Neural stem cell-derived extracellular vesicles drive early neuroprotective and anti-apoptotic responses in spinal cord injury organotypic slices

Introduction Spinal cord injury (SCI) is a devastating neurological condition with limited regenerative capacity. Stem cell–based approaches have emerged as promising strategies due to their neuroprotective and immunomodulatory properties, largely mediated by small extracellular vesicles (sEVs) and their molecular cargo, including miRNAs. In this study, we aimed to evaluate the neuroprotective and anti-apoptotic potential of sEVs derived from SPC-01 and iMR-90 neural stem cell sources using an in vitro rat model of SCI. Methods sEVs were isolated from SPC-01 and iMR-90 culture media and characterized by MADLS and Western blot. Spinal cord slices (SCS) were used as an in vitro SCI model with three groups: control, SCI, and SCI treated with sEVs. Injury was induced at 18–20 days in vitro , followed by immediate sEV application. After 72 h, tissue samples were collected and analyzed to assess proteins associated with apoptosis, cytoskeletal integrity, and survival signaling pathways. Results SCI induced cytoskeletal disruption and increased apoptotic markers. sEV treatment attenuated these changes, reducing injury-associated proteins toward baseline levels. Both SPC-01- and iMR-90-derived sEVs showed neuroprotective effects. This was associated with modulation of key pathways, including decreased PTEN, increased STAT3 phosphorylation, and elevated Bcl-xL. Reduced Nogo-A and normalized RhoA levels further indicate attenuation of inhibitory signaling and improved cytoskeletal stability. Overall, sEVs promoted early neuroprotective responses and reduced pathology-associated protein expression in the SCI model. Discussion Neural stem cell-derived sEVs promote neuroprotection in vitro by modulating PTEN/STAT3 signaling, reducing apoptosis, and stabilizing cytoskeletal dynamics. Although limited to early injury responses in an in vitro model, these findings support sEVs as a promising cell-free therapeutic strategy for SCI.