Methods of Producing RPE Cells and Compositions of RPE Cells

Abstract

The present invention provides improved methods for producing RPE cells from human embryonic stem cells or from other human pluripotent stem cells. The invention also relates to human retinal pigmented epithelial cells derived from human embryonic stem cells or other human multipotent or pluripotent stem cells. hRPE cells derived from embryonic stem cells are molecularly distinct from adult and fetal-derived RPE cells, and are also distinct from embryonic stem cells. The hRPE cells described herein are useful for treating retinal degenerative diseases.

1 . A method comprising:
 a) providing human pluripotent cells;   b) culturing the human pluripotent cells to form embryoid bodies or aggregates in a nutrient rich, low protein medium;   c) culturing the embryoid bodies or aggregates as an adherent culture in nutrient rich, low protein medium;   d) culturing the cells of step (c) in a medium capable of supporting growth of high-density somatic cell culture, whereby RPE cells appear in the culture of cells;   e) selecting RPE cells from the culture of step (d);   f) culturing the RPE cells selected in step (e) in a medium comprising one or more factors that promote growth of RPE progenitor cells; and   g) culturing the culture of step (f) in a medium that promotes formation of RPE cells, thereby producing a culture comprising RPE cells.   
     
     
         2 . The method of  claim 1 , further comprising culturing the RPE cells of step (f) to produce a culture of mature RPE cells. 
     
     
         3 . The method of  claim 1 , wherein the medium of step (b) contains serum free B-27 supplement and the medium of step (c) does not contain serum free B-27 supplement. 
     
     
         4 . The method of  claim 1 , wherein the culture medium in step (d) is a low-protein or protein-free medium. 
     
     
         5 . The method of  claim 1 , wherein the factor in step (f) is selected from the group consisting of: EGF, bFGF, VEGF, insulin-like growth factor, heparin, hydrocortisone, and ascorbic acid. 
     
     
         6 - 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the cells of step (d) are cultured for 14 or more days. 
     
     
         11 - 17 . (canceled) 
     
     
         18 . The method  claim 1 , wherein step (e) comprises contacting the culture of step (d) with an enzyme that causes RPE cells to detach from the adherent culture and selecting detached pigmented cells or detached cell clusters containing pigmented cells. 
     
     
         19 - 37 . (canceled) 
     
     
         38 . A method of treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a preparation comprising RPE cells, wherein said RPE cells are derived from human pluripotent cells according to the method of  claim 1 . 
     
     
         39 . The method of  claim 38 , wherein the condition is selected from the group consisting of: Stargardt's macular dystrophy, age related macular degeneration, or retinitis pigmentosa. 
     
     
         40 - 43 . (canceled) 
     
     
         44 . A composition comprising human RPE cells that are derived from human pluripotent cells according to the method of  claim 1 . 
     
     
         45 . The composition of  claim 44  which is a pharmaceutical composition for treating or preventing a condition characterized by retinal degeneration that is selected from the group consisting of: Stargardt's macular dystrophy, age related macular degeneration, or retinitis pigmentosa. 
     
     
         46 - 54 . (canceled) 
     
     
         55 . The method of  claim 1 , wherein the human pluripotent stem cells are induced pluripotent stem (iPS) cells or embryonic stem cells. 
     
     
         56 - 91 . (canceled) 
     
     
         92 . The composition of  claim 44 , wherein RPE cells in said preparation express one or more markers selected from the group consisting of: RPE-65, Bestrophin, PEDF, CRALBP, Otx2, Mit-F, pax-2, pax-6, and tyrosinase. 
     
     
         93 . The composition of  claim 92 , which comprises at least 75% RPE cells. 
     
     
         94 . The composition of  claim 92 , which comprises at least 30% mature RPE cells. 
     
     
         95 - 99 . (canceled) 
     
     
         100 . The composition of  claim 92 , wherein the RPE cells lack substantial expression of one or more ES cell genes selected from the group consisting of: Oct-4, nanog, and Rex-1. 
     
     
         101 - 134 . (canceled) 
     
     
         135 . The method of  claim 18 , wherein said enzyme is selected from the group consisting of collagenase IV and dispase. 
     
     
         136 . The method of  claim 135 , wherein step (e) further comprises dissociating the selected cell clusters containing pigmented cells, thereby forming a single cell suspension comprising RPE cells. 
     
     
         137 . The method of  claim 1 , wherein the medium in step (f) comprises bFGF. 
     
     
         138 . The method of  claim 1 , wherein the medium in one or more of steps (b), (c), (d), (f) and (g) comprises activin A.