Assessing the effects of peripheral immune activation on the NVU following TBI using avascularized and perfused human blood/BBB model.

项目总结 颅脑损伤是世界范围内导致死亡和致残的主要原因之一，它具有异质性。 发病机制复杂。颅脑损伤后的临床转归取决于脑损伤的性质和 原发损伤的严重程度以及外周免疫反应的激活。该项目将a)建立 制备有效的人诱导多能干细胞(IPSC)来源的脑内皮细胞的方案 形成神经血管单位(NVU)的BECs、周细胞(PC)和星形胶质细胞(ACS)，b)发展出一种新的3D 灌流血/血脑屏障(BBB)界面模型与患者来源的血浆蛋白和 C)检测脑损伤患者血液循环成分对NVU功能的影响。 在初步研究和最近的出版物中，我们已经(I)开发了使用RNA或病毒诱导的策略 转录因子(TF)将体细胞重新编程为IPSCs，(Ii)分化IPSC来源的上皮细胞 进入BEC并使用多种方法验证其身份；(Iii)生成包含 (4)开发包含血液成分和血流的单芯片脑模型。建立在 这些研究，我们假设，这种多学科的方法将建立一种新的血液灌流-血脑屏障 界面3D模型用于评估血液成分(血浆蛋白、免疫细胞) 颅脑损伤后对人类NVU的损害。我们将通过三个目标来解决这一假设。对于R61阶段的 在目标1中，我们将通过以下方式生成、表征和验证IPSC来源的成熟人NVU形成细胞 利用miRs和BBB特异的Tf调制优化已发表的协议并验证其分子同一性 和生物功能。在目标2(R61阶段)中，我们将建立血运和灌流的3D BBB模型 使用BEC、PC和ACS组合的生理相关流量，即用片上脑 设计和标记血浆代谢物和蛋白质。同时，我们将发展血管化和灌注化 具有生理相关流量和标记的血液成分的脑有机化合物。在这些模型中，我们将 通过转录学、细胞生物学、成像和功能研究鉴定血脑屏障功能。对于R33 在项目阶段(目标3)，我们将分析血液成分(血浆或免疫细胞)对以下方面的影响： 血脑屏障细胞生物学；b)标记代谢物、血浆蛋白、药物或免疫细胞的跨血脑屏障运输； C)测试血液成分对EC-PC相互作用、PC收缩、周细胞或星形胶质细胞的影响 血管覆盖率、星形胶质细胞钙信号；以及d)评估免疫细胞的转运(巨噬细胞、T 细胞)和免疫细胞对血液/BBB 3D模型的影响。拟议的研究将建立一个创新的 灌流的血液-BBB 3D界面模型，将允许我们检查血液成分之间的关系 (血浆、免疫细胞)和健康条件下的血脑屏障和脑损伤。这一模型可能有助于发现 或分析潜在的生物标记物，并评估针对系统性红斑狼疮的潜在疗法的疗效 脑损伤中炎症驱动的神经病理生理学。

PROJECT SUMMARY Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide due to its heterogeneity and complex mechanisms of pathogenesis. Clinical outcomes following TBI are determined by the nature and severity of the primary injury as well as activation of the peripheral immune response. This project will a) establish protocols to generate validated human induced pluripotent stem cells (iPSC)-derived brain endothelial cell (BECs), pericytes (PCs) and astrocytes (ACs) that form a neurovascular unit (NVU), b) develop a novel 3D perfused blood / blood-brain barrier (BBB) interface model together with patient-derived plasma proteins and immune cells and c) examine the effects of the blood components circulating in TBI patients on the NVU function. In preliminary studies and recent publications, we have (i) developed strategies using RNA or viral-induced transcription factors (TFs) to reprogram somatic cells into iPSCs, (ii) differentiate iPSC-derived epithelial cells into BECs and validate their identity using multiple approaches, (iii) generate brain organoids that incorporate blood vessels, (iv) develop brain-on-a-chip models that incorporate blood components and flow. Building upon these studies, we hypothesize that this multi-disciplinary approach will establish a novel perfused blood-BBB interface 3D model to evaluate the mechanisms by which blood components (plasma proteins, immune cells) impair the human NVU after TBI. We will address this hypothesis with three aims. For the R61 phase of the proposal in Aim 1, we will generate, characterize & validate iPSC-derived mature human NVU-forming cells by optimizing the published protocols using miRs and BBB-specific TF modulation and verify their molecular identity and biological function. In Aim 2 (R61 phase), we will establish vascularized and perfused 3D BBB models with physiological relevant flow rates using a combination of BECs, PCs and ACs, ready-to-use brain-on-a-chip devises and labelled plasma metabolites and proteins. In parallel we will develop vascularized and perfused brain organoids with physiological relevant flow and labelled blood components. In these models, we will characterize BBB function using transcriptomics, cell biological, imaging and functional studies. For the R33 phase of the project (Aim 3), we will analyze the effects of blood components (plasma or immune cells) on: a) BBB cell biology; b) transport of labelled metabolites, plasma proteins, drugs or immune cells across the BBB; c) test the effects that blood components have on EC - PC interactions, PC contractility, pericyte or astrocyte coverage of blood vessels, astrocyte Ca++ signaling; and d) evaluate immune cell trafficking (macrophage, T cells) and effects of immune cells on the blood/BBB 3D model. The proposed studies will establish an innovative perfused blood-BBB 3D interface model that will allow us to examine the relationship between blood components (plasma, immune cells) and the BBB in healthy conditions and brain injury. This model may facilitate discovery or analysis of potential biomarkers and evaluate the efficacy of potential therapeutics that target the systemic inflammatory-driven neuropathophysiology in TBI.