Synergistic induction of blood–brain barrier properties

Porkoláb, Gergő and Mészáros, Mária and Szecskó, Anikó and Vigh, Judit Piroska and Walter, Fruzsina and Figueiredo, Ricardo and Kálomista, Ildikó and Hoyk, Zsófia and Vizsnyiczai, Gaszton and Gróf, Ilona and Jan, Jeng-Shiung and Gosselet, Fabien and Pirity, Melinda and Vastag, Monika and Hudson, Natalie and Campbell, Matthew and Veszelka, Szilvia and Deli, Mária Anna (2024) Synergistic induction of blood–brain barrier properties. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 121 (21). ISSN 0027-8424

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Abstract

Blood–brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell–derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/β-catenin signaling and inhibition of the TGF-β pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/β-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell–derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

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