Compositions and methods for generating hematopoietic stem cells (hscs)
AssigneesWashington University, Ospedale San Raffaele S.R.L., Fondazione Telethon
InventorsChristopher Sturgeon, Andrea Ditadi
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Abstract
The present disclosure provides methods for generating hematopoietic progenitor cells. In some embodiments, the methods involve an in vitro or ex vivo cell culture model utilizing retinoic acid signaling for producing hematopoietic progenitor cells from pluripotent stem cells.
Claims37
A method of generating a population of hematopoietic progenitor cells, the method comprising:
- (i) culturing a population of pluripotent stem cells in a mesoderm differentiation medium;
- and
- (ii) culturing a population of cells obtained from step (i) in a hematopoietic specification medium to produce a population of hematopoietic progenitor cells.
The method of any of the proceeding claims, wherein the base media is a IMDM+F12.
The method of any of the proceeding claims, wherein the PS cells are cultured in the mesoderm differentiation medium for about 3 days.
The method of any of the proceeding claims, wherein the hematopoietic specification medium comprises a base media supplemented with:
- a. a FGF,
- b. VEGF, and
- c. a retinoic acid signaling agent.
The method of any of the proceeding claims, wherein the PS cells are genetically modified.
A method of generating hematopoietic progenitor cells comprising:
- (i) providing human pluripotent stem cells (hPS cells);
- (ii) dissociating the hPSCs into embryoid bodies;
- (iii) culturing the embryoid bodies under hypoxic conditions in defined serum-free differentiation media on day 0 of differentiation;
- (iv) introducing recombinant human BMP4 to the embryoid bodies on day 0 through day 3 of differentiation;
- (v) introducing bFGF to the differentiation media on day 1 through day 3 of differentiation;
- (vi) introducing a WNT signaling stimulating agent (e.g., a GSK3b antagonist or GSK3b inhibitor, such as CHIR99021 or analogs thereof, such as CHIR98014, a recombinant WNT protein, or a WNT agonist) sufficient for emergence of a CXCR4+ population (e.g., on day 2, 3, or 4 of differentiation;
- between day 2 and day 3 of differentiation;
- or between day 2 and day 4 of differentiation);
- (vii) introducing an ACTIVIN/NODAL signaling suppressing agent (e.g., an ALK inhibitor, such as SB-431542 or a small molecule TGFb inhibitor) (e.g., on day 2, 3, or 4 of differentiation;
- between day 2 and day 3 of differentiation;
- or between day 2 and day 4 of differentiation), resulting in a culture;
- and/or
- (viii) allowing the culture to incubate for a period of time sufficient to produce a mesodermal population identified by expression of KDR+CD235aneg and mesodermal subsets identified by the expression of CXCR4/CD184 (e.g., between day 3 and day 4 of differentiation;
- or day 3 or day 4 of differentiation).
A method of generating an RA-dependent HE comprising:
- (i) providing a differentiation culture comprising a KDR+CXCR4+ mesoderm;
- and
- (ii) contacting the differentiation culture and the RA signaling agent (e.g., retinol (ROH)) at a time point sufficient to specify a CD34+HE population (e.g., on day 3 of differentiation).
A method of generating, enriching, or selecting RA-dependent definitive hematopoietic progenitors comprising:
- (i) providing a culture comprising hPSCs;
- (ii) contacting the culture with BMP4 between day 0 and day 3, bFGF between day 1 and day 3, WNT signaling stimulating agent on day 2, and ACTIVIN/NODAL signaling suppressing agent on day 2, and RA signaling agent on day 3 of differentiation, resulting in a CD34+CD43negCD73negCD184neg hemogenic endothelial population, wherein the HE population has hematopoietic potential.
Dependencies are inferred from the claim text.