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 ETV2 and NKX3.1 using Dox-inducible or modRNA systems. This approach enables efficient co-differentiation of endothelial (iECs) and mural cells (iMCs), producing functional 3D VOs in five 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 transcription factor 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. Overall design: Using bulk RNA sequencing, we compared gene expression profiles of induced endothelial cells between 2D (2D-iEC) and VO (VO-iEC), and also the gene expression profiles of induced mural cells between 2D (2D-iMC) and VO (VO-iMC). By comparing these samples, we can assess the maturation potential of VO-derived iECs and iMCs compared to monolayer differentiation protocols. Employing single cell RNA sequencing (scRNA-seq), we investigated Dox1-VOs (dox1_S6) and Dox3-VOs (dox3_S7), as well as Dox3-VOs (S3-7_S8) cultured for an additional seven days without Dox exposure, to further investigate the vascular cell heterogeneity within VOs.