Mechanisms of Shear Induction of Blood-Brain Barrier Phenotypes in Human iPSC-derived Brain Endothelial Progenitors

血脑屏障（BBB）代表血流之间的物理、运输和代谢屏障， 而大脑及其功能对维持大脑内环境稳定至关重要。血脑屏障功能障碍是许多 神经系统疾病和紊乱。此外，BBB阻止中枢神经系统疾病的治疗 通过限制大脑对小分子和蛋白质药物的摄取。BBB的体外模型提供了 了解发育和疾病期间BBB结构和功能的工具，并促进发现 将药物输送到大脑的策略。建立的体外BBB模型通常缺乏关键的生理学特性， 然而，体内BBB的表型限制了它们的应用。前面，我们描述了一个定义的方法， 人诱导多能干细胞（iPSC）向脑微血管内皮细胞的定向分化 （BMEC），其包括BBB。这些iPSC-BMEC表达BBB特异性标志物，并表现出屏障和 转运蛋白的性质类似于体内BBB中的转运蛋白，尽管水平降低。我们的初步数据 证明对iPSC-BMEC祖细胞施加剪切流诱导p21中的BBB表型， TGFβ信号通路依赖的方式。这些数据激发了我们的中心假设： 增强iPSC-BMEC中BBB屏障和转运蛋白表型的发育和维持 通过p21和TGFβ信号传导。为了验证这一假设，我们将剪切流应用于iPSC 区分不同发育阶段的BMEC，并量化剪切应力对BBB屏障的影响 和转运蛋白表型。我们将使用遗传和生化抑制剂， 阐明p21和TGFβ通路诱导BBB表型的作用。基于这些基础研究， 我们将构建由剪切条件iPSC组成的同基因神经血管单位（NVU）模型， 衍生的BMEC，神经元，星形胶质细胞和周细胞，这将使我们能够更好地了解人类BBB 发展和疾病，并促进神经治疗的发展。我们的具体目标是： 1.量化施加于iPSC-BMEC祖细胞的剪切应力对BBB表型诱导的影响。 我们将评估发育阶段中剪切诱导屏障和转运蛋白表型， 区分iPSC-BMEC。 2.阐明p21和TGFβ信号传导在iPSC中剪切介导的BBB表型诱导中的作用- BMEC祖细胞。我们将采用p21和TGFβ通路的化学和遗传抑制来测试 这些途径在iPSC-BMEC祖细胞中BBB表型的剪切诱导中的必要性。 3.在接触和非接触等基因神经血管单元模型中评价剪切调节iPSC-BMEC。 我们将构建由在剪切存在下分化的iPSC-BMEC组成的NVU模型， iPSC衍生的神经元、星形胶质细胞和周细胞，以检验剪切施加到iPSC- BMEC祖细胞将产生具有增强的、持续的BBB表型的高稳定性NVU模型。

The blood-brain barrier (BBB) represents a physical, transport, and metabolic barrier between the bloodstream and the brain and its function is crucial to maintain brain homeostasis. BBB dysfunction is a hallmark of many neurological diseases and disorders. Moreover, the BBB prevents treatment of central nervous system diseases by limiting brain uptake of small molecule and protein-based pharmaceuticals. In vitro models of the BBB provide tools to understand BBB structure and function during development and disease and facilitate discovery of strategies to delivery pharmaceuticals to the brain. Established in vitro BBB models often lack key physiologic phenotypes of the in vivo BBB, however, limiting their utility. Previously, we described a defined method for directed differentiation of human induced pluripotent stem cells (iPSCs) to brain microvascular endothelial cells (BMECs) that comprise the BBB. These iPSC-BMECs express BBB-specific markers and exhibit barrier and transporter properties similar to those in the BBB in vivo, albeit at reduced levels. Our preliminary data demonstrate that application of shear flow to iPSC-BMEC progenitors induces BBB phenotypes in a p21 and TGFβ signaling pathway dependent manner. These data motivate our central hypothesis: Shear stress enhances development and maintenance of BBB barrier and transporter phenotypes in iPSC-BMEC progenitors via p21 and TGFβ signaling. To test this hypothesis, we will apply shear flow to iPSCs differentiating to BMECs at different stages of development and quantify effects of shear stress on BBB barrier and transporter phenotypes in the resulting BMECs. We will employ genetic and biochemical inhibitors to elucidate the roles of p21 and TGFβ pathway induction of BBB phenotypes. Based on these fundamental studies, we will construct isogenic, neurovascular unit (NVU) models comprised of shear-conditioned iPSC- derived BMECs, neurons, astrocytes and pericytes that will enable a better understanding of human BBB development and disease and facilitate neurotherapeutic development. Our specific aims are: 1. Quantify the effects of shear stress applied to iPSC-BMEC progenitors on induction of BBB phenotypes. We will assess the developmental stages at which shear induces barrier and transporter phenotypes in differentiating iPSC-BMECs. 2. Elucidate the roles of p21 and TGFβ signaling in shear-mediated induction of BBB phenotypes in iPSC- BMEC progenitors. We will employ chemical and genetic inhibition of p21 and TGFβ pathways to test the necessity of these pathways in shear induction of BBB phenotypes in iPSC-BMEC progenitors. 3. Evaluate shear-conditioned iPSC-BMECs in contact and noncontact isogenic neurovascular unit models. We will construct NVU models consisting of iPSC-BMECs differentiated in the presence of shear, and iPSC-derived neurons, astrocytes, and pericytes, to test the hypothesis that shear application to iPSC- BMEC progenitors will yield a high-fidelty NVU model with enhanced, sustained BBB phenotypes.