ES Cell Differentiation as a Model to Study Cell Biological Regulation of Vascular Development

The organized vascular system is established through three successive steps, vasculogenesis, angiogenesis and vascular remodeling. Vasculogenesis is the initiation of nascent embryonic vessel formation by the differentiation of mesoderm-derived angioblasts into endothelial cells. The nascent primitive vascular plexus then expands and reorganizes into hierarchical vascular structures by the process of angiogenesis, which involves sprouting, bridging, and intussusceptive division of preexisting vessels. Vascular smooth muscle cells are then recruited to the vessels and the highly organized mature vascular system is established after vascular remodeling. By using gene disruption strategies, a number of molecules have been identified as being involved in the development of the vascular system. These identified molecules include transcription factors, cell adhesion molecules, secretory molecules and their receptors, of which deprivation lead to failure of angiogenesis and/ or vascular remodeling in most cases. Despite the substantial expansion of the list of molecules that are essential for angiogenesis, we do not yet understand sufficiently how these molecules regulate the discrete processes of angiogenesis and vascular remodeling. Indeed, we know very little how normal endothelial cells behave in response to various angiogenic stimuli and cooperate to generate blood vessels during embryogenesis. It is critically important to establish an experimental model to study the functions of key molecules at the cellular level by monitoring the behavior of normal endothelial cells involving in angiogenesis. In vitro differentiation systems of embryonic stem (ES) cells have been serving as an excellent experimental maneuver of developmental biology since Doetschman et al. reported that hematopoietic cells were generated in vitro from ES cells (Doetschman et al., 1985). The ES cell differentiation systems have three important features. First, as differentiation of cells and tissues takes place in a culture dish instead of uterus, the process of differentiation can be directly monitored. Second, intermediate stages of differentiation can be identified and isolated as a cell population. Third, cell differentiation and proliferation can be controlled by either adding exogenous signaling molecules, introducing exogenous genes or modifying endogenous genes. Following the pioneering report of angiogenic differentiation from mouse ES cells engrafted onto quail chorioallantoic membrane (Risau et al., 1988), in vitro derivation of vascular endothelial cells from ES cells has a history of almost two decades (Bloch et al., 1997; Feraud et al., 2001; Vittet et al., 1996; Wang et al., 1992). These earlier studies relied upon formation of cell aggregates termed cystic embryoid bodies from ES cells, whereby real-time monitoring of cell differentiation with a high resolution was