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

项目摘要 创伤性脑损伤（TBI）是世界范围内死亡和残疾的主要原因之一，由于其异质性 发病机制复杂。TBI后的临床结果取决于TBI的性质， 原发性损伤的严重程度以及外周免疫应答的激活。该项目将：a） 建立产生经验证的人诱导多能干细胞（iPSC）衍生的脑内皮细胞的方案 细胞（BEC）、周细胞（PC）和星形胶质细胞（AC）形成神经血管单位（NVU），B）开发新的3D 灌注的血液/血脑屏障（BBB）界面模型以及患者来源的血浆蛋白，以及 免疫细胞，和c）检查TBI患者中循环的血液成分对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， （ii）使iPSC衍生的细胞分化成BEC 并使用多种方法验证其身份，（iii）生成包含血液的脑类器官 血管，（iv）开发包含血液成分和流动的芯片脑模型。在此基础上， 研究中，我们假设这种多学科的方法将建立一种新的灌注血液-血脑屏障界面 3D模型，以评估血液成分（血浆蛋白，免疫细胞）损害 TBI后的人NVU。我们将以三个目标来讨论这个假设。对于建议书的R61阶段， 目的1，我们将通过优化来产生、表征和验证iPSC衍生的成熟人NVU形成细胞， 使用miR和BBB特异性TF调节的公开方案，并验证其分子身份， 生物功能。在目标2（R61期），我们将建立血管化和灌注的3D BBB模型， 使用BEC、PC和AC组合的生理相关流速，即用型脑芯片 设计并标记血浆代谢物或蛋白质。与此同时，我们将开发血管化和灌注的大脑 具有生理相关流动和标记的血液成分的类器官。在这些模型中，我们将描述 BBB功能使用转录组学，细胞生物学，成像和功能研究。对于R33阶段， 项目（目标3），我们将分析血液成分（血浆或免疫细胞）对：a）BBB细胞生物学的影响; B）标记的代谢物、血浆蛋白、药物或免疫细胞穿过BB B的转运; c）BEC-PC 相互作用、PC收缩性、血管的周细胞或星形胶质细胞覆盖、星形胶质细胞Ca++信号传导;以及 免疫细胞（巨噬细胞、T细胞）穿过BBB的运输以及免疫细胞对血液/BBB 3D的影响 模型拟议的研究将建立一个创新的灌注血液-BBB 3D界面模型， 我们检查血液成分（血浆，免疫细胞）和健康人血脑屏障之间的关系， 条件和脑损伤。该模型可以促进潜在生物标志物的发现或分析，并评估 靶向全身炎症驱动的TBI神经病理生理学的潜在治疗方法的疗效。

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 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 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 and 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 or 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) BEC - PC interactions, PC contractility, pericyte or astrocyte coverage of blood vessels, astrocyte Ca++ signaling; and d) immune cell trafficking (macrophage, T cells) across the BBB 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.