Rapid generation of functional vascular organoids via simultaneous transcription factor activation of endothelial and mural lineages.

Vascular organoids (VOs) are valuable tools for studying vascular development, disease, and regenerative medicine. However, controlling endothelial and mural compartments independently remains challenging. Here, we present a streamlined method to generate VOs from induced pluripotent stem cells (iPSCs) via orthogonal activation of the transcription factors (TFs) ETV2 and NKX3.1 using Dox-inducible or modRNA systems. This approach enables efficient co-differentiation of endothelial cells (iECs) and mural cells (iMCs), producing functional 3D VOs in 5 days without ECM embedding. VOs matured further upon ECM exposure, forming larger, structured vessels. Single-cell RNA sequencing revealed vascular heterogeneity, and temporal regulation of TF expression allowed modulation of arterial and angiogenic iEC phenotypes. In vivo, VOs engrafted into immunodeficient mice, formed perfused vasculature, and promoted revascularization in models of hind limb ischemia and pancreatic islet transplantation. These findings establish a rapid and versatile VO platform with broad potential for vascular modeling, disease studies, and regenerative cell therapy.