Human Endothelial Cell Differentiation

描述（由申请人提供）：我们研究的长期目标是有效地从多能干细胞（如hES和iPS细胞）中获得人类内皮细胞类型，用于人类血管再生和临床治疗。要做到这一点，我们需要了解支配人类内皮细胞发育的机制，但它们在很大程度上是不明确的。在我们的初步研究中，我们已经开始分析调节人类内皮细胞从多能干细胞分化的信号传导结构，并发现参与这一过程的一些因素与小鼠内皮细胞分化所需的因素不同。利用hES细胞培养系统，我们发现印度刺猬（Ihh）促进内皮细胞分化，基因/蛋白表达、形态和DiI-AcLDL摄取证明了这一点。此外，骨形态发生蛋白4 （BMP4）的抑制抑制了内皮细胞的形成，而在hh抑制剂环巴胺处理的细胞中添加rhBMP4促进了内皮细胞的发育。因此，我们的初步研究表明，Ihh通过BMP信号促进人类内皮细胞从hES细胞分化，为人类临床治疗提供了适用于调节血管形成的新见解。拟议的研究将建立在我们的初步数据基础上，并进一步剖析调节人类内皮细胞分化的分子信号传导体系。我们还将研究促进特化人类内皮细胞类型产生的分子调节因子。最后，我们将优化我们的hES细胞培养系统，使中胚层祖细胞更有效地产生内皮细胞类型。我们的首要假设是，在我们的多能干细胞模型系统中，导致人类内皮细胞谱系承诺、分化和特化的步骤是可分解和可定义的。此外，通过了解这些过程所需的分子事件的序列和动力学，我们将深入了解如何更有效地获得用于组织工程和流行病理临床治疗的人类内皮细胞类型。以下具体目标将验证这些假设：1)进一步确定Ihh和BMP信号调节人内皮细胞分化的机制。2)研究人内皮细胞特化的分子调控；3)建立无饲料培养系统，使内皮细胞从人中胚层祖细胞高效分化和特化。

DESCRIPTION (provided by applicant): The long term goal of our studies is to efficiently derive human endothelial cell types from pluripotent stem cells, such as hES and iPS cells, for human vascular regeneration and clinical therapies. To do so, we need to understand the mechanisms that govern human endothelial cell development, but they have been largely undefined. In our Preliminary Studies, we have begun to dissect the signaling hierarchy that modulates the differentiation of human endothelial cells from pluripotent stem cells, and found that some factors involved in this process differ from those required for murine endothelial cell differentiation. Using a hES cell culture system, we found that Indian Hedgehog (Ihh) promoted endothelial cell differentiation, as evidenced by gene/protein expression, morphology and DiI-AcLDL uptake. Furthermore, inhibition of bone morphogenic protein 4 (BMP4) suppressed endothelial cell formation whereas addition of rhBMP4 to cells treated with the hh inhibitor cyclopamine promoted endothelial cell development. Thus, our Preliminary Studies reveal that Ihh promotes human endothelial cell differentiation from hES cells via BMP signaling, providing novel insights applicable to modulating blood vessel formation for human clinical therapies. The proposed studies will build on our Preliminary Data and further dissect the molecular signaling hierarchy that regulates human endothelial cell differentiation. We will also investigate molecular regulators that promote the generation of specialized human endothelial cell types. Finally, we will optimize our hES cell culture system to enable more efficient generation of endothelial cell types from mesodermal progenitors. Our overarching hypotheses are that the steps that lead to human endothelial cell lineage commitment, differentiation and specialization are dissectible and definable in our pluripotent stem cell model system. Furthermore, by understanding the sequence and kinetics of the molecular events needed for these process, we will gain insight into how to more efficiently derive human endothelial cell types for tissue engineering and clinical therapies for prevalent pathologies. The following Specific Aims will test these hypotheses: 1) further define the mechanism(s) by which Ihh and BMP signaling regulate human differentiation of human endothelial cells. 2) investigate the molecular regulation of human endothelial cell specialization; and 3) establish a feeder-free culture system for efficient differentiation and specialization of endothelial cells from human mesodermal progenitors.