A fully iPSC-derived 3D model of the human blood-brain barrier for exploring neurovascular disease mechanisms and therapeutic interventions

Integrity of the blood-brain barrier (BBB) is critical for brain homeostasis, and its malfunction contributes to neurovascular and neurodegenerative disorders. So far, mechanistic studies on BBB function have been mostly conducted in rodent and non-physiological in vitro models, which recapitulate some disease features, but have limited translatability to humans and pose challenges for drug discovery. Here we report on a fully human iPSC-derived, microfluidic 3D BBB model consisting of endothelial cells (EC), mural cells, and astrocytes. Our model expresses typical cell fate markers, forms a barrier in vessel-like tubes, and enables perfusion, including with human blood. We optimized iPSC differentiations and validated cellular fates by comparison to published datasets and extensive benchmarking vs. primary cells with proteomic profiles provided in an online database. To demonstrate suitability for translational research, we applied the model to investigate deficiency of FOXF2, a major risk gene for cerebral small vessel disease. Deletion of FOXF2 in EC induced key features of BBB dysfunction, including compromised cell junction integrity and enhanced caveolae formation. Proteomic analysis further revealed dysregulation of endocytosis and cell junction pathways. Disease features phenocopied those seen in mice with endothelial cell-specific Foxf2 deficiency, validating the relevance of our in vitro model to investigate in vivo phenotypes of neurovascular disease. Moreover, lipid-nanoparticle-based treatment with Foxf2 mRNA rescued BBB deficits in our FOXF2 KO model, demonstrating its potential for drug development.