Feeder-Independent Maintenance of Human Embryonic Stem Cells and Directed Differentiation into Endothelial Cells Under Hypoxic Condition

Human embryonic stem cells (hESCs) represent a promising source of cells for modern regenerative medicine due to their highly self-renewal capability and pluripotency to differentiate into almost all cell types from three different germ layers. Recent advances in the stem-cell field have enabled generation of hESC-derived endothelial cells (or endothelial progenitor cells) that might be potentially employed to build up a vascular network in engineered tissue or reconstruct artificial vascular grafts to repair ischemic tissue. However, the differentiation efficiency is relatively low. Considering the important roles of both growth factors and “physiological hypoxia” environment in facilitating endothelial-lineage differentiation and development in the embryos, we studied combinatorial effect of VEGFs and a hypoxic culture condition on in vitro differentiation of hESCs toward endothelial cells. We show that hESC endothelial-lineage differentiation efficiency can be significantly improved under hypoxic conditions, indicated by the increased expression of marker proteins and genes. These experimental results suggest a synergy effect of VEGF and hypoxic environment on the generation of hESC-derived endothelial cells. In this chapter, we describe our protocols for the maintenance of undifferentiated hESCs with feeder-free medium (mTeSR^TM 1 medium), and for the directed differentiation of hESCs toward endothelial cells with specific reference to growth factor supplementation under hypoxic culture condition in vitro.