Shear regulated differentiation of hPSCs to brain endothelial cells

项目摘要 人多能干细胞（hPSC），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 强大的系统，用于生成正常的人体细胞，用于发育研究，毒性测试， 细胞疗法脑微血管内皮细胞（BMEC）是一种特别有前途的细胞类型， 由于BMEC不能容易地从人组织或成体干细胞获得， 在神经系统疾病和药物评估中具有巨大的重要性， 血脑屏障（BBB）。最近，我们的团队开发了一种方案，通过共培养将hPSC分化为BMEC， 分化神经祖细胞和内皮祖细胞的混合群体，然后选择性地继代培养神经祖细胞和内皮祖细胞。 内皮祖细胞，其获得BMEC表型。这些hPSC衍生的BMEC表达脑特异性 标记物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， 衍生的BMEC单层产生与体内发现的相当的跨内皮电阻， 在动物模型中显示与BBB转运相关的营养物和药物的极化转运。这些 hPSC衍生的BMEC提供了第一个体外人BBB模型，其重现了关键的体内BBB 表型，并提供了一个新的平台，了解BMEC的发展和调控。但 hPSC衍生的BMEC缺乏体内水平的BBB标志物表达和转运蛋白活性，这可能是由于 体外分化微环境未能纳入BBB期间存在的关键线索的结果 发展一些研究已经暗示流体流动是血管功能的重要调节器， 包括在BMEC中形成屏障。在这个建议中，我们将测试剪切应力提供的假设， 在特定的发育阶段对BBB分化的诱导线索，并在维持BBB分化中起重要作用。 hPSC衍生的BMEC的分化表型。我们团队在机械传导、多能性 干细胞生物学和血脑屏障建模将使我们能够系统地评估切应力对BMEC的作用 BBB表型的分化和维持。这项研究将激发进一步的机制研究 在BBB发育过程中的机械转导中，并导致用于药物的人BBB建模的改进 筛选申请。 我们检验这一提议假设的具体目标是： 1.确定剪切应力对BMEC和BMEC分化命运的阶段特异性影响 祖细胞 2.确定切应力对hPSC衍生的BMEC表型诱导和维持的影响 3.确定PECAM-1和VE-钙粘蛋白在BMEC剪切诱导分化中的作用

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