Background: Extracellular vesicles derived from stem cells (SC-EVs) show promise in regenerative medicine and inflammation resolution because of their low immunogenicity and translational potential. However, low SC-EV yields hinder their clinical scalability and therapeutic utility. Therefore, we aimed to enhance SC-EV production.

Methods: We explored strategies to enhance SC-EV production using Bafilomycin A1 (Baf-A1). The data are expressed as mean ± standard deviation (SD) values, and differences with p < 0.05 were considered significant.

Results: Baf-A1 increased the production of EVs from mesenchymal stem cells derived from induced pluripotent stem cells (iPSC-MSC-EVs) (p < 0.01). Mechanistically, Baf-A1 promotes iPSC-MSC-EV secretion while inhibiting their reuptake by the parent cells (p < 0.0001). Baf-A1 treatment preserved the gene and protein expression profiles of iPSC-MSC-EVs, maintaining their intrinsic biological properties and ensuring their reliability and safety for application. Furthermore, Baf-A1-treated induced pluripotent stem cell-derived mesenchymal stem cell extracellular vesicles (iPSC-MSC-EVs) demonstrated comparable therapeutic efficacy to untreated iPSC-MSC-EVs in acute liver injury (ALI) and inflammatory bowel disease (IBD) models, confirming their retained ability to promote tissue regeneration and anti-inflammation in these models.

Conclusions: These findings highlight Baf-A1 as a safe and potent modulator of iPSC-MSC-EV production, offering a strategy to overcome yield limitations and advance the clinical translation of iPSC-MSC-EVs in regenerative medicine and inflammatory disease therapy.