Abstract: Endothelial cells (ECs) line the blood and lymphatic vessels and are essential for organ-specific metabolic, endocrine, and vascular functions. The high mortality of ischemic vascular diseases highlights the need for innovative strategies to promote neovascularization and generate functional blood vessels. Reprogramming pluripotent stem cells (PSCs) via transcription factor (TF) modulation offers a promising approach for regenerative medicine. In this study, we developed highly efficient, doxycycline-inducible embryonic stem cell lines expressing Etv2, Erg and Fli1(Etv2-ESCs, Erg-ESCs and Fli1-ESCs), three critical TFs for endothelial lineage specification, whose deletion causes embryonic lethality due to vascular defects. We demonstrate that Etv2, Erg, and Fli1 rapidly induce ESC differentiation into endothelial-like cells within 12 hours by activating conserved gene regulatory networks (GRN), critical for EC specification. Time-course analysis revealed inter- and auto-regulatory feedback loops among these TFs, which suppress pluripotent enhancers, while activating endothelial enhancers. GATA and FOXO family members were identified as co-regulators, enhancing subtype-specific endothelial differentiation. Forced expression of the three TFs in naïve ESCs bypassed capacitation, silenced pluripotency genes, and upregulated mesodermal and endothelial markers driving differentiation. We addressed endothelial subtype plasticity, by mapping chromatin accessibility and gene expression in ~14,000 single endothelial cells from adult mouse liver and lung. This revealed organ-specific chromatin remodeling, with activation or repression of cis-regulatory elements, highlighting endothelial subtype heterogeneity. Using this data, we identified novel TFs that drive organotypic endothelial subtype specification via arrayed TF library screening. Additionally, we demonstrated that extracellular signals could direct progenitor cells toward specific subtypes, such as arterial endothelial cells (AECs), enhancing their identity and function. Collectively, our study provides a framework for endothelial cell engineering by elucidating how Etv2, Erg, and Fli1, along with specific TF partners, drive endothelial specification at the tissue and subtype levels.
Funding Source: This work is supported by NIH R01HL170286 and P01 HL160469 grants.
