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

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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.