Targeted Interrogation of Blood-Brain Barrier Biology

Abstract

The blood-brain barrier (BBB) is formed by endothelial cells that line brain vasculature and separate the blood stream and central nervous system parenchyma. The BBB functions as an exceptionally dynamic molecular gatekeeper, coordinating the delivery of nutrients, clearance of waste, and exclusion of potential neurotoxins from the central nervous system. BBB dysfunction is increasingly associated with many neurodegenerative disorders, but the mechanisms by which this breakdown occurs are largely unknown.

The work presented in this dissertation seeks to address this lack of understanding through the development of representative, reproducible in vitro model systems, followed by targeted and high throughput interrogation of BBB properties. We detail improvements made in the differentiation of human induced pluripotent stem cells to BBB endothelial cells through the use of fully defined media and the exclusion of serum. We leverage these advancements to construct a biomimetic 3D neurovascular model and use RNA sequencing to identify potential contributors to this specialized phenotype. Finally, we detail a high-throughput CRISPR screen to identify potential modulators of BBB paracellular permeability. These collective works provide robust tools for interrogating the molecular underpinnings of BBB function and identifying neurodegenerative disease-modifying therapies.