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的动态平衡至关重要。血脑屏障功能障碍是许多 神经系统疾病和障碍。此外，血脑屏障还可以预防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类药物的摄取。血脑屏障的体外模型提供了 了解BBB在发育和疾病过程中的结构和功能的工具，并促进发现 将药物输送到大脑的策略。已建立的体外血脑屏障模型往往缺乏关键的生理功能 然而，体内血脑屏障的表型限制了它们的用途。在此之前，我们描述了一种定义的方法 人诱导多能干细胞定向分化为脑微血管内皮细胞 (BMEC)组成BBB。这些IPSC-BMEC表达BBB特异性标记，并表现出屏障和 转运蛋白的性质类似于体内的血脑屏障，尽管水平降低。我们的初步数据 证明应用剪切流对IPSC-BMEC前体细胞诱导p21和bbb表型 转化生长因子β信号通路依赖方式。这些数据激发了我们的中心假设：剪切力 增强IPSC-BMEC血脑屏障和转运体表型的发育和维持 前体细胞通过p21和转化生长因子β信号转导。为了验证这一假设，我们将剪切流应用于IPSC 不同发育阶段BMEC的分化及切应力对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物来 阐明p21和转化生长因子β途径诱导血脑屏障表型的作用。在这些基础研究的基础上， 我们将构建等基因神经血管单位(NVU)模型，包括剪切条件下的IPSC- 衍生的骨髓微血管内皮细胞、神经元、星形胶质细胞和周细胞，将使更好地了解人类的血脑屏障 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祖细胞的剪切力对BBB表型诱导的影响。 我们将评估在不同发育阶段剪切力诱导的屏障和转运蛋白表型。 IPSC-BMEC的分化。 2.阐明p21和转化生长因子β信号在剪切法诱导IPSC-BBB表型中的作用。 BMEC的祖细胞。我们将使用化学和遗传抑制p21和转化生长因子β途径来测试 这些途径在IPSC-BMEC祖细胞BBB表型剪切诱导中的必要性。 3.在接触性和非接触性等基因神经血管单位模型中评价剪切条件下的IPSC-BMECs。 我们将构建由剪切作用下分化的IPSC-BMEC组成的NVU模型，并 IPSC来源的神经元、星形胶质细胞和周细胞，以检验剪切应用于IPSC- BMEC的前体细胞将产生高保真的NVU模型，具有增强的、持续的BBB表型。

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.