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.
