This is a monocentric, translational, procedure-based interventional study investigating the ex vivo effects of human amniotic mesenchymal stromal cell (hAMSC)-derived secretome in inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn's disease. The study is designed to evaluate the immunomodulatory, anti-inflammatory, anti-fibrotic, and pro-regenerative properties of a cell-free biologic product on patient-derived biological systems.
A total of 40 adult patients (≥18 years) with confirmed IBD are prospectively enrolled and stratified into four clinical subgroups based on disease activity, treatment exposure, and fibrostenotic phenotype. Intestinal biopsies (ileal and/or colonic) and peripheral blood mononuclear cells (PBMCs) are collected and processed within 7 days of sampling.
Patient-derived biological samples are exposed ex vivo to hAMSC-derived secretome, which contains soluble bioactive factors and extracellular vesicles. Experimental platforms include intestinal tissue explants, PBMC cultures, intestinal organoids, and PBMC-organoid co-culture systems. Organoids are derived from Crohn's disease biopsies and cultured under basal conditions and under transforming growth factor beta 1 (TGF-β1)-induced pro-fibrotic conditions to model disease-relevant microenvironments.
Immunological effects are assessed by multicolor flow cytometry, including characterization of T helper 1 (Th1), T helper 17 (Th17), regulatory T cells (Treg), macrophages, dendritic cells, and innate lymphoid cells. Cytokine profiling is performed using multiplex bead-based assays to quantify pro- and anti-inflammatory mediators, including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 6 (IL-6), interleukin 10 (IL-10), and interleukin 17 (IL-17).
Fibrotic remodeling is evaluated through analysis of collagen type I alpha 1 chain (COL1A1) and actin alpha 2 smooth muscle (ACTA2) expression using quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence. Epithelial barrier integrity is assessed by quantification of claudin-1, claudin-2, and mucin 2 (MUC2) expression, as well as functional measurement of transepithelial electrical resistance (TEER) in organoid-based systems.
Advanced multi-omics analyses are performed to characterize the immune-epithelial-stromal niche. Spatial transcriptomics (10x Genomics Visium) is used to map tissue-level transcriptional changes, integrated with single-cell RNA sequencing data to identify cell-type-specific responses and molecular pathways associated with treatment exposure. Bioinformatic analyses are conducted to define differentially regulated pathways and candidate biomarkers of response, with particular focus on refractory disease and fibrostenotic Crohn's disease phenotypes.
The study duration is 36 months, including patient recruitment and sample collection (months 4-18), ex vivo experimental procedures (months 6-20), multi-omics analyses (months 18-30), and data integration, interpretation, and dissemination (months 30-36).
Statistical analyses include assessment of normality using the Shapiro-Wilk test and homogeneity of variance using the Levene test. Group comparisons are performed using Student's t-test or Mann-Whitney U test for pairwise analyses, and one-way or two-way analysis of variance (ANOVA) with appropriate post hoc corrections (Bonferroni or Tukey). Non-parametric alternatives (Kruskal-Wallis test) are applied where appropriate. Statistical significance is defined as p < 0.05 (two-sided). Data analysis is performed using GraphPad Prism and R-based computational pipelines for high-dimensional biological data.
