An Optimized Protocol for the Generation of Vascular Organoids from Human Induced Pluripotent Stem Cells

Vascular organoids derived from human pluripotent stem cells (hPSCs) have emerged as powerful three-dimensional models for studying vascular development, disease mechanisms, and drug responses. Current vascular organoid protocols enable the generation of self-organizing endothelial-pericyte networks; however, batch-to-batch variability, inconsistent organoid formation, and inadequate pericyte coverage can compromise reproducibility and scalability. This study presents an optimized protocol for generating vascular organoids from human induced pluripotent stem cells (hiPSCs) with improved reproducibility and structural consistency. Key modifications include refined embryoid body formation conditions, optimized growth factor concentrations and timing during mesoderm induction and vascular specification, and improved three-dimensional culture conditions that promote robust pericyte recruitment and endothelial-pericyte interactions. The optimized organoids exhibit highly branched CD31-positive endothelial networks consistently ensheathed by PDGFR-β-positive pericytes, with significantly reduced batch-to-batch variability compared with the original protocol. Detailed, step-by-step procedures are provided for organoid generation, whole-mount immunofluorescence characterization, and quality control assessment. This optimized method enables the scalable production of high-quality vascular organoids suitable for studying vascular cell interactions, modeling disease, and screening compounds, thereby lowering the technical barrier for laboratories seeking to adopt this model system.