A16 ESOPHAGEAL ORGANOID PROLIFERATION AND DIFFERENTIATION ARE ALTERED BY LOSS OF MSH2. (21st February 2022)

• Record Type: Journal Article
• Authors: Rolland, M; Gonneaud, A; Jean, D; Giroux, V
• Is Part Of: Journal of the Canadian Association of Gastroenterology. Volume 5(2022)Supplement 1
• Page Start: 19
• Page End: 20
• Publication Date: 2022-02-21
• Subjects: Gastroenterology -- Periodicals; 616.33005
• DOI: 10.1093/jcag/gwab049.015
• Languages: English
• ISSNs: 2515-2084

Abstract

Abstract: Background: The stratified epithelium of the esophagus includes Krt15+ basal stem cells that display self-renewing and regenerative capacity, and multipotency. However, the mechanisms that specifically control their functions remain unknown. Interestingly, RNA sequencing and GSEA revealed an enrichment of a gene set associated with DNA repair in Krt15+ cells vs Krt15 - cells. We also observed that Msh2 (DNA mismatch repair pathway) is the most significantly upregulated gene in Krt15+ stem cells. Aims: To determine the effect of Msh2 loss on self-renewal and differentiation of esophageal organoids. Methods: Esophageal epithelial cells were isolated from a wild-type mouse. Using flow cytometry, esophageal Krt15+ (GFP+) and Krt15 - (GFP-) cells were sorted from Krt15-CrePR1 (R26 mT/mG ) mice. All cell populations were grown as organoids and Msh2 was depleted using a CRISPR/Cas9 approach. Impact of Msh2 loss on self-renewal and differentiation in esophageal epithelial organoids was evaluated through organoid formation assays, WST-1 proliferation assays and histological analysis. Results: At baseline, organoids depleted for Msh2 formed more poorly differentiated and less well-differentiated organoids than controls. Lower expression of differentiation gene Krt13 was also observed in Msh2 -depleted organoids, confirming an altered differentiation pattern. Furthermore, these organoids showed a higher organoid formation rate and proliferation by WST-1 assay, suggesting that self-renewal capacity and viability are increased when Msh2 is depleted. Interestingly, following radiation, organoids depleted for Msh2 showed higher residual levels of p-H2AX (DNA damage marker), suggesting that their capacity to cope with DNA damages is altered. As mentioned above, we previously reported that Msh2 is the most upregulated gene in Krt15+ vs Krt15 - cells. Therefore, to determine if Msh2 role is distinct in both populations, we depleted Msh2 in Krt15+ and Krt15 - cells-derived organoids. Interestingly, our preliminary results suggest that Msh2 deletion led to increased p-H2AX and decreased Krt13 levels in Krt15+ organoids but not in Krt15 - organoids. Conclusions: Our results show that Msh2 is potentially a key contributor of esophageal stemness in homeostatic and injured conditions. Funding Agencies: CIHRCanada Research Chair