Methods of producing rpe cells and compositions of rpe cells
AssigneeAdvanced Cell Technology, Inc.
InventorsChristopher Malcuit, Linda Lemieux, William Holmes, Pedro Huertas, Lucy Vilner
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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.
Claims30
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 as embryoid bodies or aggregates in nutrient rich, low protein medium that comprises less than 5% protein;
- c) culturing the embryoid bodies or aggregates as an adherent culture in nutrient rich, low protein medium that comprises less than 5% protein;
- d) culturing the cells of (c) in medium capable of supporting growth of high-density somatic cell culture, whereby RPE cells appear in the culture of cells;
- e) contacting the culture of (d) with an enzyme;
- f) 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.
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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 that comprises less than 5% protein;
- c) culturing the human pluripotent stem cells as an adherent culture in nutrient rich, low protein medium that comprises less than 5% protein;
- d) culturing the cells of (c) in medium capable of supporting growth of high-density somatic cell culture, whereby RPE cells appear in the culture of cells;
- e) contacting the culture of (d) with an enzyme;
- f) 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
- g) propagating the enriched culture on RPE cells to produce a substantially purified culture of RPE cells.
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A method comprising:
- a) culturing the human pluripotent cells to form embryoid bodies or aggregates in a nutrient rich, low protein medium, wherein the low protein medium comprises less than 5% protein;
- b) culturing the embryoid bodies or aggregates of step (a) as an adherent culture in a nutrient rich, low protein medium, wherein the low protein medium comprises less than 5% protein;
- c) culturing the cells of step (b) in a medium capable of supporting growth of high-density somatic cell culture, whereby RPE cells appear in the culture of cells;
- d) selecting RPE cells from the culture of step (c);
- e) culturing the RPE cells selected in step (d) in a medium comprising one or more factors;
- and
- f) culturing the culture of step (e) in a medium that promotes formation of RPE cells, thereby producing a culture comprising RPE cells.
Dependencies are inferred from the claim text.