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bioRxiv · 10.1101/2025.09.26.678752

Pericytes and Wnt signaling induce functional blood-brain barrier phenotype in human iPSC-based model

Nogueira Pinto, H.·Kok, N. R.·Hauger, P. C.·Karsten-van Diepen, M.·de Kok, M.·Paauw, N. J.·van der Pol, S. M.·Nugteren-Boogaard, J. P.·Hordijk, P. L.·Beekhuis-Hoekstra, S. D.·Gibbs, S.·de Wit, N. M.·De Vries, H. E.
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Abstract

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/{beta}-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/{beta}-catenin activation on ETS1, SMAD3/4, and PPAR{gamma} transcriptional networks, establishing a gene signature closely matching the adult human BBB. Downstream analysis revealed that hiBPC cues engaged sphingosine-1-phosphate, TGF-{beta}, and angiopoietin/Tie2 pathways, which were further regulated by canonical Wnt activation. These findings uncover a synergistic mechanism by which brain pericytes and Wnt/{beta}-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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=36 SRC="FIGDIR/small/678752v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@186ba4corg.highwire.dtl.DTLVardef@310cc7org.highwire.dtl.DTLVardef@3e1970org.highwire.dtl.DTLVardef@f80f5e_HPS_FORMAT_FIGEXP M_FIG C_FIG The paper explainedO_ST_ABSPROBLEMC_ST_ABSThe blood-brain barrier (BBB) protects the brain by tightly regulating the passage of molecules and cells. Its dysfunction contributes to disorders such as stroke, dementia, and multiple sclerosis. Yet, existing human in vitro models fail to capture the full complexity of BBB biology, limiting our ability to study disease mechanisms or test brain-targeted drugs. RESULTSWe discovered that two signals are essential for generating functional human BBB endothelial cells from stem cells: cues from brain pericytes and activation of the Wnt/{beta}-catenin pathway. Together, these signals enabled endothelial cells to form tight barriers, operate transporters, and mount appropriate responses to inflammation. Transcriptomic analyses of the resulting cells revealed a gene signature closely matching the adult human BBB and identified how pericyte- and Wnt-activated pathways converge on specific transcriptional programs driving BBB identity. IMPACTThis study provides both a molecular framework for in vitro BBB development and a reliable and reproducible human BBB model. This platform can be applied to explore BBB dysfunction in neurological disease and to accelerate the development of drugs that need to reach the brain or target the brain vasculature.

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Nogueira Pinto, H., Kok, N. R., Hauger, P. C., Karsten-van Diepen, M., de Kok, M., Paauw, N. J., van der Pol, S. M., Nugteren-Boogaard, J. P., Hordijk, P. L., Beekhuis-Hoekstra, S. D., Gibbs, S., de Wit, N. M., De Vries, H. E.. 2025-09-26. Pericytes and Wnt signaling induce functional blood-brain barrier phenotype in human iPSC-based model. https://doi.org/10.1101/2025.09.26.678752

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