Rpe cell populations and methods of generating same

Abstract

A population of human polygonal RPE cells is disclosed. At least 95% of the cells thereof co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP), wherein the trans-epithelial electrical resistance of the cells is greater than 100 ohms. Methods of generating same are also disclosed.

1 . (canceled) 
     
     
         2 . A method of treating age-related macular degeneration in a subject comprising:
 administering into the subretina of the subject, a composition comprising human retinal pigment epithelium (RPE) cells,   wherein at least 95% of the cells co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP),   wherein the cells have a trans-epithelial electrical resistance (TEER) greater than 100 ohms and, thereby treating the subject.   
     
     
         3 . The method of  claim 2 , wherein the purity of the RPE cells is greater than 98%. 
     
     
         4 . The method of  claim 2 , wherein the PMEL17 and CRABP co-expression in the RPE cells are increased by at least two fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, or at least 50 as compared to control cells. 
     
     
         5 . The method of  claim 2 , wherein the cells have a TEER greater than 300 ohms, greater than 400 ohms, greater than 500 ohms, or greater than 600 ohms. 
     
     
         6 . The method of  claim 2 , wherein the cells secrete pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in a polarized manner, and wherein the ratio of apical/basal secretion of PEDF or VEGF is greater than 1. 
     
     
         7 . The method of  claim 6 , wherein the ratio of apical/basal secretion of PEDF is between 1 and 9. 
     
     
         8 . The method of  claim 6 , wherein the ratio of apical/basal secretion of VEGF is between 1 and 3. 
     
     
         9 . The method of  claim 6 , wherein the secretion of PEDF and VEGF remains stable following incubation of the cells at 2-8° C. for 6 hours, 8 hours, 10 hours, 12 hours or 24 hours. 
     
     
         10 . The method of  claim 2 , wherein the TEER of the cells remains stable in the cells following their incubation at 2-8° C. for 6 hours, 8 hours, 10 hours, 12 hours or 24 hours. 
     
     
         11 . The method of  claim 2 , wherein the number of RPE cells increases after administration to the subretina of the subject. 
     
     
         12 . The method of  claim 2  wherein the composition is administered in an amount from about 50×103 cells to about 100×103 cells per administration. 
     
     
         13 . The method of  claim 2 , wherein the cells secrete angiogenin, tissue inhibitor metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp 130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor-α (sTNF-R1) in a polarized manner. 
     
     
         14 . The method of  claim 2 , wherein the cells are capable of rescuing visual acuity. 
     
     
         15 . The method of  claim 2 , wherein the cells are generated by ex-vivo differentiation of human embryonic stem cells. 
     
     
         16 . The method of  claim 2 , wherein the RPE cells are generated by: (a) culturing human embryonic stem cells or induced pluripotent stem cells in a medium comprising nicotinamide, wherein said medium is devoid of activin A; (b) culturing the differentiating cells in a medium comprising nicotinamide and activing A to generate cells which are further differentiated towards the RPE lineage; and (c) culturing the further differentiated cells in a medium comprising nicotinamide, and wherein said medium is devoid of activin A. 
     
     
         17 . The method of  claim 16 , wherein said embryonic stem cells or induced pluripotent stem cells are propagated in a medium comprising bFGF and TGFβ. 
     
     
         18 . The method of  claim 2 , wherein the RPE cells comprise a polygonal shape morphology. 
     
     
         19 . The method of  claim 2 , wherein the RPE cells comprise less than 1:250,000 of Oct4+TRA-1-60+ cells. 
     
     
         20 . The method of  claim 2 , wherein at least 80% of the RPE cells express Bestrophin 1, PAX-6 or microphthalmia-associated transcription factor (MITF). 
     
     
         21 . A method of treating a retinal disease in a subject comprising:
 administering into the subretina of the subject, a composition comprising human retinal pigment epithelium (RPE) cells,   wherein at least 95% of the cells co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP),   wherein the cells have a trans-epithelial electrical resistance greater than 100 ohms.   
     
     
         22 . The method of  claim 21 , wherein the retinal disease comprises age related macular degeneration (AMD), retinitis pigmentosa (RP), retinoschisis, lattice degeneration, or Best disease.