Rapid vascular formation and growth in embryos and fetuses are required for transportation of respiratorygases, nutrients, and metabolic wastes into or out of fetuses to support the rapidly growing fetuses. However, themechanisms underlying early vasculogenesis and angiogenesis in the human embryo remain poorly defined due toobvious ethical reasons. Recent studies demonstrating the successful differentiation of endothelial cells in vitrofrom mouse and rhesus monkey embryonic stem (ES) cells has provided an important tool for investigatingsuch mechanisms. Nonetheless, reports on endothelial differentiation from human ES cells are limited, andthe successful establishment of stable human ES cell-derived endothelial cell lines with potent angiogenicactivities or vasodilator has not been reported. More importantly, the mechanisms controlling endothelialdifferentiation from ES cells may differ among species. Thus, we propose to establish human ES cellderivedendothelial (HESCDE) cell lines with angiogenic capacity in vitro and in vivo.
Four SpecificAims 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 fromhuman ES cells in vitro by treating human ES cell-derived embryoid bodies (EBs) and human ES cells with adefined differentiation medium;
AIM 2. To determine whether these HESCDE cells have in vitro angiogeniccapacities and produce nitric oxide, a vasodilator, in response to basic Fibroblast Growth Factor (bFGF) andVascular Endothelial Growth Factor (VEGF) in vitro;
AIM 3. To determine whether these HESCDE cells arecapable of forming blood vessels and integrate into pre-existing blood vessels after transplantation intosevere combined immunodeficient (SCID) beige mice;
and AIM 4. To compare global gene expression inHESCDE 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 theseHESCDE cells for Projects I, II & IV to compare their behaviors with UAEC in static (Project I), underdynamic shear stress (Project II) conditions, and used as effector cells for trophoblast differentiation (ProjectIV). Thus, these studies will glean potentially important information regarding the mechanisms that regulateendothelial 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 studyingmechanisms that regulate angiogenesis but also for clinical applications such as tissue transplantationtherapies and as a vehicle for more enduring and sophisticated gene therapies.
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