Methods and compositions for the differentiation of stem cells
First Claim
1. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising:
- a) culturing a pluripotent stem cell in a first culture medium that is free or essentially free of feeder cells, the culture comprising a matrix component, wherein said culturing includesi) dispersing a pluripotent stem cell colony or clonal cell grouping to form dispersed essentially individual cells, andii) seeding the dispersed cells into the first culture medium at a density of from about 10,000 stem cells per square centimeter of culturing surface to less than 50,000 stem cells per square centimeter of culturing surface; and
b) differentiating the dispersed cells in a differentiation culture medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, under a hypoxic atmosphere having less than or equal to 5.5% oxygen for a period of time to provide the CD34+ progenitor cells.
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Accused Products
Abstract
The present invention provides methods and compositions for the production of hematopoietic progenitor cells or endothelial progenitor cells from human pluripotent stem cells using a defined cell culture medium without the need to utilize feeder cells or serum. In some embodiments, differentiation is accomplished using hypoxic atmospheric conditions. The defined medium of the present invention may contain growth factors and a matrix component. The hematopoietic progenitor cells may be further differentiated into cell lineages including red blood cells, macrophages, granulocytes, and megakaryocytes. The endothelial progenitor cells may be further differentiated into endothelial cells. Also disclosed are screening assays for identification of candidate substances that affect differentiation of pluripotent stem cells into progenitor cells.
12 Citations
36 Claims
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1. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising:
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a) culturing a pluripotent stem cell in a first culture medium that is free or essentially free of feeder cells, the culture comprising a matrix component, wherein said culturing includes i) dispersing a pluripotent stem cell colony or clonal cell grouping to form dispersed essentially individual cells, and ii) seeding the dispersed cells into the first culture medium at a density of from about 10,000 stem cells per square centimeter of culturing surface to less than 50,000 stem cells per square centimeter of culturing surface; and b) differentiating the dispersed cells in a differentiation culture medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, under a hypoxic atmosphere having less than or equal to 5.5% oxygen for a period of time to provide the CD34+ progenitor cells. - View Dependent Claims (2, 3, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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4. A method for differentiating a human pluripotent stem cell into a CD43+ progenitor cell comprising:
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a) culturing a pluripotent stem cell in a first culture medium that is free or essentially free of feeder cells, the culture comprising a matrix component, wherein said culturing includes i) dispersing a pluripotent stem cell colony or clonal cell grouping to form dispersed essentially individual cells, and ii) seeding the dispersed cells into the first culture medium at a density of from about 10,000 stem cells per square centimeter of culturing surface to less than 50,000 stem cells per square centimeter of culturing surface; and b) differentiating the dispersed cells in a differentiation culture medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, under a hypoxic atmosphere having less than or equal to 5.5% oxygen for a period of time to provide the CD43+ progenitor cells. - View Dependent Claims (5, 6)
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7. A method for differentiating a human pluripotent stem cell into a CD31+ progenitor cell comprising:
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a) culturing a pluripotent stem cell in a first culture medium that is free or essentially free of feeder cells, the culture comprising a matrix component, wherein said culturing includes i) dispersing a pluripotent stem cell colony or clonal cell grouping to form dispersed essentially individual cells, and ii) seeding the dispersed cells into the first culture medium at a density of from about 10,000 stem cells per square centimeter of culturing surface to less than 50,000 stem cells per square centimeter of culturing surface; and b) differentiating the dispersed cells in a differentiation culture medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, under a hypoxic atmosphere having less than or equal to 5.5% oxygen for a period of time to provide the CD31+ progenitor cells. - View Dependent Claims (8, 9)
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Specification