Synergistic induction of blood-brain barrier properties
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 propertiesin vitro. To target this novel 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 cARLAviathe tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells towards thein vivobrain 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.
Significance Statement
The blood-brain barrier (BBB) hinders drug delivery to the brain and is implicated in neurological diseases. To better understand these processes in humans, there is a need for culture models that mimic the complexity of the BBB. However, state-of-the-art human BBB models either suffer from a non-physiological, mixed epithelial-endothelial identity or have weak barrier tightness, which greatly limits their usability. We identified a molecule combination that synergistically enhances barrier tightness in severalin vitromodels and induces complex BBB properties in human stem cell-derived endothelial cells by targeting a novel link between three signaling pathways. The molecule combination has the potential to improve BBB culture models across laboratories to advance both basic research and drug development for the human brain.
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