Stepwise Differentiation of Mouse Embryonic Stem Cells Into Murine Blood Vessel Organoids With Endothelial Lineage...

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.