IBD-Derived Colonic Fibroblasts Exhibit an Osteopontin-Enriched Secretome, and Osteopontin Restrains Human Colonic Organoid Maturation
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Background
Intestinal fibroblasts are extensively remodeled in inflammatory bowel disease (IBD), yet the soluble stromal signals that directly influence epithelial maturation remain incompletely understood. We examined whether fibroblasts derived from inflamed IBD colon display an osteopontin (OPN; SPP1)-enriched secretory phenotype and whether extracellular OPN directly modifies non-neoplastic human colonic epithelium.
Methods
Conditioned media from 5 noninflamed-associated fibroblast (NAF) and 4 inflammatory-associated fibroblast (IAF) cultures were analyzed in the validated multi-donor cytokine-array matrix, with orthogonal SPP1 RT-qPCR validation in a complementary fibroblast cohort. Recombinant OPN was then tested in human colonic organoids from 3 donors using donor-resolved molecular and functional analyses under standard, fibroblast-conditioned, and WNT-modified culture conditions. Donor identity defined biological replication.
Results
OPN showed the strongest positive rank-based separation between IAF and NAF cultures: all 4 IAF values were higher than all 5 NAF values (Cliff’s delta=1.00; exact Mann-Whitney P=.0159; median ratio=3.64; Benjamini-Hochberg q=.19). Fibroblast RT-qPCR showed approximately 10-fold higher mean SPP1 expression in IAF than NAF cultures (P<.05). In organoids, OPN consistently reduced KRT20, FABP1, CA2, and MUC2 from Day 5 to Day 9. SOX9, HES1, and NOTCH1 increased at Day 9, whereas LGR5 and ALDH provided no evidence of canonical stem-cell expansion. Organoid-area and EdU responses were modest and donor dependent.
Conclusions
IBD-derived colonic fibroblasts can display an OPN-enriched secretory phenotype. In human colonic organoids, OPN is sufficient to impair epithelial maturation, whereas its effects on growth and proliferation are variable and depend on the surrounding niche.
Key Messages
What is already known?
IBD remodels intestinal fibroblasts, however the direct effects of fibroblast-associated osteopontin on human colonic epithelial maturation are poorly defined.
What is new here?
In a validated multi-donor NAF/IAF matrix, OPN was the only analyte with complete positive separation between groups, was supported by SPP1 RT-qPCR, and induced sustained loss of maturation markers followed by a later SOX9/HES1/NOTCH1-associated state in human colonoids.
How can this study help patient care?
The data identify stromal OPN as a candidate contributor to incomplete epithelial normalization in IBD and provide a human experimental framework for testing whether endogenous OPN blockade can restore epithelial maturation.
