Generating vascular smooth muscle cells in vitro from ES cells

Abstract

A simplified and inexpensive method for the in-vitro identification, isolation and culture of human vasculogenic progenitor cells is provided. The method and the progenitor cells provided herein can be used for in-vitro vascular engineering, treatment of congenital and acquired vascular and hematological abnormalities, for evaluation and development of drugs affecting vasculo- and angiogenic processes, and for further investigation into tissue differentiation and development.

1 . A method of preparing somatic cells from a population of vasculogenic progenitor cells, the method comprising:
 (a) obtaining a population of vasculogenic progenitor cells; and   (b) culturing the population of vasculogenic progenitor cells in the presence of at least one growth factor suitable for inducing somatic cell differentiation, thereby preparing the somatic cells.   
     
     
         2 . A method of preparing endothelial cells from vascular tissue, the method comprising:
 (a) subjecting the vascular tissue to conditions designed for dissociating cells from the vascular tissue, thereby obtaining a mixed population of dissociated cells; and   (b) isolating cells smaller than 50 μm from said mixed population of cells, said cells smaller than 50 μm being endothelial cells.   
     
     
         3 . The method of  claim 2 , wherein said cells smaller than 50 μm are isolated by filtration, morphometry and/or densitometry. 
     
     
         4 . The method of  claim 3 , wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μm. 
     
     
         5 . The method of  claim 2 , wherein said vascular tissue is human vascular tissue. 
     
     
         6 . A method of preparing epithelial cells from vascular tissue, the method comprising:
 (a) subjecting the vascular tissue to conditions designed for dissociating cells from the vascular tissue, thereby obtaining a mixed population of dissociated cells; and   (b) isolating cells larger than 50 μm from said mixed population of cells, said cells larger than 50 μm being epithelial cells.   
     
     
         7 . The method of  claim 6 , wherein said cells larger than 50 μm are isolated by filtration, morphometry and/or densitometry. 
     
     
         8 . The method of  claim 7 , wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μm. 
     
     
         9 . The method of  claim 6 , wherein said vascular tissue is human vascular tissue. 
     
     
         10 . A method of generating epithelial cells from vasculogenic progenitor cells, comprising culturing the vasculogenic progenitor cells in a differentiating medium including a serum volume concentration lower than 5% for a time period sufficient to induce differentiation of the vasculogenic progenitor cells into endothelial cells. 
     
     
         11 . The method of  claim 10 , wherein said vasculogenic progenitor cells are derived from ES cells. 
     
     
         12 . The method of  claim 11 , wherein said ES cells are human ES cells. 
     
     
         13 . A method of enhancing differentiation, maturation and/or functionality of vasculogenic cells, comprising exposing the vasculogenic cells to a shear force of at least 1 dyne/cm 2  for a time period sufficient to enhance differentiation, maturation and/or functionality of the vasculogenic cells. 
     
     
         14 . The method of  claim 13 , wherein said vasculogenic cells are vascular smooth muscle cells or endothelial cells derived from ES cells.