Prolonged culturing of iPSC-derived brain endothelial-like cells is associated with quiescence, downregulation of glycolysis, and resistance to disruption by an Alzheimer's brain milieu.

Background: Human induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (iBECs) are a robust, scalable, and translatable model of the human blood-brain barrier (BBB). Prior works have shown that high transendothelial electrical resistance (TEER) persists in iBECs for at least 2 weeks, emphasizing the utility of the model for longer term studies. However, most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. In this study, we characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points.

Methods: hiPSCs were differentiated into iBECs using fully defined, serum-free medium. The proportion of proliferating cells was determined by BrdU assays. We evaluated TEER, expression of glycolysis enzymes and tight and adherens junction proteins (TJP and AJP), and glucose transporter-1 (GLUT1) function by immunoblotting, immunofluorescence, and quantifying radiolabeled tracer permeabilities. We also compared barrier disruption in response to TNF-α and conditioned medium (CM) from hiPSC-derived neurons harboring the Alzheimer's disease (AD)-causing Swedish mutation (APP).

Results: A significant decline in iBEC proliferation over time in culture was accompanied by adoption of a more quiescent endothelial metabolic state, indicated by downregulation of glycolysis-related proteins and upregulation GLUT1. Interestingly, upregulation of GLUT1 was associated with reduced glucose transport rates in more quiescent iBECs. We also found significant decreases in claudin-5 (CLDN5) and vascular endothelial-cadherin (VE-Cad) and a trend toward a decrease in platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas zona occludens-1 (ZO-1) increased and occludin (OCLN) remained unchanged. Despite differences in TJP and AJP expression, there was no difference in mean TEER on day 2 vs. day 9. TNF-α induced disruption irrespective of iBEC proliferative state. Conversely, APP CM disrupted only proliferating iBEC monolayers.

Conclusion: iBECs can be used to study responses to disease-relevant stimuli in proliferating vs. more quiescent endothelial cell states, which may provide insight into BBB vulnerabilities in contexts of development, brain injury, and neurodegenerative disease.