Methods and materials for hematoendothelial differentiation of human pluripotent stem cells under defined conditions

Abstract

Methods and compositions for differentiating pluripotent stem cells into cells of endothelial and hematopoietic lineages are disclosed.

1 - 15 . (canceled) 
     
     
         16 . A method of treating a blood disorder in a subject, the method comprising administering therapeutic cells comprising CD43 +  hematopoietic progenitors composed of CD43 + CD235a + CD41a +  erythromegakaryocytic progenitors and lin−CD34 + CD43 + CD45 −/−  multipotent hematopoietic progenitors to treat the blood disorder, the therapeutic cells made by the method comprising the steps of:
 (a) exposing human pluripotent stem cells to a xenogen-free and serum albumin-free mixture comprising components of about 25 ng/ml to about 50 ng/ml FGF2, at least 50 ng/ml to about 250 mg/ml of bone morphogenetic protein 4 (BMP4), 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 therapeutic  EMH lin − KDR + APLNR + PDGFRalpha +  primitive mesoderm cells with mesenchymoangioblast potential without the formation of embryoid bodies or coculture with stromal cell lines; 
 (b) exposing cells at the primitive mesoderm stage of step (a) 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; 
 (c) exposing the cells at the hematovascular mesoderm stage of step (b) 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 −  angiogenic hematopoietic progenitors (AHP), and CD43 + CD41a +  hematopoietic progenitor cells; 
 (d) exposing 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; wherein the population of CD43 +  hematopoietic progenitors comprise the therapeutic cells. 
 
     
     
         17 . The method of  claim 16 , wherein the xenogen-free and serum-albumin free mixture consists essentially of the components. 
     
     
         18 . The method of  claim 16 , wherein in step (b), 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.   
     
     
         19 . The method of  claim 16 , wherein the xenogen-free and serum-albumin free mixture of step (c) 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.   
     
     
         20 . The method of  claim 16 , wherein the cells are seeded on a substrate. 
     
     
         21 . The method of  claim 20 , wherein the substrate is treated with Tenascin-C. 
     
     
         22 . The method of  claim 20 , wherein the substrate is collagen IV (Col IV). 
     
     
         23 . The method of  claim 16 , wherein the cells are administered by transfusion or infusion.