Assessing the effects of peripheral immune activation on the NVU following TBI using avascularized 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 （b）形成神经血管单位（NVU）的BEC（BEC）、周细胞（PC）和星形胶质细胞（AC），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， 转录因子（TF）将体细胞重编程为iPSC，（ii）分化iPSC衍生的上皮细胞 （iii）生成脑类器官， 血管，（iv）开发包含血液成分和流动的脑芯片模型。基础上 通过这些研究，我们假设这种多学科的方法将建立一种新的灌注血-BBB 接口3D模型，以评估血液成分（血浆蛋白，免疫细胞） 损伤TBI后的人NVU。我们将以三个目标来讨论这个假设。对于R61阶段， 在目标1的建议中，我们将通过以下方式产生、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调节优化已发表的方案，并验证其分子身份 和生物功能。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和蛋白质。与此同时，我们将发展血管化和灌注 具有生理相关流动和标记的血液成分的脑类器官。在这些模型中，我们将 使用转录组学、细胞生物学、成像和功能研究表征BBB功能。对于R33 在项目的第三阶段（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： BB B细胞生物学; 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）测试血液成分对EC-PC相互作用、PC收缩性、周细胞或星形胶质细胞的影响， 血管覆盖率、星形胶质细胞Ca++信号传导;和d）评估免疫细胞运输（巨噬细胞，T细胞）。 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 灌注血液-BBB 3D界面模型，使我们能够检查血液成分之间的关系 （血浆、免疫细胞）和BBB在健康状况和脑损伤中的作用。这个模型可以帮助发现 或分析潜在的生物标志物，并评估靶向全身性肿瘤的潜在治疗剂的功效。 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.