Ex Vivo Study of hAMSC Secretome in Inflammatory Bowel Disease: Effects on Inflammation and Fibrosis (TARGET)

Inflammatory Bowel Diseases (IBD), including ulcerative colitis and Crohn's disease, are chronic immune-mediated disorders characterized by relapsing gastrointestinal inflammation driven by genetic, immune, microbial, and environmental factors. Despite advances in biologic therapies and small molecules, a substantial proportion of patients exhibit incomplete response, loss of response over time, or progression toward structural bowel damage, including fibrosis, for which no approved anti-fibrotic therapies are currently available. Current treatments mainly target single inflammatory pathways and are insufficient to restore the complex immune, epithelial, and stromal network dysfunction underlying disease persistence and progression, particularly in refractory disease and fibrostenotic Crohn's disease. This study investigates the ex vivo effects of human amniotic mesenchymal stromal cell (hAMSC)-derived secretome, a cell-free biologic product containing bioactive mediators and extracellular vesicles with immunomodulatory, anti-inflammatory, anti-fibrotic, and pro-regenerative properties. The secretome is hypothesized to modulate immune responses, epithelial barrier integrity, mucosal repair, and fibrotic pathways simultaneously. Preliminary data in peripheral blood mononuclear cells (PBMCs) show reduced T helper 1 (Th1) polarization with decreased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and increased regulatory T cells (FOXP3+). Ex vivo experiments in Crohn's disease biopsies indicate a shift toward a more tolerogenic and reparative cytokine profile. This monocentric translational study includes 40 adult patients (≥18 years) with confirmed IBD, stratified into four clinical subgroups based on disease activity, treatment exposure, and fibrostenotic phenotype. Intestinal biopsies and PBMCs are collected prospectively and analyzed within 7 days of sampling. The primary objective is to evaluate ex vivo modulation of inflammatory, immune, epithelial, and fibrotic pathways after exposure to hAMSC secretome. Secondary objectives include assessment of fibrosis markers (COL1A1, ACTA2), epithelial barrier proteins (claudin-1, claudin-2, MUC2), barrier function (TEER), and molecular pathway changes using patient-derived organoids and co-culture systems under basal and pro-fibrotic conditions. The study duration is 36 months, including sample collection, laboratory experiments, multi-omics analyses, and data integration.

Inclusion Criteria: * Age ≥ 18 years * Confirmed diagnosis of inflammatory bowel disease (IBD), including Crohn's disease or ulcerative colitis * Classification into one of the predefined clinical subgroups (remission, active treatment-naïve disease, refractory disease defined as failure of ≥2 lines of therapy, or fibrostenotic Crohn's disease phenotype) * Availability of intestinal biopsies and/or peripheral blood mononuclear cells (PBMCs) for ex vivo analyses * Written informed consent for collection, storage, and use of biological samples for research purposes, in accordance with the Declaration of Helsinki and local Ethics Committee approval Exclusion Criteria: * Age \< 18 years * Presence of systemic diseases not related to inflammatory bowel disease (IBD) that may interfere with immunological or molecular analyses * Inadequate or unavailable biological material for ex vivo experiments, including inability to generate patient-derived intestinal organoids