Graphene oxide-induced multiple iPSC spheroid formation for enhanced production of angiogenic exosomes accelerating tissue regeneration.

Exosome-based therapeutics have emerged as a promising cell-free strategy for tissue regeneration by coordinating angiogenesis and tissue repair. Induced pluripotent stem cell (iPSC)-derived exosomes are particularly attractive because of their regenerative potential and scalability; however, the reproducible and scalable production of functionally potent exosomes remains a major barrier to clinical translation. Here, we developed a graphene oxide (GO)-mediated nanoengineering strategy to establish a multi-spheroid platform that simultaneously regulates spheroid architecture, enhances exosome production, and improves angiogenic functionality in both iPSC spheroids and iPSC-derived vascular endothelial cell (vEC) spheroids. GO reproducibly reorganized cellular aggregation from a single dominant spheroid into multiple uniformly sized spheroids per well and increased exosome production while preserving characteristic vesicle properties. GO-engineered iPSC spheroid-derived exosomes exhibited distinct angiogenesis-related miRNA profiles and significantly enhanced endothelial cell migration and capillary-like network formation in vitro. Extending this strategy to iPSC-vEC spheroids further enhanced endothelial differentiation and generated lineage-specific exosomes with greater pro-angiogenic activity. In a rat full-thickness skin wound model, GO-engineered spheroid-derived exosomes accelerated wound closure and promoted vascularized skin regeneration. Collectively, these findings establish GO-mediated spheroid nanoengineering as a reproducible and potentially scalable strategy for producing therapeutically potent exosomes. By integrating controlled spheroid formation, enhanced exosome productivity, preserved vesicle characteristics, and improved angiogenic efficacy, this platform provides a practical framework for standardized exosome manufacturing and supports its future clinical translation toward vascular regenerative therapies.