Spheroid construction using iPS cell-derived mesenchymal stem and endothelial cells

Induced pluripotent stem cells (iPSCs) are a promising source for regenerative medicine due to their multilineage differentiation ability. We established a feeder-free xeno-reduced workflow to merge iPSC-derived mesenchymal stem cells (MSCs) and endothelial cells (ECs) into uniform hybrid spheroids for bone repair. iPSCs were differentiated into MSCs (CD73⁺/CD90⁺/CD105⁺; osteogenic, adipogenic, and chondrogenic) and ECs using a defined kit with brief fetal bovine serum exposure, yielding CD31⁺ cells that formed tube-like networks on the Matrigel. MSCs and ECs (5:1) were seeded in low-adhesion MicoCell^® microwells, self-assembled within 4 h into hundreds of compact size-controlled spheroids, and maintained for 72 h under serum-free conditions. After clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC) clearing and confocal immunofluorescence, CD90 and CD31 signals showed a distinct spatial organization within the three-dimensional structures, indicating the coexistence of both lineages. Notably, lumen formation was not observed at this stage. Overall, our feeder-free single-source system provides a reproducible platform for subsequent vascular maturation studies.