# Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

## TL;DR

This study shows that Notch signaling lowers vesicular endocytosis in human brain-like endothelial cells, helping to understand how the blood-brain barrier forms.

## Contribution

The study demonstrates that Notch signaling reduces vesicular endocytosis in a human in vitro model of the blood-brain barrier.

## Key findings

- N1ICD overexpression upregulated GLUT-1 and reduced PLVAP and caveolin-1 expression.
- Combined Wnt/β-catenin activation and N1ICD overexpression reduced vesicles and albumin uptake.
- Notch signaling contributes to reduced transcytosis in human CNS-like endothelial cells.

## Abstract

Mechanisms guiding the induction of blood-brain barrier (BBB) properties in central nervous system (CNS) endothelial cells during human development are incompletely understood. For example, there is a limited understanding of signaling pathways that influence the unique property of low vesicular endocytosis and transcytosis in brain microvascular endothelial cells (BMECs) relative to peripheral endothelial cells. Mouse studies suggest the importance of BBB-relevant developmental pathways, including Wnt and Notch signaling, for the induction of this BBB feature in developing BMECs.

To explore induction of reduced vesicular endocytosis and transcytosis in human in vitro model of the BBB, we used human pluripotent stem cell (hPSC)-derived endothelial progenitor cells (EPCs) in which Wnt/β-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 resulted in upregulation of GLUT-1, a BBB-enriched glucose transporter, and decreased expression of both PLVAP and caveolin-1, two vesicular endocytosis-associated proteins. The combination of Wnt/β-catenin activation and N1ICD overexpression resulted in fewer vesicles and reduced albumin uptake.

These findings indicate that Notch signaling reduces vesicular endocytosis and transcytosis in a human model of the developing BBB and contribute to our understanding of how Notch signaling induces these specific BBB properties in this model of human CNS EC development.

The online version contains supplementary material available at 10.1186/s12987-025-00754-6.