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 through tight junctions, selective transporters, and low transcytosis. BBB dysfunction contributes to cerebrovascular and neurodegenerative disease, yet current human in vitro models recapitulate only a subset of BMEC features. Here, we describe a strategy generate BMECs (hiBMECs) from human induced pluripotent stem cell-derived endothelial cells by co-culture with isogenic brain pericytes and activation of Wnt/β-catenin signaling. The resulting hiBMECs display barrier properties, active efflux transporters, and appropriate inflammatory responses. Transcriptomic profiling revealed convergence of pericyte-derived cues and Wnt/β-catenin activation on ETS1, SMAD3/4, and PPARγ transcriptional networks, establishing a gene signature closely matching the adult human BBB. Downstream analysis revealed that hiBPC cues engaged sphingosine-1-phosphate, TGF-β, and angiopoietin/Tie2 pathways, which were further regulated by canonical Wnt activation. These findings uncover a synergistic mechanism by which brain pericytes and Wnt/β-catenin signaling orchestrate 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.