Generating populations of human blood and blood vessel progenitors from pluripotent stem cells
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.
1 . 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.
2 . The method of claim 1 , wherein the method further comprises contacting the artery progenitor cells in step (c) with a DLL4 binding agent and isolating the artery progenitor cells by binding of the DLL4 binding agent to the artery progenitor cells and selecting for DLL4 binding cells to produce a purified population of artery progenitor cells.
3 . A substantially pure population of hematopoietic stem cells produced by the method according to claim 1 .
4 . A method of treatment, comprising administering to an individual the population of cells according to claim 3 .
5 . A method of screening a substantially pure population of hematopoietic stem cells for a cellular response, comprising contacting a population of substantially pure population of hematopoietic stem cells of claim 3 with a pharmacological agent and evaluating the population of cells for a cellular response induced by the pharmacological agent.
6 . The method of claim 1 , wherein the media in step (b) comprises a retinoic acid agonist.
7 . The method of claim 1 , wherein the media in step (c) comprises a retinoic acid agonist.
8 . The method of claim 1 , wherein the substantially pure population of hematopoietic stem cells (HSC) comprises greater than 90% HSC.
9 . The method of claim 1 , wherein the HSC express core HSC markers HLF and HOXA5-10.