METHOD FOR GENERATING RETINAL PIGMENT EPITHELIUM (RPE) CELLS FROM INDUCED PLURIPOTENT STEM CELLS (IPSCs)

Abstract

High efficiency methods for producing retinal pigment epithelial cells from induced pluripotent stem cells (iPSCs) are disclosed herein. The iPSCs are produced from somatic cells, including retinal pigment epithelial (RPE) cells, such as fetal RPE stem cells. In some embodiments, the iPSC include a tyrosinase promoter operably linked to a marker. Methods are disclosed for using the RPE cells, such as for treatment. Methods for screening for agents that affect RPE differentiation are also disclosed.

1 - 22 . (canceled) 
     
     
         23 . A method for authenticating that a cell of interest is a retinal pigment epithelial cell, comprising
 culturing an induced pluripotent stem cell under conditions sufficient to form a retinal pigment epithelial cell to produce a cell of interest;   assaying expression of a set of markers comprising MITF, PAX6 LHX2, TFEC, CDH1, CDH3, CLDN 10, CLDN 16, CLDN 19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143 (NM 000273), GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR, in the cell of interest;   assaying the polarization of the cell of interest;   patch clamping the cell of interest and determining the resting potential of the cell of interest; and   evaluating the fluid transport rate of the cell of interest;   wherein expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR, a resting potential of about −50 to about −60 mV, and a fluid transport rate of about 5 to about 10 μl cm −2 h −1  indicates that the cell of interest is a retinal pigment epithelial cell.   
     
     
         24 . The method of  claim 23 , further comprising:
 assaying expression of miR204 and miR211 in the cell of interest;   wherein expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR, expression of miR204 and miR211, a resting potential of about −50 to about −60 mV and a fluid transport rate of about 5 to about 10 μl cm −2 h −1  indicates that the cell is a retinal pigment epithelial cell.   
     
     
         25 - 45 . (canceled) 
     
     
         46 . The method of  claim 23 , wherein assaying expression of a set of markers comprises MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR comprises detecting MITF mRNA, PAX6 mRNA, LHX2 mRNA, TFEC mRNA, CDH1 mRNA, CDH3 mRNA, CLDN10 mRNA, CLDN16 mRNA, CLDN19 mRNA, BEST1 mRNA, TIMP3 mRNA, TRPM1 mRNA, TRPM3 mRNA, TTR mRNA, VEGFA mRNA, CSPG5 mRNA, DCT mRNA, TYRP1 mRNA, TYR mRNA, SILV mRNA, SIL1 mRNA, MLANA mRNA, RAB27A mRNA, OCA2 mRNA, GPR143 mRNA, GPNMB mRNA, MYO6 mRNA, MYRIP mRNA, RPE65 mRNA, RBP1 mRNA, RBP4 mRNA, RDH5 mRNA, RDH11 mRNA, RLBP1 mRNA, MERTK mRNA, ALDH1A3 mRNA, FBLN1 mRNA, SLC16A1 mRNA, KCNV2 mRNA, KCNJ13 mRNA, and CFTR mRNA. 
     
     
         47 . The method of  claim 23 , wherein assaying expression of a set of markers comprises assaying MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1 MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR comprises detecting MITF protein, PAX6 protein, LHX2 protein, TFEC protein, CDH1 protein, CDH3 protein, CLDN10 protein, CLDN16 protein, CLDN19 protein, BEST1 protein, TIMP3 protein, TRPM1 protein, TRPM3 protein, TTR protein, VEGFA protein, CSPG5 protein, DCT protein, TYRP1 protein, TYR protein, SILV protein, SIL1 protein, MLANA protein, RAB27A protein, OCA2 protein, GPR143 protein, GPNMB protein, MYO6 protein, MYRIP protein, RPE65 protein, RBP1 protein, RBP4 protein, RDH5 protein, RDH11 protein, RLBP1 protein, MERTK protein, ALDH1A3 protein, FBLN1 protein, SLC16A1 protein, KCNV2 protein, KCNJ13 protein, and CFTR protein. 
     
     
         48 . A method for authenticating that a cell of interest is a retinal pigment epithelial cell, comprising
 culturing an induced pluripotent stem cell under conditions sufficient to form a retinal pigment epithelial cell to form a cell of interest; and   assaying expression of a set of markers comprising MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DC1, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK in the cell of interest;   wherein expression of MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK indicates the cell of interest is a retinal pigment epithelial cell.   
     
     
         49 . The method of  claim 48 , further comprising
 assaying expression of miR204 and miR211 in the cell of interest;   wherein expression of MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, miR204 and miR211 indicates that the cell of interest is a retinal pigment epithelial cell.   
     
     
         50 . The method of  claim 49 , further comprising
 assaying the polarization of the cell of interest;   wherein expression of MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK, and expression of miR204 and miR211 indicates the cell of interest is a retinal pigment epithelial cell.   
     
     
         51 . The method of  claim 50 , wherein the cell of interest has an apical surface and a basal surface, and wherein assaying polarization comprises measuring selective CO 2  permeability of the apical surface of the cell of interest. 
     
