Low oxygen tension enhances endothelial fate of human pluripotent stem cells

Abstract

Low oxygen tension is a critical regulator of the developing or regenerating vasculature. The present invention is based on the determination that low oxygen tension during early stages of early vascular cell (EVC) derivation induces endothelial commitment and maturation of pluripotent stem cells. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogates the endothelial inductive effects of the low oxygen environments. Methods of generating various types of cells from pluripotent stem cells (PSCs) are described, as well as compositions and methods of use thereof. In particular, generation of EVCs, bicellular vascular populations, early endothelial cells (ECs) and pericytes via culture in a low oxygen environment is described.

What is claimed is: 
     
         1 . A method of generating a bicellular population of vascular cells capable of self-organizing into vascular networks comprising:
 a) culturing pluripotent stem cells (PSCs) under hypoxic conditions on a first culture substrate in a first growth medium suitable to induce differentiation of the PSCs toward early vascular cells (EVCs);   b) harvesting the cells of (a); and   c) culturing the harvested cells under hypoxic conditions on a second culture substrate in a second growth medium suitable to induce differentiation toward cells capable of self-organizing into vascular networks, thereby generating a population of cells capable of self-organizing into vascular networks.   
     
     
         2 . The method of  claim 1 , wherein hypoxic conditions are from about 1-5% oxygen. 
     
     
         3 . The method of  claim 2 , wherein hypoxic conditions are about 5% oxygen. 
     
     
         4 . The method of  claim 1 , wherein the first and second culture substrates are two-dimensional. 
     
     
         5 . The method of  claim 1 , wherein the first culture substrate is selected from the group consisting of type I collagen, type IV collagen and fibronectin. 
     
     
         6 . The method of  claim 1 , further comprising addition of vascular endothelial growth factor (VEGF), a transforming growth factor-β (TGF-β) inhibitor, or a combination thereof, to the first and second growth medium. 
     
     
         7 . The method of  claim 1 , wherein the cells of (c) exhibit increased expression of VEcad and CD31 as compared to corresponding cells generated in oxygen conditions of about 15-20%. 
     
     
         8 . The method of  claim 1 , wherein the population generated comprises VEcad+ cells and PDGFRβ+ cells. 
     
     
         9 . The method of  claim 1 , wherein the PSCs are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).