Shear regulated differentiation of hPSCs to brain endothelial cells

描述：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，这两个特性为发育研究、毒性测试和细胞治疗提供了一个强大的系统来产生正常的人类体细胞。脑微血管内皮细胞(BMECs)是一种特别有前景的细胞类型，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，并且在神经系统疾病和跨血脑屏障(BBB)转运的药物评估中具有极其重要的作用。最近，我们的团队开发了一种方案，通过共分化混合的神经和内皮祖细胞群体，然后选择性地传代获得BMEC表型的内皮祖细胞，来区分hPSCs和BMECs。这些hPSC来源的BMEC表达大脑特异的标记，包括紧密连接蛋白和分子转运蛋白。当与星形胶质细胞共同培养时，hPSC来源的BMEC单层产生的跨内皮细胞电阻与体内发现的相当，并在动物模型中显示出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BMEC提供了第一个概括体内关键BBB表型的体外人类BBB模型，并为了解BMEC的发育和调控提供了一个新的平台。然而，hPSC来源的BMECs体内缺乏BBB标志物的表达和转运蛋白活性，可能是由于体外分化微环境未能整合BBB发育过程中的关键信号。一些研究表明，流体流动是血管功能的重要调节因素，包括BMECs中屏障的形成。在这一应用中，我们将检验这样一种假设，即剪切力在特定发育阶段提供了诱导血脑屏障分化的线索，并对维持hPSC来源的BMEC的分化表型很重要。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应力对BMEC分化和维持BBB表型的作用。这项研究将推动在血脑屏障发育过程中机械转导的进一步机械学研究，并导致用于药物筛选应用的人血脑屏障模型的改进。我们的具体目的是验证这一应用假说：1.确定剪应力对BMEC和BMEC祖细胞分化命运的阶段性影响2.确定剪应力对hPSC来源的BMEC表型诱导和维持的影响3.确定PECAM-1和VE-cadherin在剪切诱导的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