A27 msh2 contributes to self-renewal of esophageal organoids

Abstract Background The esophagus is lined by a stratified epithelium in which basal cells can proliferate and undergo differentiation while migrating towards the lumen. In the basal layer, we also find Krt15+ stem cells that are multipotent, self-renewing and that have regenerative capacity. However, mechanisms that specifically control their functions remain unknown. Interestingly, RNA sequencing and gene set enrichment analysis (GSEA) revealed an enrichment of a gene set associated with DNA repair in Krt15+ cells in comparison to Krt15- cells. We also observed that Msh2 (MutS homolog 2), a gene associated with the DNA mismatch repair (MMR) mechanism, is the most significantly upregulated gene in Krt15+ stem cells. Aims To determine the impact of Msh2 loss on self-renewal of esophageal organoids under normal and stress conditions. Methods Esophageal epithelial cells were isolated from a wild type mouse and grown as organoids, a 3D culture model that supports stem cell growth and morphologically reproduces the tissue of origin. To determine Msh2 role in esophageal epithelium, this gene was deleted through a CRISPR/Cas9 approach in mouse esophageal organoids. Invalidation was confirmed by Western Blot and immunofluorescence. Impact of Msh2 loss on self-renewal was measured under normal condition and following radiation. Results At baseline, loss of Msh2 decreases the organoid formation rate of esophageal organoids. Furthermore, following high-dose radiation, Msh2 deficient cells form less organoids than control cells. These results suggest that self-renewal capacity is reduced when Msh2 is depleted. Interestingly, following radiation, organoids depleted for Msh2 show higher residual levels of p-H2AX, a DNA damage marker, and p-ATM, a key kinase in DNA damage response, suggesting that their capacity to cope with DNA damages is reduced. Conclusions Our results suggest that Msh2 contributes to maintaining genomic integrity in esophageal cells and that contributes to maintaining self-renewal capacity of basal cells and possibly esophageal stem cells. Funding Agencies Canada Research Chair.