# Identification of Cell Fate Determining Transcription Factors for Generating Brain Endothelial Cells

## TL;DR

This study identifies key transcription factors that can guide stem cells to become brain endothelial cells, which are crucial for modeling the blood-brain barrier.

## Contribution

The study identifies specific transcription factors that can reprogram immature endothelial cells into brain endothelial-like cells.

## Key findings

- ETV2 overexpression generates immature endothelial cells from stem cells.
- ZIC3, TFAP2C, TFAP2A, and DLX2 are enriched in cells resembling brain endothelial cells.
- These transcription factors could be used to create in vitro models of the blood-brain barrier.

## Abstract

Reliable models of the blood-brain barrier (BBB), wherein brain microvascular endothelial cells (BMECs) play a key role in maintenance of barrier function, are essential tools for developing therapeutics and disease modeling. Recent studies explored generating BMEC-like cells from human pluripotent stem cells (hPSCs) by mimicking brain-microenvironment signals or genetic reprogramming. However, due to the lack of comprehensive transcriptional studies, the exact cellular identity of most of these cells remains poorly defined. In this study we aimed to identify the most likely master transcription factors (TFs) for inducing brain endothelial cell (EC) fate and assess the transcriptomic changes following their introduction into immature ECs. Therefore, we first generated PSC-derived immature ECs by transient overexpression of the TF, ETV2. Subsequently, by performing an extensive meta-analysis of transcriptome studies of brain and non-brain ECs, 12 candidate TFs were identified, which might fate immature ECs towards cells with brain EC features. Following combinatorial overexpression of these 12 TFs tagged with unique barcodes, single cell transcriptomics identified a subset of transduced cells that resembled mid-gestational human brain ECs. Assessment of the TF barcodes present in these cells revealed significant enrichment of the TFs ZIC3, TFAP2C, TFAP2A, and DLX2. These TFs might be useful to fate PSC-EC to BMEC-like cells, which could be incorporated in human in vitro BBB models.

The online version contains supplementary material available at 10.1007/s12015-025-10842-7.