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Differentiation of pluripotent cells

  • US 10,100,282 B2
  • Filed: 02/01/2013
  • Issued: 10/16/2018
  • 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 (HPCs) 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, wherein said cells are cultured or maintained in said media on a matrix-coated surface;

    (b) culturing individualized pluripotent cells from step a) under aggregate forming conditions in a second defined, feeder-free media comprising a ROCK inhibitor, for a period of time sufficient to form cell aggregates;

    (c) culturing the cell aggregates of step (b) in a third defined media comprising an amount of BMP4, VEGF, and FGF-2 or an FGF-2 mimic, for a period of time sufficient to expand or promote differentiation of cells to a mesodermal lineage;

    (d) culturing the cells of step (c) in a fourth defined media comprising an amount of IL-3 and Flt3 ligand, for a period of time sufficient to further expand or promote differentiation of the cells to HPCs and endothelial cells;

    (e) optionally, where a further enrichment of endothelial cells is desired, the fourth defined media of step (d) further includes VEGF and FGF; and

    wherein a plurality of the pluripotent cells are differentiated into hematopoietic precursor cells or endothelial cells, defined as cells that express CD34 and CD3.

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