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

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.

Grant Number: 5R33HL159949-05
NIH Institute/Center: NIH
Principal Investigator: Dritan Agalliu