Introduction: Amniotic epithelial cell (AEC)-derived secretomes play a pivotal role in intercellular communication by modulating key biological processes, thanks to their enrichment in growth, trophic, and protective factors. While their cell-free regenerative potential has been widely demonstrated in vitro, in vivo evidence remains limited. Recent studies suggest that perinatal stem cell secretomes exert antioxidant and anti-apoptotic effects in zebrafish. Our group previously showed that the microvesicle (MV)-enriched fraction of both ovine and human immortalized-AEC (iAEC) secretomes display significant anti-inflammatory and immunomodulatory properties. The present study investigated the regenerative potential of the iAEC-derived MV fraction using a zebrafish model of caudal fin regeneration.

Methods: The regenerative potential of iAEC-derived MVs was evaluated in zebrafish larvae subjected to caudal fin resection at 72 hours post-fertilization (hpf), followed by exposure to MVs isolated by ultracentrifugation. Fin regrowth was assessed morphometrically at 6, 24, and 48 hours post-treatment using an inverted microscope and quantified with ImageJ. Expression of pro-regenerative genes (has3, msxb, fgf20a, mmp9) was evaluated by qPCR, and cell proliferation visualized using Click-iT® EdU staining under confocal microscopy. Mitochondrial uptake was assessed via TurboRFP-transfected mitochondria in Tg(mpx:GFP) larvae at confocal microscopy, and human mtDNA quantified by qPCR. Apoptosis was evaluated through Acridine Orange staining. Immune cell dynamics were monitored in Tg(lyz:DsRED2) larvae using time-lapse imaging and fluorescence quantification.

Results: MV treatment significantly enhanced fin regrowth in treated larvae compared to controls, with measurable differences emerging as early as 24 hours post-treatment. Exposure to MVs sustained the activation of key regenerative pathways, as supported by the upregulation of pro-regenerative genes, with further enhancement observed upon progesterone-mediated amplification of iAECs. Mitochondrial transfer was confirmed, indicating effective internalization of MV cargo by zebrafish larvae. Moreover, MV-treated larvae exhibited reduced apoptosis compared to controls. An accelerated acute inflammatory response was also observed during the early phases post-injury, suggesting a role for MVs in priming the regenerative process. Collectively, these findings indicate that iAEC-derived MVs orchestrate a coordinated regenerative program involving enhanced proliferation, reduced apoptosis, and dynamic immune modulation.

Conclusion: These findings validate the regenerative and immunomodulatory potential of iAEC-derived MVs, supporting their development as a cell-free therapeutic strategy. By actively enhancing regeneration in vivo, iAEC-derived MVs demonstrate translational relevance in regenerative medicine, potentially overcoming well-known issues of cell-based approaches such as safety and immunogenicity.