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Differentiation of pluripotent cells

  • US 8,372,642 B2
  • Filed: 03/01/2010
  • Issued: 02/12/2013
  • Est. Priority Date: 02/27/2009
  • Status: Active Grant
First Claim
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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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