Brain pericytes and Wnt/β-catenin signaling induce functional blood-brain barrier phenotype in human iPSC-derived model

The blood-brain barrier (BBB), formed by brain microvascular endothelial cells (BMECs), restricts vascular permeability by establishing tight junctions, reducing vesicular transport, and regulating molecular trafficking via dedicated transporters. In disease, BBB dysfunction leads to barrier disruption and neuroinflammation. Existing human in vitro BBB models recapitulate some but not all of these key BMEC features. Here, we present a method to differentiate human induced pluripotent stem cells (hiPSCs) into BMECs (hiBMECs) by co-culturing hiPSC-derived endothelial cells with brain pericytes (hiBPCs) and activating the Wnt/β-catenin pathway. The resulting hiBMECs exhibit barrier properties, functional efflux transporters, and appropriate inflammatory responses. RNA sequencing revealed a transcriptomic profile in hiBMECs closely matching the adult human BBB. Mechanistically, hiBPCs and Wnt/β-catenin signaling provided complementary cues that converged on ETS1, SMAD3/4, and PPARγ transcriptional networks. These findings reveal a synergistic mechanism by which brain pericytes and Wnt/β-catenin signaling orchestrate human BMEC differentiation and function, providing mechanistic insight into human BBB development and an improved hiPSC-derived BBB model for future drug screening and disease modeling.