Murine vascular organoids are responsive and adaptable 3D systems with cellular heterogeneity and dynamic plasticity

Blood vascular organoids (BVOs) have emerged as powerful tools for interrogating endothelial heterogeneity, function, and organ-specific cues during development, homeostasis, and disease. Here, we describe the stepwise generation and characterization of murine BVOs from different embryonic stem cells and introduce a fluorescent tracer system to follow their formation and maturation, visualizing functional and perfused vessels after transplantations into mice. Single-cell RNA sequencing, FACS, and whole-mount immunohistochemistry revealed a heterogeneous and dynamic trajectory of multiple endothelial, mesenchymal, and macrophage clusters in mBVOs at growth spike and full maturation. Endothelial cells inferred high plasticity toward arterial, venous, capillary, and tip/stalk cells and a neural-like identity at full maturation state. mBVOs responded to inflammatory cues and VEGF blockade in vitro and could be assembled with tumor spheroids in which tumor cells mimicked the same vascular growth pattern as observed in vivo. Our results highlight mBVOs as adaptable in vitro platforms, unlocking various applications and avenues for discovery in vascular biology.