Generating populations of human blood and blood vessel progenitors from pluripotent stem cells
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsKyle M. Loh, Lay Teng Ang, Alana T. Nguyen, Jonas Fowler, Irving L. Weissman
Life of this patent
Estimating the term…
Abstract
Methods are provided for the efficient differentiation of hPSCs into HSC-like cells and endothelial cells in defined, monolayer conditions solely using extracellular signals to guide differentiation. The instant disclosure also provides methods of screening for cellular responses of the generated hematopoietic stem cells, endothelial cells and derivatives thereof. Treatment methods making use of the generated hematopoietic stem cells and endothelial cells are also provided. The instant disclosure also provides systems, compositions, and kits for practicing the methods of the disclosure.
Claims9
A method of producing a substantially pure population of hematopoietic stem cells (HSC) in defined monolayer conditions in media comprising extracellular signaling agents to guide differentiation, the method comprising:
- (a) differentiating human pluripotent stem cells by culture in media comprising a BMP agonist, an FGF agonist, a WNT agonist, and TGFβ agonist for a period of about 1 day to generate a population of primitive streak cells;
- (b) differentiating the primitive streak cells by culture in media comprising a WNT inhibitor, BMP agonist, VEGF agonist, PI3K inhibitor, CAMP agonist, TGFβ inhibitor, and Vitamin C for a period of about 1 day to 36 hours or longer to generate a population of dorsal lateral mesoderm;
- (c) differentiating the dorsal lateral mesoderm cells by culture in media comprising:
- (i) a VEGF agonist, TGFβ agonist, WNT inhibitor, PI3K inhibitor, BMP inhibitor, and Vitamin C;
- or (ii) a VEGF agonist, TGFβ agonist, WNT inhibitor, PI3K inhibitor, BMP inhibitor, and Vitamin C for a period of about 1 day to produce a population of artery progenitor cells;
- (d) differentiating the artery progenitor cells by culture at high density in media comprising an OSM agonist, LIF agonist, CAMP agonist, TGFβ inhibitor, Notch agonist, and serum replacement for about 2 to about 3 days to produce a population of hemogenic endothelium cells;
- and
- (e) differentiating the hemogenic endothelium in media comprising an OSM agonist, LIF agonist, CAMP agonist, IL-1 agonist, aryl hydrocarbon receptor inhibitor, TGFβ inhibitor, UM171, Notch agonist, and serum replacement for a period of from about 2 to about 3 days to generate a population of HSC.
Dependencies are inferred from the claim text.