Optimization of a Canine Colon Organoid Platform for Modeling Intestinal Injury and Mesenchymal Stem Cell-Mediated Regeneration.

Canine chronic enteropathy (CE) is characterized by persistent mucosal inflammation and impaired barrier function. Despite its high clinical prevalence, research has been hindered by a lack of host-specific intestinal models. Here, we present an optimized canine-derived colon organoid (cCO) culture platform designed for assessing drug toxicity and regenerative potential. Combining Wnt/EGF activation with TGF-β/p38 MAPK inhibition supported the long-term maintenance of stem cell-enriched organoids. Upon maturation, differentiated cCOs exhibited both absorptive and secretory cells, validated by lineage-specific marker expression and functional P-glycoprotein efflux activity. While pro-inflammatory cytokines exerted non-specific toxicity, diclofenac-induced injury was significantly more pronounced in differentiated organoids than in expanding counterparts. This indicates that our platform effectively recapitulates the cell type-specific sensitivity of the intestinal epithelium to pharmacological insults. Next, to determine whether the cCOs could model epithelial repair process, we examined the therapeutic effects of canine mesenchymal stem cells (MSCs), a representative cell-based therapy for CE, on damaged organoids. Notably, treatment with MSC-conditioned media (MSC-CM) triggered a morphological transition into cystic spheroids with the upregulation of pro-regenerative stem cell markers, suggesting the induction of a fetal-like regenerative reprogramming. Indeed, MSC-CM treatment restored growth and proliferation while preserving barrier integrity in diclofenac-treated organoids. Collectively, these findings demonstrate the advantages of cCO system for modeling intestinal injury and repair, supporting the development of targeted therapeutic strategies for canine gastrointestinal disorders.