     
         52 . The method of  claim 50 , further comprising
 patch clamping the cell of interest and determining the resting potential of the cell;   wherein expression of MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK, expression of miR204 and miR211, and a resting potential of about −50 to about −60 mV indicates the cell of interest is a retinal pigment epithelial cell.   
     
     
         53 . The method of  claim 52 , further comprising
 evaluating the fluid transport rate of the cell of interest;   wherein expression of MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK, expression of miR204 and miR211, a resting potential of about −50 to about −60 mV, and a fluid transport rate of about 5 to about 10 μl cm −2 h −1  indicates that the cell of interest is a retinal pigment epithelial cell.   
     
     
         54 . The method of  claim 53 , wherein evaluating the fluid transport rate of the cell of interest comprises measuring the fluid transport rate with a capacitance probe. 
     
     
         55 . The method of  claim 48 , wherein assaying the expression of a set of markers comprises assaying MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK comprises detecting MITF mRNA, PAX6 mRNA, TFEC mRNA, CLDN16 mRNA, CLDN19 mRNA, BEST1 mRNA, TIMP3 mRNA, DCT mRNA, TYRP1 mRNA, TYR mRNA, OCA2 mRNA, GPNMB mRNA, MYO6 mRNA, MYRIP mRNA, RPE65 mRNA, RBP1 mRNA, RBP4 mRNA, RDH5 mRNA, RDH11 mRNA, RLBP1 mRNA, and MERTK mRNA. 
     
     
         56 . The method of  claim 48 , wherein assaying the expression of a set of markers comprises assaying MITF, PAX6, TFEC, CLDN16, CLDN19, BEST1, TIMP3, DCT, TYRP1, TYR, OCA2, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, and MERTK comprises detecting MITF protein, PAX6 protein, TFEC protein, CLDN16 protein, CLDN19 protein, BEST1 protein, TIMP3 protein, DCT protein, TYRP1 protein, TYR protein, OCA2 protein, GPNMB protein, MYO6 protein, MYRIP protein, RPE65 protein, RBP1 protein, RBP4 protein, RDH5 protein, RDH11 protein, RLBP1 protein, and MERTK protein. 
     
     
         57 . A method of determining if an induced pluripotent stem cell has differentiated into a retinal pigment epithelial cell;
 culturing an induced pluripotent stem cell under conditions sufficient to form a retinal pigment epithelial cell to produce a cell of interest;   assaying expression of SOX2, PAX6, RPE65, RDH5, TRPM1, and BEST1 in the cell of interest;   assaying the polarization of the cell of interest;   patch clamping the cell of interest to determine the resting potential; and   evaluating the fluid transport rate of the cell;   wherein a decrease in expression of SOX2 and an increase in the expression of PAX6, RPE65, RDH5, TRPM1, and BEST1 in the cell of interest as compared to the induced pluripotent stem cell, a resting potential of about −50 to about −60 mV and a fluid transport rate of about 5 to about 10 μl cm −2 h −1  indicates that the cell derived from the induced pluripotent stem cell is a retinal pigment epithelial cell.   
     
     
         58 . The method of  claim 57 , wherein evaluating the fluid transport rate of the cell of interest comprises measuring the fluid transport rate with a capacitance probe. 
     
     
         59 . The method of  claim 57 , wherein the cell of interest has an apical surface and a basal surface, and wherein assaying polarization comprises measuring selective CO 2  permeability of the apical surface of the cell of interest. 
     
     
         60 . The method of  claim 57 , further comprising
 assaying expression of miR204 and miR211 in the cell of interest;   wherein the decrease in expression of SOX2 and the increase in the expression of PAX6, RPE65, RDH5, TRPM1, and BEST1 as compared to the induced pluripotent stem cell, expression of miR204 and miR211, a resting potential of about −50 to about −60 mV and the fluid transport rate of about 5 to about 10 μl cm −2 h −1  indicates that the cell derived from the induced pluripotent stem cell is a retinal pigment epithelial cell.   
     
     
         61 . The method of  claim 23 , wherein the cell of interest is transformed with a heterologous nucleic acid molecule encoding a marker, wherein the heterologous nucleic acid molecule is operably linked to a retinal pigment epithelial cell specific promoter. 
     
     
         62 . The method of  claim 61 , wherein the retinal specific promoter is a tyrosinase promoter. 
     
     
         63 . The method of  claim 61 , further comprising measuring transepithelial potential of the cell of interest using calomel electrodes in an Üssing chamber. 
     
     
         64 . The method of  claim 63 , wherein assaying expression of expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR comprises performing a multiplex assay to quantitate expression of expression of all of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR. 
     
     
         65 . The method of  claim 63 , wherein assaying expression of expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SU, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR comprises performing quantitative polymerase chain reaction (qPCR) to detect expression of MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR mRNA. 
     
     
         66 . The method of  claim 64 , wherein assaying expression of miR204 and miR211 comprises performing qPCR to detect expression of miR204 and miR211.