Methods and materials for hematoendothelial differentiation of human pluripotent stem cells under defined conditions
Methods and compositions for differentiating pluripotent stem cells into cells of endothelial and hematopoietic lineages are disclosed.
1. A method of differentiating human pluripotent stem cells into EMH lin − KDR + APLNR + PDGFRalpha + primitive mesoderm cells, comprising the steps of:
(a) providing human pluripotent stem cells; and
(b) exposing the stem cells to a xenogen-free and serum albumin-free mixture comprising components of about 25 ng/ml to about 50 ng/ml FGF2, BMP4 of at least 50 ng/ml to about 250 mg/ml, Activin A of less than 15 ng/ml, and about 1 mM to about 2 mM LiCl under hypoxic conditions for a period of about two days to form a population of EMH lin − KDR+APLNR+PDGFRalpha+primitive mesoderm cells with mesenchymoangioblast potential without the formation of embryoid bodies or coculture with stromal cell lines.
2. The method of claim 1 , additionally comprising the step of:
(c) exposing cells at the primitive mesoderm stage of step (b) to a mixture comprising components FGF2 and VEGF under hypoxic conditions for a period of about 1-2 days to obtain a population comprising EMH lin-KDR + APLNR + PDGFRalpha + primitive mesoderm with hemangioblast (HB-CFC) potential and hematovascular mesoderm cells ( EMH lin-KDR hi APLNR + PDGFRalpha lo/− ) enriched in cells with a potential to form hematoendothelial clusters when cultured on OP9 cells.
3. The method of claim 2 , additionally comprising the step of:
(d) exposing the cells at the hematovascular mesoderm stage of step (c) to a mixture comprising components FGF2, VEGF, IL6, SCF, TPO, and IL3 for about one day to achieve formation of CD144 + CD73 + CD235a/CD43 − non-hemogenic endothelial progenitors (non-HEP), CD144 + CD73 − CD235a/CD43 − hemogenic endothelial progenitors (HEPs), CD144 + CD73 − CD235a/CD43 + 41a − a ngiogenic hematopoietic progenitors (AHP), and CD43 + CD41a + hematopoietic progenitor cells.
4. The method of claim 3 , additionally comprising the step of:
(e) continuing to expose the HEPs and emerging hematopoietic progenitor cells to a mixture comprising components FGF2, VEGF, IL6, SCF, TPO, IL3 under normoxia for about three days resulting in hematopoietic expansion to obtain a population of CD43 + hematopoietic progenitors composed of CD43 + CD235a + CD41a + erythromegakaryocytic progenitors and lin-CD34 + CD43 + CD45 +/− multipotent hematopoietic progenitors.
5. The method of claim 1 , wherein the xenogen-free and serum-albumin free mixture consists essentially of the components.
6. The method of claim 1 , wherein the cells are seeded on a substrate treated with Tenascin-C.
7. The method of claim 1 , wherein the xenogen-free and serum albumin-free mixture comprises:
about 50 to about 250 ng/ml BMP4;
about 10 to about 15 ng/ml Activin A;
about 10 to about 50 ng/ml FGF2; and
about 1 to about 2 mM LiCl.
8. The method of claim 2 , wherein in step c, the xenogen-free and serum-albumin free mixture is supplemented with:
about 10 to about 50 ng/ml FGF2; and
about 20 to about 50 ng/ml VEGF.
9. The method of claim 4 , wherein the xenogen-free and serum-albumin free mixture comprises:
about 50 to about 100 ng/ml SCF,
about 50 to about 100 ng/ml TPO,
about 50 to about 100 ng/ml IL-6, and
about 5 to about 15 ng/ml IL-3.
10. The method of claim 1 , wherein the human pluripotent stem cells are seeded as a single cell suspension on a substrate comprising a layer of Tenascin C at a concentration of at least about 0.25 μg/cm 2 to about 1 μg/cm 2 ; and the xenogen-free serum albumin-free mixture comprises:
about 50 to about 250 ng/ml BMP4;
about 10 to about 15 ng/ml Activin A;
about 10 to about 50 ng/ml FGF2; and
about 1 to about 2 mM LiCl.
11. The method of claim 6 , wherein the layer of Tenascin C is at a concentration of at least about 0.25 ug/cm 2 to about 1 ug/cm 2 .
12. The method of claim 6 , wherein the layer of Tenascin C is at a concentration of about 0.5 ug/cm 2 .
13. The method of claim 1 , wherein the hypoxic conditions comprise a level of environmental oxygen of about 3% to about 10% O 2 .
14. The method of claim 1 , wherein the cells are seeded on a substrate comprising Collagen IV.