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 population of human RPE cells, wherein at least 80% of the cells thereof co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP) and wherein cells of the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor-α (sTNF-R1). 
     
     
         3 . (canceled) 
     
     
         4 . The cell population of  claim 2 , wherein the cells secrete said angiogenin, said TIMP2, said sgp130 or said sTNF-R1 in a polarized manner. 
     
     
         5 . The cell population of  claim 2 , wherein the cells secrete each of said angiogenin, said TIMP2, said sgp130 and said sTNF-R1 in a polarized manner. 
     
     
         6 . The cell population of  claim 4 , wherein the ratio of apical secretion of sgp130:basal secretion of sgp130 is greater than 1. 
     
     
         7 . The cell population of  claim 4 , wherein the ratio of apical secretion of sTNF-R1:basal secretion of sTNF-R1 is greater than 1. 
     
     
         8 . The cell population of  claim 4 , wherein the ratio of basal secretion of angiogenin:apical secretion of angiogenin is greater than 1. 
     
     
         9 . The cell population of  claim 4 , wherein the ratio of apical secretion of TIMP2:basal secretion of TIMP2 is greater than 1. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The cell population of  claim 2 , wherein at least 80% of the cells express Microphthalmia-associated transcription factor (MITF), as measured by immunostaining. 
     
     
         13 . The cell population of  claim 2 , wherein more than 50% of the cells express paired box gene 6 (PAX-6) as measured by FACS. 
     
     
         14 . The cell population of  claim 2 , wherein the cells secrete greater than 750 ng of Pigment epithelium-derived factor (PEDF) per ml per day. 
     
     
         15 . The cell population of  claim 2 , wherein the cells secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner. 
     
     
         16 . The cell population of  claim 15 , wherein the ratio of apical secretion of PEDF:basal secretion of PEDF is greater than 1. 
     
     
         17 . The cell population of  claim 16 , wherein said ratio remains greater than 1 following incubation for 8 hours at 2-8° C. 
     
     
         18 . The cell population of  claim 2 , wherein the trans-epithelial electrical resistance of the population of cells is greater than 100 ohms. 
     
     
         19 . The cell population of  claim 18 , wherein said trans-epithelial electrical resistance of the cells remains greater than 100 ohms following incubation for 8 hours at 2-8° C. 
     
     
         20 . The cell population of  claim 15 , wherein the ratio of basal secretion of VEGF:apical secretion of VEGF is greater than 1. 
     
     
         21 . The cell population of  claim 20 , wherein said ratio remains greater than 1 following incubation for 8 hours at 2-8° C. 
     
     
         22 . The cell population of  claim 2 , being capable of rescuing visual acuity in the RCS rat following subretinal administration. 
     
     
         23 . The cell population of  claim 2 , being capable of rescuing photoreceptors for at least 180 days post-subretinal administration in the RCS rat. 
     
     
         24 . The cell population of  claim 2 , being generated by ex-vivo differentiation of human embryonic stem cells. 
     
     
         25 . (canceled) 
     
     
         26 . The cell population of  claim 24 , wherein said embryonic stem cells are propagated in a medium comprising bFGF and TGFβ. 
     
     
         27 . The cell population of  claim 24 , wherein said embryonic stem cells are cultured on human cord fibroblasts. 
     
     
         28 . (canceled) 
     
     
         29 . The cell population of  claim 27 , wherein the method further comprises culturing said differentiated cells in a medium under conditions wherein the atmospheric oxygen level is greater than about 10% in the presence of nicotinamide following step (c). 
     
     
         30 . A pharmaceutical composition comprising the cell population of  claim 2 , as the active agent and a pharmaceutically acceptable carrier. 
     
     
         31 . Use of the cell population of  claim 2 , for treating a retinal degeneration. 
     
     
         32 . A method of generating RPE cells comprising:
 (a) culturing pluripotent stem cells in a medium comprising a differentiating agent so as to generate differentiating cells, wherein said medium is devoid of a member of the transforming growth factor β (TGF β) superfamily;   (b) culturing said differentiating cells in a medium comprising said member of the transforming growth factor β (TGF β) superfamily and said differentiating agent to generate cells which are further differentiated towards the RPE lineage;   (c) culturing said cells which are further differentiated towards the RPE lineage in a medium comprising a differentiating agent so as to generate RPE cells, wherein said medium is devoid of a member of the transforming growth factor β (TGF β) superfamily, wherein steps (a)-(c) are effected under conditions wherein the atmospheric oxygen level is less than about 10%.   
     
     
         33 . The method of  claim 32 , wherein step (a) is effected under non-adherent conditions. 
     
     
         34 . The method of  claim 33 , wherein said non-adherent conditions comprise a non-adherent culture plate. 
     
     
         35 . The method of  claim 32 , wherein step (a) comprises:
 i) culturing said cultured population of human pluripotent stem cells in a medium comprising nicotinamide, in the absence of activin A; under non-adherent conditions to generate a cluster of cells comprising differentiating cells; and subsequently   ii) culturing said differentiating cells of (i) in a medium comprising nicotinamide, in the absence of activin A under adherent conditions.   
     
     
         36 . The method of  claim 35 , further comprising dissociating said cluster of cells prior to step (ii) to generate clumps of cells or a single cell suspension of cells. 
     
     
         37 . The method of  claim 32 , further comprising culturing said differentiated cells in a medium under conditions wherein the atmospheric oxygen level is greater than about 10% in the presence of a differentiating agent following step (c). 
     
     
         38 . The method of  claim 32 , wherein said member of the transforming growth factor β (TGF β) superfamily is selected from the group consisting of TGF 62  1, TGFβ3 and activin A. 
     
     
         39 . The method of  claim 32 , wherein said differentiating agent of step (a) and said differentiating agent of step (c) are identical. 
     
     
         40 . The method of  claim 32 , wherein said differentiating agent of step (a) is nicotinamide (NA) or 3-aminobenzamide. 
     
     
         41 . The method of  claim 32 , further comprising selecting polygonal cells following step (c). 
     
     
         42 . The method of  claim 41 , further comprising propagating said polygonal cells. 
     
     
         43 . The method of  claim 42 , wherein said propagating is effected on an adherent surface. 
     
     
         44 . The method of  claim 32 , wherein said pluripotent stem cells comprise embryonic stem cells. 
     
     
         45 . The method of  claim 44 , wherein said embryonic stem cells are propagated in a medium comprising bFGF and TGFβ. 
     
     
         46 . The method of  claim 44 , wherein said embryonic stem cells are cultured on human cord fibroblasts.