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Updated: Aug 12, 2026

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation
Published on: April 1, 2021
TNF Receptor 1 regulates colonic mesenchymal cell diversity and the epithelial stem cell niche
Safina Gadeock1, Nandini Girish1, Cambrian Y Liu2
1Division of Pediatric Gastroenterology, Hepatology, and Nutrition, and The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, 90027, USA; Department of Pediatrics, University of California San Diego School of Medicine, La Jolla, CA, 92093, USA.
Background & Aims:
Tumor necrosis factor receptor 1 (TNFR1) regulates intestinal epithelial survival and repair, yet its role in the colonic mesenchyme remains unclear. We investigated how mesenchymal TNFR1 signaling controls stromal cell identity, stem cell niche function, and epithelial homeostasis.
Methods:
Colonic mesenchymal populations from germline TNFR1-deficient (TNFR1-/-) and mesenchymal-specific Pdgfra-Cre;TNFR1fl/fl mice were analyzed using immunostaining, western blotting, bulk and single-cell RNA sequencing, and organoid-mesenchymal co-culture systems. Functional studies included wound healing assays, recombinant RSPO3 rescue, and ITGA6 blockade.
Results:
TNFR1 deletion altered mesenchymal composition, reducing PDGFRα expression while increasing αSMA expression in vivo and in vitro. Single-cell RNA sequencing demonstrated loss of mesenchymal heterogeneity following TNFR1 deletion, including depletion of a distinct TNF and interferon responsive mesenchymal subpopulation enriched for Mmp9, Cd200, Traf1, and Tnfrsf9. Transcriptomic profiling revealed downregulation of stem cell niche factors including Rspo3, Wnt2b, and Fgf2, alongside increased extracellular matrix and proliferative signaling pathways, particularly Itga6, PI3K, MAPK, and RAP1 signaling. Mesenchymal TNFR1 deletion impaired epithelial proliferation and reduced Lgr5+ and Ephb3+ intestinal stem cell expression in vivo. In organoid co-culture systems, TNFR1-deficient mesenchymal cells suppressed epithelial stem cell marker expression and altered colonoid morphology, effects which were rescued by recombinant RSPO3. ITGA6 blockade restored PDGFRα and RSPO3 expression and normalized proliferative signaling and migration in TNFR1-deficient myofibroblasts. Similarly, TNFR1-dependent regulation of ITGA6, PDGFRA, and RSPO3 was observed in human colonic myofibroblasts.
Conclusions:
Mesenchymal TNFR1 preserves colonic mesenchymal diversity, sustains RSPO3-dependent stem cell niche signaling, and maintains intestinal stem cell homeostasis.
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