Stepwise Differentiation of Mouse Embryonic Stem Cells Into Murine Blood Vessel Organoids With Endothelial Lineage Tracing for Quality Control

In vitro vascular models are most informative when they recapitulate endothelial assembly within a 3D microenvironment. Blood vessel organoids (BVOs) enable the study of vascular heterogeneity, function, and organ-instructive cues in development, homeostasis, and disease. Here, we present a robust stepwise method to generate murine blood vessel organoids (mBVOs) from feeder-dependent mouse embryonic stem cells (mESCs) of common genetic backgrounds. Embryoid bodies (EBs) are formed using strain-specific seeding densities (day 0-3), followed by mesoderm induction (day 3-6) and vascular induction (day 6-8). Induced EBs are embedded in collagen I with Geltrex to drive sprouting and network formation (day 8-13). Vascular networks are microdissected and grown in suspension to yield mature mBVOs (day 21-30). The inclusion of a Cre-inducible VE-cadherin-GFP reporter line enables a quantitative quality control, reducing variability by excluding poorly differentiated organoids. The protocol reliably produces ~100 mBVOs per differentiation and is compatible with engineered mouse strains for gain- and loss-of-function studies, functional assays of vascular plasticity, and syngeneic grafting to assess perfusion. Thus, mBVOs provide a scalable and traceable 3D platform that bridges endothelial assays, mouse models, and human organoid systems. Key features • A detailed timeline to differentiate feeder-dependent mESCs into mBVOs, with key success readouts and troubleshooting. • Efficient across three genetic backgrounds with strain-specific EB seeding densities and typical yields of ~100 mBVOs per differentiation. • The inducible VE-cadherin-GFP lineage tracing/reporter system provides an endothelial quality control to quantify efficiency and exclude poorly differentiated organoids. • Compatible with engineered mouse strains for gain/loss-of-function, with in vitro assays of vascular plasticity/remodeling, and with syngeneic in vivo validation.