Notch signaling activation in hPSC-derived CNS-like endothelial cells induces differential expression of blood-brain barrier-related genes

Mechanisms guiding the induction of blood-brain barrier (BBB) properties in central nervous system (CNS) endothelial cells during human development are incompletely understood. To explore induction of reduced vesicular endocytosis and transcytosis properties in a human in vitro model of the BBB, we used human pluripotent stem cell (hPSC)-derived endothelial progenitor cells (EPCs) in which Wnt/ß-catenin signaling was activated to generate hPSC-derived CNS-like ECs (hPSC-CECs). We assessed the effects of Notch signaling through overexpression of the Notch1 receptor intracellular domain (N1ICD). N1ICD overexpression in hPSC-CECs was induced by treatment with doxycycline (Dox), and hPSC-CECs were sorted into subpopulations by FACS based on high or low surface expression of CD144/VE-cadherin (CD144hi or CD144lo, respectively). Several negative control replicates treated with PBS (nearly all of which were CD144hi), were included as well. Our findings indicate that Notch signaling differentially regulates vesicular endocytosis and transcytosis-related genes in a human model of the developing BBB, contributing to our understanding of how Notch signaling induces these specific BBB properties in this model of human CNS EC development.