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

使用血管化和灌注的人血/BBB 模型评估 TBI 后外周免疫激活对 NVU 的影响

• 批准号: 10897345
• 负责人: Dritan Agalliu
• 金额: $ 56.54万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10897345
• 关键词: 3-Dimensional; Astrocytes; Biological; Biological Process; Blood; Blood - brain barrier anatomy; Blood Vessels; Blood brain barrier dysfunction; Brain; Brain Injuries; Cause of Death; Cell Line; Cell physiology; Cells; Cellular biology; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Diffusion; Disease; Extravasation; Foundations; Functional disorder; Heterogeneity; Human; Image; Immune; Immune response; Immune system; Impairment; Incidence; Inflammatory; Injury; Label; Macrophage; Maintenance; Mediating; Methods; Military Personnel; Modeling; Molecular; Nature; Neuroprotective Agents; Nutrient; Organoids; Outcome; Pathogenesis; Patients; Perfusion; Pericytes; Peripheral; Pharmaceutical Preparations; Phase; Physiological; Plasma; Plasma Proteins; Population; Property; Proteins; Protocols documentation; Publications; Publishing; RNA; Role; Severities; Signal Pathway; Signal Transduction; Somatic Cell; Source; Structure; System; T-Lymphocyte; TBI Patients; Testing; Tight Junctions; Traumatic Brain Injury; Treatment Efficacy; Vascularization; Viral; blood perfusion; blood-brain barrier function; blood-brain barrier permeabilization; brain endothelial cell; cytokine; disability; efficacy evaluation; immune activation; induced pluripotent stem cell; innovation; interdisciplinary approach; nervous system disorder; neurovascular unit; novel; potential biomarker; programs; seal; single-cell RNA sequencing; therapeutic target; three-dimensional modeling; tool; trafficking; transcription factor; transcriptome sequencing; transcriptomics; transcytosis

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.

项目摘要 由于其异质性，创伤性脑损伤（TBI）是全球死亡和残疾的主要原因之一 和发病机理的复杂机制。 TBI之后的临床结果由性质和 原发性损伤的严重程度以及外周免疫反应的激活。这个项目将：a） 建立协议以生成经过验证的人类诱导的多能干细胞（IPSC）衍生的脑内皮 细胞（BEC），周细胞（PC）和星形胶质细胞（AC）形成神经血管单元（NVU），b）发展了新型的3D 灌注血 /血脑屏障（BBB）界面模型以及患者来源的血浆蛋白和 免疫细胞和c）检查TBI患者循环的血液成分对NVU功能的影响。 在初步研究和最新出版物中，我们有：（i）使用RNA或病毒引起的制定策略 转录因子（TF）将体细胞重编程为IPSC，（ii）将IPSC衍生的细胞区分为BEC 并使用多种方法验证其身份 血管，（iv）开发出脑线芯片模型，这些模型结合了血液成分和流动。基于这些 研究，我们假设这种多学科方法将建立一种新型的灌注血液BBB界面 3D模型评估血液成分（血浆蛋白，免疫细胞）损害的机制 TBI之后的人类NVU。我们将以三个目标解决这一假设。提案的R61阶段 AIM 1，我们将通过优化生成，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调制的已发布协议，并验证其分子身份和 生物功能。在AIM 2（R61阶段）中，我们将建立具有的血管化和灌注的3D BBB模型 使用BEC，PC和ACS的生理相关流速 设计并标记为血浆代谢物或蛋白质。同时，我们将发展血管和灌注大脑 具有生理相关流和标记为血液成分的类器官。在这些模型中，我们将表征 使用转录组学，细胞生物学，成像和功能研究的BBB功能。对于R33阶段 项目（AIM 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）在BBB上运输标记的代谢物，血浆蛋白，药物或免疫细胞； c）BEC -PC 血管的相互作用，PC收缩性，周细胞或星形胶质细胞覆盖范围，星形胶质细胞CA ++信号传导；和D） 免疫细胞运输（巨噬细胞，T细胞）跨BBB以及免疫细胞对血液/BBB 3D的影响 模型。拟议的研究将建立创新的灌注血液BBB 3D接口模型，该模型将允许 我们检查健康中的血液成分（血浆，免疫细胞）与BBB之间的关系 条件和脑损伤。该模型可以促进或分析潜在的生物标志物并评估 针对全身性炎症驱动的神经病理生理学的潜在疗法的功效。