Shear regulated differentiation of hPSCs to brain endothelial cells

项目总结 人类多能干细胞(HPSCs)，包括胚胎干细胞和诱导多能干细胞， 提供了无限自我更新潜力和多能性的独特组合，这两个特性赋予了 用于发育研究、毒性测试和生产正常人体细胞的强大系统 细胞疗法。脑微血管内皮细胞(BMECs)是一种特别有前途的细胞类型，它可以 因为骨髓间充质干细胞不容易从人体组织或成人干细胞中获得，而且 在神经疾病和药物转运的药物评估中具有极其重要的意义 血脑屏障(BBB)。最近，我们的团队开发了一种方案，通过共同作用来区分hPSC和BMEC 分化神经和内皮祖细胞的混合群体，然后选择性地传代 内皮祖细胞，获得BMEC表型。这些hPSC来源的骨髓间充质干细胞表达脑特异性 标记包括紧密连接蛋白和分子转运蛋白。与星形胶质细胞共培养时，hPSC- 衍生的BMEC单层产生的跨内皮细胞电阻与体内发现的相当 在动物模型中展示与血脑屏障转运相关的营养物质和药物的极化运输。这些 HPSC来源的BMECs提供了第一个重现体内关键BBB的体外人类BBB模型 表型，为了解BMEC的发育和调控提供了一个新的平台。然而， HPSC来源的BMEC在体内缺乏BBB标志物的表达和转运蛋白活性，可能作为一种 体外分化微环境未能纳入血脑屏障期间出现的关键线索的后果 发展。一些研究表明，液体流动是血管功能的重要调节因素， 包括在BMEC中形成屏障。在这个提案中，我们将检验剪切力提供的假设 在特定发育阶段的血脑屏障分化的诱导线索，并在维持 HPSC来源的骨髓间充质干细胞的分化表型。我们团队在机械转导方面的专业知识，多功能 干细胞生物学和BBB模型将使我们能够系统地评估切应力对BMEC的作用 血脑屏障表型的分化和维持。这项研究将推动进一步的机械论研究。 在血脑屏障发育过程中的机械转导，并导致对药物的人血脑屏障模型的改进 筛选申请。 我们测试这项建议的假设的具体目标是： 1.确定切应力对BMEC和BMEC分化命运的阶段性影响 先祖 2.确定切应力对hPSC来源的BMEC表型诱导和维持的影响 3.确定PECAM-1和VE-钙粘蛋白在剪切诱导BMEC分化中的作用

PROJECT SUMMARY Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating normal human somatic cells for developmental studies, toxicity testing, and cellular therapies. Brain microvascular endothelial cells (BMECs) are a particularly promising cell type that can be derived from hPSCs since BMECs cannot easily be obtained from human tissue or adult stem cells and are of tremendous importance in neurological disease and pharmaceutical evaluation of transport across the blood-brain barrier (BBB). Recently, our team developed a protocol to differentiate hPSCs to BMECs by co- differentiating a mixed population of neural and endothelial progenitors, then selectively subculturing the endothelial progenitors, which acquire BMEC phenotypes. These hPSC-derived BMECs express brain-specific markers including tight junction proteins and molecular transporters. When co-cultured with astrocytes, hPSC- derived BMEC monolayers generate transendothelial electrical resistance comparable to that found in vivo and exhibit polarized transport of nutrients and drugs that correlate with BBB transport in an animal model. These hPSC-derived BMECs provide the first in vitro human BBB model that recapitulates key in vivo BBB phenotypes, and provide a novel platform for understanding BMEC development and regulation. However, the hPSC-derived BMECs lack in vivo levels of BBB marker expression and transporter activity, perhaps as a consequence of the in vitro differentiation microenvironment failing to incorporate key cues present during BBB development. Several studies have implicated fluid flow as an important regulator of vascular function, including barrier formation in BMECs. In this proposal we will test the hypothesis that shear stress provides inductive cues on BBB differentiation at specific developmental stages and is important in maintaining the differentiated phenotypes of hPSC-derived BMECs. Our team's expertise in mechanotransduction, pluripotent stem cell biology, and BBB modeling will permit us to systematically assess the role of shear stress on BMEC differentiation and maintenance of BBB phenotypes. This study will then motivate further mechanistic research in mechanotransduction during BBB development and lead to improvements in human BBB modeling for drug screening applications. Our specific aims to test the hypothesis of this proposal are: 1. Identify stage-specific effects of shear stress on differentiation fates of BMECs and BMEC progenitors 2. Ascertain the effects of shear stress on hPSC-derived BMEC phenotype induction and maintenance 3. Determine the roles of PECAM-1 and VE-cadherin in shear-induced differentiation of BMECs