Shear regulated differentiation of hPSCs to brain endothelial cells

产品说明：人多能干细胞（hPSC），包括胚胎干细胞和诱导多能干细胞，提供无限自我更新潜力和多能性的独特组合，这两种特性赋予用于产生正常人类体细胞以用于发育研究、毒性测试和细胞疗法的强大系统。脑微血管内皮细胞（BMEC）是一种特别有前途的细胞类型，其可以源自hPSC，因为BMEC不能容易地从人组织或成体干细胞获得，并且在神经系统疾病和跨血脑屏障（BBB）运输的药物评价中具有巨大的重要性。最近，我们的团队开发了一种方案，通过共分化神经和内皮祖细胞的混合群体，然后选择性传代培养获得BMEC表型的内皮祖细胞，将hPSC分化为BMEC。这些hPSC衍生的BMEC表达脑特异性标志物，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，hPSC衍生的BMEC单层产生与体内发现的相当的跨内皮电阻，并且在动物模型中表现出与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，该模型概括了关键的体内BBB表型，并为理解BMEC的发育和调控提供了新的平台。然而，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结合BBB发育期间存在的关键线索。一些研究已经暗示流体流动作为血管功能的重要调节器，包括BMEC中的屏障形成。在本申请中，我们将测试剪切应力在特定发育阶段提供BBB分化的诱导线索并且在维持hPSC衍生的BMEC的分化表型中是重要的假设。我们团队在机械转导、多能干细胞生物学和BBB建模方面的专业知识将使我们能够系统地评估剪切应力对BMEC分化和BBB表型维持的作用。这项研究将进一步激发BBB发育过程中机械转导的机制研究，并改善药物筛选应用中的人类BBB建模。我们检验本申请假设的具体目的是：1。确定剪切力对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

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