In vitro derivation and expansion of endothelial cells from embryonic stem cells

Vascular endothelial cells or endothelial progenitor cells derived from stem cells could potentially lead to a variety of clinically relevant applications, including cell-based therapies and tissue engineering. Embryonic stem (ES) cells serve as an excellent in vitro system for studying differentiation events and for developing methods of generating various specialized cells for future regenerative therapeutic applications. Two obstacles associated with using embryonic stem cells include (1) isolating homogeneous populations of differentiated cells and (2) obtaining terminally differentiated cell populations that are capable of proliferating further. Here, we describe methods for isolating purified proliferating populations of endothelial cells from mouse ES cells using Flk-1-positive cells, vascular endothelial growth factor supplementation, and a highly selective manual selection technique. This methodology, although rigorous, overcomes two current obstacles in ES derivation and culture by generating highly purified (>96%) populations of actively proliferating endothelial cells from mouse ES cells. Using this in vitro derivation procedure, millions of cells at various stages of differentiation may be obtained and expanded up to 25 population doublings.