Shear regulated differentiation of hPSCs to brain endothelial cells

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，这两种特性赋予了一个强大的系统，可以产生用于发育研究、毒性测试和细胞治疗的正常人类体细胞。脑微血管内皮细胞（BMECs）是一种特别有前途的细胞类型，可以从hPSCs中获得，因为BMECs不容易从人体组织或成人干细胞中获得，并且在神经疾病和血脑屏障运输（BBB）的药物评估中具有巨大的重要性。最近，我们的团队开发了一种方案，通过共分化神经祖细胞和内皮祖细胞的混合群体，然后选择性地传代内皮祖细胞，使其获得BMEC表型，从而将hPSCs分化为BMEC。这些hpsc衍生的bmec表达脑特异性标记，包括紧密连接蛋白和分子转运蛋白。当与星形胶质细胞共培养时，hPSC衍生的BMEC单层产生与体内相当的跨内皮电阻，并且在动物模型中表现出与血脑屏障运输相关的营养物质和药物的极化运输。这些hpsc衍生的BMEC提供了第一个体外人血脑屏障模型，概括了体内关键的血脑屏障表型，并为理解BMEC的发育和调控提供了一个新的平台。然而，hpsc衍生的bmec缺乏血脑屏障标志物的体内表达水平和转运蛋白活性，这可能是体外分化微环境未能纳入血脑屏障发育过程中存在的关键线索的结果。一些研究表明，流体流动是血管功能的重要调节因子，包括bmec中屏障的形成。在本应用中，我们将验证剪切应力在特定发育阶段对血脑屏障分化提供诱导线索的假设，并在维持hpsc来源的bmec分化表型中发挥重要作用。我们的团队在机械转导、多能干细胞生物学和血脑屏障建模方面的专业知识将使我们能够系统地评估剪切应力在BMEC分化和血脑屏障表型维持中的作用。这项研究将进一步推动血脑屏障发育过程中机械转导的机制研究，并改进人类血脑屏障模型，用于药物筛选应用。我们测试这个应用程序的假设的具体目的是：1。确定剪切应力对BMEC和BMEC祖细胞分化命运的阶段特异性影响2。确定剪切应力对hpsc源性BMEC表型诱导和维持的影响确定PECAM-1和VE-cadherin在剪切诱导bmec分化中的作用

DESCRIPTION: 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, and 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 application we will test the hypothesis tha 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 application 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