Background and aims:

Current single-target biologics for Crohn’s disease (CD) fail to adequately address mucosal repair and immune dysregulation. The human amniotic mesenchymal stromal cell-derived secretome (hAMSC secretome) contains soluble factors and extracellular vesicles with immunomodulatory and regenerative properties; however, its effects on intestinal tissue from CD patients are still poorly defined. This study aimed to assess whether hAMSC-derived secretome modulates cytokine responses in CD patient-derived biopsies and promotes epithelial repair in vitro.

Methods:

Paired inflamed and macroscopically non-inflamed biopsies from CD patients (n=7) were cultured for 24 h with control medium or hAMSC secretome at 25% and 50%, and supernatants were analysed using a 27-plex cytokine assay. Caco-2 cells were stimulated with TNF-α/IFN-γ, treated with hAMSC secretome, and assessed by scratch assay over 48 h. Data were analysed using repeated-measures ANOVA or one-way ANOVA with appropriate multiple-comparison corrections.

Results:

In diseased mucosa, MCP-1/CCL2 levels increased after treatment with both 25% and 50% secretome compared with control conditions (p<0.01), together with a significant upregulation of G-CSF (p<0.05), suggesting activation of pathways involved in immune cell recruitment, tissue remodelling and mucosal repair. Trends toward increased IL-5 and IL-13 were also observed, while no exacerbation of pro-inflammatory cytokine release was detected in either inflamed or macroscopically non-inflamed biopsies. In the scratch assay, TNF-α/IFN-γ stimulation impaired epithelial wound closure in Caco-2 cells. hAMSC-derived secretome restored epithelial repair under inflammatory conditions, with 25% secretome inducing 79.5% wound closure at 24 h and 99.4% at 48 h, and 50% secretome reaching 64.5% and 94.8%, respectively. Both concentrations significantly improved overall wound closure compared with cytokine-stimulated controls by AUC₀₋₄₈ₕ analysis (p<0.001). Secretome alone also accelerated wound closure compared with untreated cells (98.2% vs 54.2% at 48 h, p<0.05), indicating an intrinsic pro-migratory effect on intestinal epithelial cells.

Conclusions:

hAMSC-derived secretome modulates cytokine responses in CD patient-derived biopsies and enhances epithelial repair in vitro. By promoting mediators associated with mucosal remodelling and restoring wound closure under inflammatory conditions, hAMSC-derived secretome may represent a promising cell-free approach to support mucosal healing in Crohn’s disease.

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