Retinal pigment epithelial cells differentiated from embryonic stem cells with nicotinamide and activin a

Abstract

The present invention concerns RPE cells obtainable by directed differentiation from stem cell, particularly, human stem cells. It has been specifically found that culturing stem cells in the presence of one or more member of the TGFβ superfamily, such as Activin A) induced directed differentiation into mature and functional RPE cells. This was evidenced by the expression of markers specific to mature RPE cells, including MiTF-A, RPE65 or Bestrophin). In accordance with one particular embodiment, the cells are a priori cultured with nicotinamide (NA) which was found to augment the cells' response to the inductive effect of the one or more member of the TGFβ superfamily. The invention also provides methods of performing the directed differentiation, as well as methods for use of the resulting RPE cells.

1 . A method of transplanting retinal pigment epithelial (RPE) cells into a subject's eye, comprising:
 (a) culturing human stem cells in a medium comprising a differentiating agent to obtain differentiating cells;   (b) culturing said differentiating cells in a medium comprising one or more members of the TGFβ superfamily to obtain differentiated RPE cells;   (c) harvesting said differentiated RPE cells; and   (d) transplanting said differentiated RPE cells into the subject's eye, thereby transplanting RPE cells into the subject's eye.   
     
     
         2 . The method of  claim 1 , wherein said transplanting of said differentiated RPE cells is effected at the subretinal space of the eye. 
     
     
         3 . The method of  claim 1 , wherein said RPE cells are transplanted in a suspension, or as a monolayer of cells immobilized on a matrix or a substrate. 
     
     
         4 . The method of  claim 1 , wherein said human stem cells comprise human pluripotent stem cells. 
     
     
         5 . The method of  claim 4 , wherein said human pluripotent stem cells comprise human embryonic stem cells. 
     
     
         6 . The method of  claim 1 , wherein said differentiating agent comprises nicotinamide. 
     
     
         7 . The method of  claim 1 , wherein said member of the TGFβ superfamily is TGFβ1, TGFβ3 or activin A. 
     
     
         8 . The method of  claim 6 , wherein said medium of step (b) comprises nicotinamide and activin A. 
     
     
         9 . A method of treating a retinal disease or disorder in a subject in need thereof, comprising:
 (a) culturing human stem cells in a medium comprising a differentiating agent to obtain differentiating cells;   (b) culturing said differentiating cells in a medium comprising one or more members of the TGFβ superfamily to obtain differentiated RPE cells;   (c) harvesting said differentiated RPE cells; and   (d) transplanting said differentiated RPE cells into the subject's eye, thereby treating the retinal disease or disorder.   
     
     
         10 . The method of  claim 9 , wherein said transplanting of said differentiated RPE cells is effected at the subretinal space of the eye. 
     
     
         11 . The method of  claim 9 , wherein said RPE cells are transplanted in a suspension, or as a monolayer of cells immobilized on a matrix or a substrate. 
     
     
         12 . The method of  claim 9 , wherein said human stem cells comprise human pluripotent stem cells. 
     
     
         13 . The method of  claim 12 , wherein said human pluripotent stem cells comprise human embryonic stem cells. 
     
     
         14 . The method of  claim 9 , wherein said differentiating agent comprises nicotinamide. 
     
     
         15 . The method of  claim 9 , wherein said member of the TGFβ superfamily is TGFβ1, TGFβ3 or activin A. 
     
     
         16 . The method of  claim 14 , wherein said medium of step (b) comprises nicotinamide and activin A. 
     
     
         17 . The method of  claim 9 , wherein said retinal disease or disorder is selected from at least one of retinitis pigmentosa, lebers congenital amaurosis, hereditary or acquired macular degeneration, age related macular degeneration (AMD), Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE dystrophies, Stargardt disease, RPE and retinal damage due to damage caused by any one of photic, laser, inflammatory, infectious, radiation, neovascular or traumatic injury.