Early-passage human umbilical cord MSC-derived exosomes exhibit enhanced neurofunctional activity in neuronal cells

Mesenchymal stem cell (MSC)-derived exosomes are promising mediators of neuroregeneration, but their functional heterogeneity remains incompletely understood. We investigated whether parental-cell passage and vesicle integrity influence the characteristics and biological activity of exosomes derived from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSC identity was confirmed by spindle-shaped morphology and flow cytometric analysis demonstrating high expression of CD73, CD90, and CD105 together with minimal expression of CD34 and CD45. Exosomes isolated from passages 1 (P1), 3 (P3), and 5 (P5) were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, scanning electron microscopy, and Western blotting. Their biological effects were evaluated in Neuro-2a (N2A) cells by immunofluorescence analysis of βIII-tubulin, MAP2, and NeuN together with the CCK-8 assay. P1-derived exosomes were associated with increases in neuronal-associated marker expression, neurite-like morphology, and CCK-8 metabolic activity, whereas heat-treated exosomes exhibited attenuated biological activity. These findings indicate that parental-cell passage and vesicle integrity influence the biological activity of hUC-MSC-derived exosomes and highlight the importance of standardized MSC characterization, passage control, and exosome quality assessment for the development of reproducible neuroregenerative products.