A novel human blood-brain barrier model reveals pericytes as critical regulators of viral neuroinvasion

The blood-brain barrier (BBB) plays a vital role in regulating the passage of biomolecules between the bloodstream and the central nervous system (CNS) while also protecting the CNS from pathogens. Pericytes reside at the interface between the endothelial cells that form the vessel walls and the brain parenchyma. These cells are critical for maintaining BBB integrity and regulate vessel permeability, blood flow, and immune cell migration. In this study, we developed a novel serum-free protocol to generate neural crest cell-derived pericytes (NCC-PCs) from human pluripotent stem cells (hPSCs). These NCC-PCs enhance BMEC barrier function and can be co-cultured with hPSC-derived brain microvascular endothelial cells (BMECs) in a co-culture BBB model that recapitulates the in vivo cellular interactions at the BBB. We used this model to evaluate the pathological consequences of BBB exposure to highly neuroinvasive flaviviruses. Our results identify a previously undescribed role for NCC-PCs in maintaining BMEC barrier integrity during infection and reducing the spread of viral infection to the CNS. Overall design: Human brain microvascular endothelial cells (BMECs), pericytes (PCs), and astrocytes were differentiated from human pluripotent stem cells (hPSCs). Cells were infected with neuroinvasive flaviviruses including West Nile virus, Poweassan virus, and Japanese encephalitis virus. In addition, hPSC-derived BMECs and PCs were plated in a transwell co-culture system and then exposed to West Nile virus. The transcriptional response of both BMECs and PCs to West Nile virus was examined at both early (8 hours post infection) and late (48 hours post infection) time points. The hPSC-derived pereicytes were made using a novel serum-free protocol, for cell characterization experiments the transciptional profile was compared to both primary brain pericytes as well as hPSCs, hPSC-derived neural crest cells, and hPSC-derived PCs generated from previously published protocols.