Differentiation of pluripotent cells
First Claim
1. A method of differentiating pluripotent cells into hematopoietic precursor cells or endothelial cells comprising the sequential steps of:
- a) culturing or maintaining a plurality of substantially undifferentiated pluripotent cells in a first defined media comprising at least one growth factor appropriate to maintain pluripotency;
b) incubating the cells in a second defined media which is free or essentially free of BMP4, VEGF, IL-3, FIG ligand, and GMCSF, wherein said media comprises about 0.1 ng/ml or less of TGFβ and
about 20 ng/ml or less of FGF-2, to precondition the cells for differentiation;
c) culturing the preconditioned cells in a third defined media comprising an amount of BMP4 and VEGF sufficient to promote differentiation in a plurality of the cells; and
d) culturing the cells in a fourth defined media comprising an amount of either (1) IL-3 and Flt3 ligand, or (2) VEGF, FGF-2 or an FGF-2 mimic, and IGF sufficient to expand and promote differentiation in a plurality of the cells;
wherein a plurality of the pluripotent cells are differentiated into hematopoietic precursor cells or endothelial cells.
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Accused Products
Abstract
Provided herein are methods for the in vitro maintenance, expansion, culture, and/or differentiation of pluripotent cells, such as human embryonic stem cells (hESC) or induced pluripotent cells (iPSC), into hematopoietic precursor cells or endothelial cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the pluripotent cells into hematopoietic precursor cells or endothelial cells. The resulting hematopoietic precursor cells may be further differentiated into various myeloid or lymphoid lineages.
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Citations
54 Claims
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1. A method of differentiating pluripotent cells into hematopoietic precursor cells or endothelial cells comprising the sequential steps of:
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a) culturing or maintaining a plurality of substantially undifferentiated pluripotent cells in a first defined media comprising at least one growth factor appropriate to maintain pluripotency; b) incubating the cells in a second defined media which is free or essentially free of BMP4, VEGF, IL-3, FIG ligand, and GMCSF, wherein said media comprises about 0.1 ng/ml or less of TGFβ and
about 20 ng/ml or less of FGF-2, to precondition the cells for differentiation;c) culturing the preconditioned cells in a third defined media comprising an amount of BMP4 and VEGF sufficient to promote differentiation in a plurality of the cells; and d) culturing the cells in a fourth defined media comprising an amount of either (1) IL-3 and Flt3 ligand, or (2) VEGF, FGF-2 or an FGF-2 mimic, and IGF sufficient to expand and promote differentiation in a plurality of the cells; wherein a plurality of the pluripotent cells are differentiated into hematopoietic precursor cells or endothelial 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
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Specification