Methods for producing enriched populations of human retinal pigment epithelium cells
First Claim
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1. A method for producing an enriched population of human retinal pigment epithelium (“
- RPE”
) cells, the method comprising;
(a) providing a multilayer population of human embryonic stem (hES) cells;
(b) culturing said multilayer population of hES cells under conditions that do not maintain the undifferentiated state of said hES cells for a sufficient time to allow for the appearance of putative human RPE cells, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm;
(c) selecting one or more of said putative human RPE cells from the culture of step (b); and
(d) culturing said one or more of said putative human RPE cells selected in step (c) to form a culture containing cells that are Pax6−
, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm.
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Abstract
This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.
42 Citations
41 Claims
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1. A method for producing an enriched population of human retinal pigment epithelium (“
- RPE”
) cells, the method comprising;(a) providing a multilayer population of human embryonic stem (hES) cells; (b) culturing said multilayer population of hES cells under conditions that do not maintain the undifferentiated state of said hES cells for a sufficient time to allow for the appearance of putative human RPE cells, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm; (c) selecting one or more of said putative human RPE cells from the culture of step (b); and (d) culturing said one or more of said putative human RPE cells selected in step (c) to form a culture containing cells that are Pax6−
, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- RPE”
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12. A method for producing an enriched population of human RPE cells, the method comprising:
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(a) providing a culture of hES cells; (b) culturing the hES cells to produce one or more embryoid bodies; (c) culturing said one or more embryoid bodies for a sufficient time to allow for the appearance of putative human RPE cells within at least one of said one or more embryoid bodies, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm, whereby one or more embryoid bodies containing putative human RPE cells are formed; (d) selecting and dissociating one or more of said embryoid bodies containing putative human RPE cells from the culture of step (c) to obtain human RPE cells; and (e) culturing said putative human RPE cells obtained in step (d) to form a culture containing cells that are Pax6−
, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm, thereby producing an enriched population of human RPE cells. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
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20. A method for producing an enriched population of human RPE cells, the method comprising:
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(a) providing a multilayer population of hES cells; (b) culturing said multilayer population of hES cells under conditions that do not maintain the undifferentiated state of said hES cells for a sufficient time for the appearance of putative human RPE cells, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm, wherein said conditions that do not maintain the undifferentiated state of said hES cells comprise media lacking exogenously added FGF and the duration of culturing is at least 6 weeks; (c) selecting one or more of said putative human RPE cells from the culture of step (b) to obtain human RPE cells; and (d) culturing said human RPE cells selected in step (c) to form a cell monolayer containing cells that are Pax6−
, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A method for producing an enriched population of human RPE cells, the method comprising:
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(a) providing a culture of hES cells; (b) culturing the hES cells to produce one or more embryoid bodies; (c) culturing said one or more embryoid bodies for a sufficient time for the appearance of putative human RPE cells within at least one of said one or more embryoid bodies, wherein said putative human RPE cells comprise brown pigment dispersed within their cytoplasm, and the duration of culturing is at least 6 weeks, whereby one or more embryoid bodies containing putative human RPE cells are formed; (d) selecting and dissociating one or more of said embryoid bodies containing putative human RPE cells from the culture of step (c) to obtain human RPE cells; and (e) culturing said human RPE cells obtained in step (d) to form a culture containing cells that are Pax6−
, bestrophin+, CRALBP+, PEDF+, and express RPE65, and exhibit a characteristic cobblestone, polygonal, epithelial-like appearance and comprise brown pigment dispersed within their cytoplasm, thereby producing an enriched population of human RPE cells. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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Specification