Abstract: While organoid-based models offer valuable insights into gut biology, their lack of key microenvironmental components—such as vasculature, mesenchyme, and immune cells—limits their physiological relevance and utility. As such, intestinal fibroblasts play essential roles in gut epithelial homeostasis, matrix remodeling, and inflammation. Here, we leveraged bioengineered human mini-colons to establish a co-culture platform with intestine-derived fibroblasts. The biomimetic extracellular matrix (ECM) surrounding mini-colon tissue readily accommodated fibroblasts, enabling direct interactions between the two compartments. Notably, fibroblast addition facilitated microchannel epithelialization and epithelium formation, mirroring colonic repair in vivo. Bulk RNAseq and immunofluorescence analysis revealed transient epithelial reprogramming into a regenerative state, with upregulation of regenerative and wound-associated epithelial (WAE) cell markers during early stages of the epithelium formation. Interestingly, this regenerative phenotype gradually diminished as cells established a tightly packed columnar epithelium and was entirely absent in mini-colons cultured without fibroblasts. Our findings demonstrate that this repair phenotype is driven by soluble factors secreted by fibroblasts, mediating epithelial changes via paracrine signaling. Proteomic profiling of fibroblast-conditioned media identified 467 unique proteins, categorized into ECM proteins, secreted growth factors, and other signaling molecules. Remarkably, the identified growth factors were conserved across the secretomes of two distinct fibroblast lines—both commercially available and in-house intestine-derived—cultured under the same conditions. Functional validation further revealed the key ligands playing a role in modulating epithelial restitution in mini-colons. Together, our results underscore the critical role of fibroblasts in epithelial repair and provide a dynamic, human-relevant co-culture system to investigate epithelial-stromal interactions observed in vivo.
Funding Source: This work was funded by support from the Swiss National Science Foundation (SNSF) research grant 310030_179447, the EU Horizon 2020 Project INTENS (#668294-2), Ecole Polytechnique Fédérale de Lausanne (EPFL), and Roche.
