Generating vasculogenic cell populations
The present invention relates generally to methods and compositions useful for therapeutic vascular tissue engineering. In particular, the present invention provides methods for generating substantially pure populations of vasculogenic cells from human mesenchymal progenitors, and methods and compositions for clinical applications in the field of regenerative medicine.
1. A method for generating an isolated population of primate vasculogenic cells, the method comprising:
(a) culturing a colony of primate mesenchymoangioblasts (MAB)-derived cells comprising PDGFRB+ (Platelet Derived Growth Factor Receptor B+)/EMCN high /CD105 low ; CD248 − /CD73 − /CD31 − mesenchymal progenitors on a culture plate having a coating comprising fibronectin, gelatin or collagen, in a serum-free culture medium comprising about 5 ng/mL to about 15 ng/mL of Platelet-Derived Growth Factor-BB (PDGF-BB) and about 20 ng/mL of fibroblast growth factor 2 (FGF2) that promotes differentiation of the MAB-derived mesenchymal progenitors to immature pericytes having a RGS5 + (Regulator of G-protein Signaling 5 + )/PDGFRβ + /ANG-1 + (angiopoietin-1 + )/CD146 + /CD44 + /CD13 + /NGY (Nerve/Glial antigen 2 + )/CD73 + expression profile;
(b) culturing the immature pericytes on the coated culture plate in the presence of maturation culture medium comprising TGFβ receptor inhibitor and PDGF-BB under conditions that promote maturation of the immature pericytes into a mature pericyte population that is capillary pericytes, venule pericytes, or arteriole pericytes; and
(c) isolating the mature pericyte cells.
2. The method of claim 1 , wherein the primate is human.
3. The method of claim 1 , wherein the TGFβ receptor inhibitor is SB-431542.
4. The method of claim 1 , wherein the mature pericyte is a NG2 high /α-SMA10 high /RGS5 + capillary pericyte.
5. The method of claim 1 , wherein the maturation culture medium further comprises 10 μM SB-431542, 25 ng/mL PDGF-BB, and 25 ng/mL of Vascular Endothelial Growth Factor-A (VEGF), and wherein the mature pericyte is a NG2 low /α-SMA high /RGS5 + venule pericyte.
6. The method of claim 1 , wherein the maturation culture medium further comprises 10 μM SB-431542, 10 ng/mL PDGF-BB, 10 ng/mL VEGF, and 5 ng/mL Epidermal Growth Factor 2 (EGF2), and wherein the mature pericyte is a NG2 high /α-SMA high /RGS5 + arteriole pericyte.
7. The method of claim 1 , wherein the colony of mesenchymal progenitors is clonal.
8. The method of claim 1 , wherein the colony of mesenchymal progenitors is polyclonal.