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 . An isolated population of cells comprising human retinal pigment epithelial (RPE) cells and a marker selected from the group consisting of bestrophin and RPE65 at a higher level than the expression of said marker in RPE cells obtained by spontaneous differentiation from embryonic stem cells. 
     
     
         2 . The isolated population of cells of  claim 1 , wherein said human retinal pigment epithelial cells express Pax6. 
     
     
         3 . The isolated population of cells of  claim 1 , obtained by directed differentiation of human stem cells in the presence of one or more members of the TGFβ superfamily. 
     
     
         4 . The isolated population of cells of  claim 1  obtained by:
 (a) culturing human stem cells in a medium comprising a differentiating agent to obtain differentiating cells; and 
 (b) culturing said differentiating cells in a medium comprising one or more members of the TGFβ superfamily to obtain differentiated RPE cells. 
 
     
     
         5 . The isolated population of cells of  claim 4 , wherein said differentiating agent comprises nicotinamide. 
     
     
         6 . The isolated population of cells of  claim 1 , wherein said human stem cells comprise human pluripotent stem cells. 
     
     
         7 . The isolated population of cells of  claim 6 , wherein said human pluripotent stem cells comprise human embryonic stem cells. 
     
     
         8 . The isolated population of cells of  claim 4 , wherein said member of the TGFβ superfamily is TGFβ1, TGFβ3 or activin A. 
     
     
         9 . The isolated population of cells of  claim 5 , wherein said medium of step (b) comprises nicotinamide and activin A. 
     
     
         10 . The isolated population of cells of  claim 1 , being terminally differentiated RPE cells. 
     
     
         11 . The isolated population of cells of  claim 1  being in a dosage form suitable for intraocular transplantation into a subject.