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

Valutazione ex Vivo Del Secretoma di Cellule Stromali Mesenchimali Amniotiche Umane in Pazienti Con Malattie Infiammatorie Croniche Intestinali: Effetti su Infiammazione, Fibrosi e Riparazione Mucosale.

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.

Study Overview

• Status: Not yet recruiting
• Conditions: Inflammatory Bowel Diseases
• Intervention / Treatment: Biological: Human amniotic mesenchymal stromal cell (hAMSC)-derived secretome

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 40
• Phase: Not Applicable

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm

Intervention / Treatment

Other: hAMSC secretome-treated samples; Ex vivo treatment of patient-derived intestinal biopsies, peripheral blood mononuclear cells (PBMCs), and organoid co-culture systems with human amniotic mesenchymal stromal cell (hAMSC)-derived secretome.

Biological: Human amniotic mesenchymal stromal cell (hAMSC)-derived secretome; Ex vivo exposure of patient-derived intestinal biopsies, peripheral blood mononuclear cells (PBMCs), and intestinal organoid co-culture systems to human amniotic mesenchymal stromal cell (hAMSC)-derived secretome. The secretome consists of bioactive soluble factors and extracellular vesicles evaluated for immunomodulatory, anti-inflammatory, anti-fibrotic, and pro-regenerative effects on immune, epithelial, and stromal pathways.; Other Names: amniotic MSC secretome; mesenchymal stromal cell secretome; conditioned medium hAMSC; extracellular vesicles hAMSC

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Ex vivo modulation of immune cell populations by hAMSC-derived secretome	Frequency and activation status of T helper 1 (Th1), T helper 17 (Th17), regulatory T cells (Treg), and innate immune populations in patient-derived intestinal biopsies and peripheral blood mononuclear cells (PBMCs) after ex vivo exposure to hAMSC-derived secretome, assessed by multicolor flow cytometry.	Ex vivo assessment performed within 7 days of sample collection.
Ex vivo modulation of inflammatory cytokines by hAMSC-derived secretome	Levels of key inflammatory and regulatory cytokines, including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 6 (IL-6), and interleukin 10 (IL-10), in supernatants of patient-derived intestinal biopsies and PBMCs after ex vivo exposure to hAMSC-derived secretome, assessed by multiplex cytokine assays.	Ex vivo assessment performed within 7 days of sample collection.
Ex vivo modulation of fibrosis- and mucosal repair-related markers by hAMSC-derived secretome.	Molecular analysis of fibrosis- and mucosal repair-related markers expression in patient-derived intestinal biopsies and PBMCs after ex vivo exposure to hAMSC-derived secretome.	Ex vivo assessment performed within 7 days of sample collection.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Intestinal epithelial barrier integrity and function	Evaluation of epithelial barrier markers including claudin-1, claudin-2, and mucin 2 (MUC2), and functional barrier assessment using transepithelial electrical resistance (TEER) measurements in organoid and PBMC co-culture systems.	Ex vivo analysis within 7 days of treatment
Transcriptomic and spatial omics profiling of immune-epithelial-fibrotic niche	Identification of transcriptional changes and signaling pathways in patient-derived tissues and organoids using spatial transcriptomics (10x Genomics Visium) and single-cell RNA sequencing integration to characterize molecular signatures associated with response to hAMSC secretome.	Within study period (months 6-30)
Immune-epithelial cross-talk modulation	Evaluation of interactions between immune cells and intestinal epithelial organoids in co-culture systems under basal and transforming growth factor beta 1 (TGF-β1)-induced pro-fibrotic conditions following exposure to hAMSC secretome.	Ex vivo analysis within 7 days of treatment

Collaborators and Investigators

• Sponsor: Fondazione Policlinico Universitario Agostino Gemelli IRCCS

Study record dates

Study Major Dates

• Study Start (Estimated): November 1, 2026
• Primary Completion (Estimated): November 1, 2027
• Study Completion (Estimated): December 1, 2027

Study Registration Dates

• First Submitted: July 1, 2026
• First Submitted That Met QC Criteria: July 20, 2026
• First Posted (Actual): July 22, 2026

Study Record Updates

• Last Update Posted (Actual): July 22, 2026
• Last Update Submitted That Met QC Criteria: July 20, 2026
• Last Verified: July 1, 2026

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Intestinal Diseases; Digestive System Diseases; Gastrointestinal Diseases; Gastroenteritis; Inflammatory Bowel Diseases
• Other Study ID Numbers: TARGET 27622

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No