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 culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and
b) differentiating the cells under a hypoxic atmosphere having less than 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.
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Citations
67 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 culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD34+ progenitor cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 63)
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34. 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 culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD43+ progenitor cells.
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35. 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 culture medium that is free or essentially free of feeder cells comprising a matrix component and at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF; and b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD31+ progenitor cells.
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36. A method for differentiating a human pluripotent stem cell into a CD31+ progenitor cell comprising growing a pluripotent stem cell in a culture comprising:
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a) a matrix component; and b) a differentiation medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, wherein the culture is free or essentially free of non-human animal serum, feeder cells, and Matrigel™
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37. (canceled)
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39. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising growing a pluripotent stem cell in a culture comprising:
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a) a matrix component; and b) a differentiation medium comprising at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, wherein the culture is free or essentially free of non-human animal serum, feeder cells, and Matrigel™
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40. (canceled)
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41. (canceled)
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42. A method for differentiating a human pluripotent stem cell into a CD31+ progenitor cell comprising culturing a pluripotent stem cell in a defined differentiation medium.
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43. A method for differentiating a human pluripotent stem cell into a CD34+ progenitor cell comprising culturing a pluripotent stem cell in a defined differentiation medium.
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44. A method for differentiating a human pluripotent stem cell into a CD43+ progenitor cell comprising culturing a pluripotent stem cell in a defined differentiation medium.
- 45. A differentiation culture medium comprising BMP-4, VEGF, bFGF, and a matrix component, wherein the medium is free or essentially free of feeder cells, serum, and Matrigel™
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46. (canceled)
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60. A method of screening a candidate substance for ability to promote differentiation of a pluripotent cell into a CD34+ progenitor cell, comprising:
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a) culturing a pluripotent stem cell in a culture medium that is free or essentially free of feeder cells, wherein the medium comprises a matrix component, at least one recombinant growth factor selected from the group consisting of BMP-4, VEGF, and bFGF, and a candidate substance; b) differentiating the cells under a hypoxic atmosphere having less than 5.5% oxygen for a period of time to provide the CD34+ progenitor cells; and c) assessing the pluripotent cell for differentiation into a CD34+ progenitor cell.
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61. (canceled)
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64. A clonal cell population comprising CD34+ progenitor cells, wherein at least 5% of the cells are CD34+ or CD43+ progenitor cells and the cell population is in a defined medium that is free or essentially free of serum and feeder cells.
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65. (canceled)
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66. A clonal cell population comprising CD31+ progenitor cells, wherein at least 20% of the cells are CD31+ or CD34+ progenitor cells and the cell population is in a defined medium that is free or essentially free of serum and feeder cells.
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67. (canceled)
Specification