METHODS OF PRODUCING RPE CELLS AND COMPOSITIONS OF RPE CELLS
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 of producing a substantially purified culture of retinal pigment epithelial (RPE) cells, comprising
a) providing human embryonic stem cells;
b) culturing the human embryonic stem cells as embryoid bodies in nutrient rich, low protein medium;
c) culturing the embryoid bodies as an adherent culture in nutrient rich, low protein medium;
d) culturing the adherent culture of cells of (c) in nutrient rich, low protein medium, which medium does not contain serum free B-27 supplement;
e) culturing the cells of (d) in medium capable of supporting growth of high-density somatic cell culture, whereby RPE cells appear in the culture of cells
f) contacting the culture of (e) with an enzyme;
g) selecting the RPE cells from the culture and transferring the RPE cells to a separate culture containing medium supplemented with a growth factor to produce an enriched culture of RPE cells; and
h) propagating the enriched culture on RPE cells to produce
(i) a substantially purified culture of RPE cells, and/or
(ii) mature RPE cells.
2 - 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, which RPE cells are derived from human pluripotent stem cells according to the method of claim 1 .
39 - 52 . (canceled)
53 . A method of producing a substantially purified culture of retinal pigment epithelial (RPE) cells, comprising
a) providing human pluripotent stem cells;
b) culturing the human pluripotent stem cells in nutrient rich, low protein medium;
c) culturing the human pluripotent stem cells as an adherent culture in nutrient rich, low protein medium;
d) culturing the adherent culture of cells of (c) in nutrient rich, low protein medium, which medium does not contain serum free B-27 supplement;
e) culturing the cells of (d) in medium capable of supporting growth of high-density somatic cell culture, whereby RPE cells appear in the culture of cells;
f) contacting the culture of (e) with an enzyme;
g) selecting the RPE cells from the culture and transferring the RPE cells to a separate culture containing medium supplemented with a growth factor to produce an enriched culture of RPE cells; and
h) propagating the enriched culture on RPE cells to produce a substantially purified culture of RPE cells.
54 . (canceled)
55 . The method of claim 53 , wherein the human pluripotent stem cells are induced pluripotent stem (iPS) cells.
56 . (canceled)
57 . The method of claim 53 , wherein the human pluripotent stem cells are human embryonic stem cells.
58 - 61 . (canceled 0
62 . The method of claim 53 , wherein the medium contains one or more of: heparin, hydrocortisone, or ascorbic acid.
63 - 71 . (canceled)
72 . The method of claim 53 , wherein the growth factor of (g) is selected from the group consisting of: EGF, bFGF, VEGF, and recombinant insulin-like growth factor.
73 . The method of claim 53 , wherein the medium of (g) contains one or more of: heparin, hydrocortisone, or ascorbic acid.
74 . The method of claim 53 , wherein the enzyme of (f) is selected from the group consisting of: trypsin, collagenase, and dispase.
75 - 76 . (canceled)
77 . 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, which RPE cells are derived from human induced pluripotent stem (iPS) cells.
78 - 123 . (canceled)
124 . A method of treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of the preparation of claim 92 , which RPE cells are derived from human pluripotent stem cells.
125 - 134 . (canceled)