Human Endothelial Cell Differentiation

描述（由申请人提供）：我们研究的长期目标是从多能干细胞（如hES和iPS细胞）中有效衍生人内皮细胞类型，用于人血管再生和临床治疗。要做到这一点，我们需要了解人类内皮细胞发育的机制，但它们在很大程度上还不确定。在我们的初步研究中，我们已经开始剖析调节人内皮细胞从多能干细胞分化的信号传导层次，并发现参与这一过程的一些因子与鼠内皮细胞分化所需的因子不同。使用hES细胞培养系统，我们发现，印度刺猬（Ihh）促进内皮细胞分化，证明了基因/蛋白质表达，形态和DiI-AcLDL摄取。此外，抑制骨形态发生蛋白4（BMP 4）抑制内皮细胞的形成，而加入rhBMP 4与hh抑制剂环巴胺处理的细胞促进内皮细胞的发展。因此，我们的初步研究表明，Ihh促进人内皮细胞分化的hES细胞通过BMP信号，提供新的见解适用于调节血管形成的人类临床治疗。拟议的研究将建立在我们的初步数据，并进一步剖析调节人类内皮细胞分化的分子信号转导层次。我们还将研究促进特化人类内皮细胞类型生成的分子调节剂。最后，我们将优化我们的hES细胞培养系统，使更有效地产生内皮细胞类型的中胚层祖细胞。我们的首要假设是，导致人内皮细胞谱系定型、分化和特化的步骤在我们的多能干细胞模型系统中是可解剖和可定义的。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人类内皮细胞类型，用于组织工程和临床治疗流行的病理。以下具体目的将检验这些假设：1）进一步确定Ihh和BMP信号传导调节人内皮细胞的人分化的机制。2)研究人内皮细胞分化的分子调控机制; 3）建立无饲养层培养体系，以促进人中胚层祖细胞内皮细胞的高效分化和特化。公共卫生相关性：我们正在研究血管形成的过程，并试图了解形成血管内膜的人类内皮细胞是如何从多能性的人类胚胎干细胞分化出来的。从我们的研究中获得的见解可能有助于制定人类组织再生和治疗常见血管疾病患者的策略。

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. PUBLIC HEALTH RELEVANCE: We are studying the process of blood vessel formation and trying to understand how human endothelial cells, that form the lining of blood vessels, are differentiated from human embryonic stem cells that are pluripotent. Insights gained from our studies may help to develop strategies for human tissue regeneration and for treating patients with common vascular diseases.