Human Embryonic Stem Cell-Derived Endothelial Cells

胚胎和胎儿中的快速血管形成和生长是呼吸道运输所必需的。 气体、营养物和代谢废物进入或排出胎儿，以支持快速生长的胎儿。但 人类胚胎中早期血管发生和血管生成的潜在机制仍然不清楚， 明显的道德原因。最近的研究证明了内皮细胞在体外的成功分化 从小鼠和恒河猴胚胎干细胞（ES细胞）中提取的胚胎干细胞为研究 这样的机制。尽管如此，关于人ES细胞内皮分化的报道有限， 成功建立稳定的人ES细胞来源的内皮细胞系， 活动或血管扩张剂尚未报告。更重要的是， ES细胞的分化在物种之间可能不同。因此，我们建议建立人ES细胞来源的 内皮细胞（HESCDE）细胞系在体外和体内具有血管生成能力。四个具体 将使用充分表征的人ES细胞系（NIH Human ES Cell Registry Code： WA 01和WA 09）：AIM 1。为了确定和优化诱导内皮细胞分化的条件， 体外培养人ES细胞，方法是用含有人ES细胞衍生的胚状体（EB）和人ES细胞的 确定的分化培养基; AIM 2.为了确定这些HESCDE细胞是否具有体外血管生成能力， 能力和产生一氧化氮，血管扩张剂，响应于碱性成纤维细胞生长因子（bFGF）， 体外血管内皮生长因子（VEGF）; AIM 3.为了确定这些HESCDE细胞是否 能够形成血管并在移植到 严重联合免疫缺陷（SCID）米色小鼠;和AIM 4.为了比较 HESCDE细胞与人脐静脉内皮细胞（HUVEC），人胎盘动脉内皮细胞（HPAE）， 和人胎盘微血管内皮（HPME）细胞。 此外，为了进一步表征和定义HESCDE细胞的功能，我们将提供这些 项目I、II和IV的HESCDE单元，将其行为与静态UAEC（项目I）进行比较， 动态剪切应力（项目II）条件下，并用作滋养层分化的效应细胞（项目 IV）。因此，这些研究将收集有关调节机制的潜在重要信息。 在早期人类胚胎发育过程中，内皮细胞分化和血管形成。 此外，它将潜在地提供高纯度和无限的内皮细胞供应，不仅用于研究， 调节血管生成的机制，但也用于临床应用， 作为一种更持久和复杂的基因疗法的载体。

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.