSCs in differentiation media to produce human RPE cells.

1 . A method for 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;   wherein the method does not comprise the formation of embryoid bodies.   
     
     
         2 . The method of  claim 1 , wherein the iPSCs in step (b) are cultured on a matrix. 
     
     
         3 . The method of  claim 2 , wherein the matrix comprises at least one recombinant cellular adhesion protein. 
     
     
         4 . The method of  claim 3 , wherein the at least one cellular adhesion protein is laminin, vitronectin or fibronectin. 
     
     
         5 . The method of  claim 3 , wherein the at least one cellular adhesion protein is human. 
     
     
         6 . The method of  claim 1 , wherein the retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor and insulin growth factor 1 (IGF1). 
     
     
         7 . The method of  claim 1 , wherein the retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor and IGF1. 
     
     
         8 . The method of  claim 1 , further comprising following step (e) dissociating the RPE cells, reseeding the RPE cells and culturing the RPE cells in the RPE maturation medium comprising a MEK inhibitor. 
     
     
         9 . The method of  claim 8 , further comprising dissociating the RPE cells and reseeding the RPE cells on a degradable scaffold in the RPE maturation medium. 
     
     
         10 . The method of  claim 1 , further comprising culturing the RPE cells in the RPE maturation medium, wherein the RPE maturation medium comprises at least one primary cilium inducer thereby producing mature RPE cells. 
     
     
         11 . The method of  claim 10 , wherein the at least one primary cilium inducer is prostaglandin E2 (PGE2) or aphidicolin. 
     
     
         12 . The method of  claim 1 , further comprising culturing the RPE cells in the RPE maturation medium, wherein the RPE maturation medium comprises N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2) and/or 4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide (endo-IWR1). 
     
     
         13 . The method of  claim 1 , further comprising cryopreserving the RPE cells. 
     
     
         14 . The method of  claim 1 , wherein the starting population of iPSCs of step (a) are pre-confluent cells that have been dissociated into single cells. 
     
     
         15 . The method of  claim 1 , wherein the iPSCs of step (b) are cultured mat an initial cell density of about 5,000 to 40,000 cells/cm 2 ; ii) without a feeder layer, iii) in a fully-defined culture medium, and/or iv) in a xeno-free culture medium. 
     
     
         16 - 18 . (canceled) 
     
     
         19 . The method of  claim 6 , wherein the WNT pathway inhibitor is N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulphonamide dihydrochloride (CKI-7), N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-Phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydrazide (PNU 74654) 2,4-diamino-quinazoline, quercetin, 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-Dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH 535), N-[4-[2-Ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK 715), Dickkopf-related protein one (DKK1), or Secreted frizzled-related protein (SFRP1) 1. 
     
     
         20 . The method of  claim 6 , wherein 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-ieri-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, left-right determination factor (Lefty), 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01), 4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D 4476), 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW 788388), 4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]quinoline (LY 364847), 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R 268712), or 2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox). 
     
     
         21 . The method of  claim 7 , wherein the MEK inhibitor N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), or 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244). 
     
     
         22 . The method of  claim 6 , wherein the BMP pathway inhibitor is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), or 5-[6-(4-Methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML 347). 
     
     
         23 . The method of  claim 6 , wherein the BMP pathway inhibitor is LDN193189 in the retinal induction medium. 
     
     
         24 . The method of  claim 7 , wherein the BMP pathway inhibitor is LDN193189 and the MEK inhibitor is PD0325901 in the retinal differentiation medium. 
     
     
         25 . The method of  claim 1 , wherein the starting population of iPSCs are MHC haplotype-matched to a subject in need thereof. 
     
     
         26 . The method of  claim 1 , wherein the starting population of iPSCs are homozygous for at least one HLA allele. 
     
     
         27 . The method of  claim 26 , wherein the at least one HLA allele is HLA-A, HLA-B or HLA-DR. 
     
     
         28 . A method for 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 in a fully defined medium;   b) culturing the iPSCs on laminin, vitronectin or a combination thereof, 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 retinal medium comprising nicotinamide and Activin A to form differentiating RPE cells; and   e) culturing the RPE cells in a RPE maturation medium, thereby producing human RPE cells;   wherein the method does not comprise the formation of embryoid bodies.