Methods of producing RPE cells and compositions of RPE cells
View Patent ↗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 retinal pigment epithelial (RPE) cells, comprising
(a) culturing human pluripotent stem cells for 7-14 days to form embryoid bodies or aggregates in suspension culture in a medium that comprises less than 5% animal-derived protein;
(b) culturing the embryoid bodies or aggregates as an adherent culture in a medium that comprises less than 5% animal-derived protein, wherein RPE cells appear within 14-28 days of adherent culture;
(c) selecting RPE cells from the culture of step (b);
(d) culturing the RPE cells selected in step (c) thereby producing a culture comprising RPE cells.
2. The method of claim 1 , wherein the RPE cells of step (d) are further cultured to produce a culture of mature RPE cells.
3. The method of claim 1 , wherein the cells of step (a) are cultured for 7-10 days.
4. The method of claim 1 , wherein the embryoid bodies or aggregates of step (b) are cultured for 28 days or more.
5. The method of claim 1 , wherein the RPE cells of step (d) are cultured with a growth factor selected from the group consisting of: EGF, bFGF, VEGF, and recombinant insulin-like growth factor.
6. The method of claim 1 , wherein the RPE cells of step (d) are cultured in the presence of one or more of: heparin, hydrocortisone, or ascorbic acid.
7. The method of claim 1 , wherein the human pluripotent stem cells are induced pluripotent stem (iPS) cells.
8. The method of claim 1 , wherein in step (a) the human pluripotent stem cells are cultured to form aggregates.
9. The method of claim 1 , wherein the human pluripotent stem cells are human embryonic stem cells.
10. The method of claim 1 , wherein step (c) comprises contacting the culture of step (b) 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.
11. The method of claim 10 , wherein said enzyme is selected from the group consisting of collagenase IV and dispase.
12. The method of claim 11 , wherein step (c) further comprises dissociating the selected cell clusters containing pigmented cells, thereby forming a single cell suspension comprising RPE cells.
13. The method of claim 1 , wherein the medium in step (b) comprises activin A.