Rapid vascular formation and growth in embryos and fetuses are required for transportation of respiratory gases, nutrients, and metabolic wastes into or out of fetuses to support the rapidly growing fetuses. However, the mechanisms underlying early vasculogenesis and angiogenesis in the human embryo remain poorly defined due to obvious ethical reasons. Recent studies demonstrating the successful differentiation of endothelial cells in vitro from mouse and rhesus monkey embryonic stem (ES) cells has provided an important tool for investigating such mechanisms. Nonetheless, reports on endothelial differentiation from human ES cells are limited, and the successful establishment of stable human ES cell-derived endothelial cell lines with potent angiogenic activities or vasodilator has not been reported. More importantly, the mechanisms controlling endothelial differentiation from ES cells may differ among species. Thus, we propose to establish human ES cellderived endothelial (HESCDE) cell lines with angiogenic capacity in vitro and in vivo. Four Specific Aims will be addressed using well-characterized human ES cell lines (NIH Human ES Cell Registry Code: WA01 and WA09): AIM 1. To determine and optimize conditions for inducing endothelial differentiation from human ES cells in vitro by treating human ES cell-derived embryoid bodies (EBs) and human ES cells with a defined differentiation medium; AIM 2. To determine whether these HESCDE cells have in vitro angiogenic capacities and produce nitric oxide, a vasodilator, in response to basic Fibroblast Growth Factor (bFGF) and Vascular Endothelial Growth Factor (VEGF) in vitro; AIM 3. To determine whether these HESCDE cells are capable of forming blood vessels and integrate into pre-existing blood vessels after transplantation into severe combined immunodeficient (SCID) beige mice; and AIM 4. To compare global gene expression in HESCDE cells with human umbilical vein endothelial, (HUVEC), human placental artery endothelial (HPAE), and human placental microvascular endothelial (HPME) cells using human cDNA microarray analysis. Additionally, to further characterize and define the functions of HESCDE cells, we will provide these HESCDE cells for Projects I, II & IV to compare their behaviors with UAEC in static (Project I), under dynamic shear stress (Project II) conditions, and used as effector cells for trophoblast differentiation (Project IV). Thus, these studies will glean potentially important information regarding the mechanisms that regulate endothelial differentiation and formation of blood vessels during early human embryonic development. Moreover, it will potentially provide highly purified and unlimited endothelial cell supply not only for studying mechanisms that regulate angiogenesis but also for clinical applications such as tissue transplantation therapies and as a vehicle for more enduring and sophisticated gene therapies.