Epimorphin Regulates the Mouse Intestinal Stem Cell Niche via the Stromal Microenvironment
Authors: Vishy Courtney C , Swietlicki Elzbieta C. , Heslop Gabriela C. , Lyons Lydia C. , Levin Marc S. , Rubin Deborah C. 
Published: November 2023

Intestinal resection for disorders such as Crohn's disease or bowel ischemia may result in short bowel syndrome, with nutrient malabsorption and parenteral nutrition dependence. There are few effective treatments. Stem cell therapies represent a novel potential therapeutic approach. We have previously shown that gut stem cell derived enteroids have increased growth and budding (increased crypt formation) when co‐cultured with intestinal subepithelial myofibroblasts (ISEMFS) vs. enteroids grown alone. Epimorphin (Epim), a syntaxin homolog expressed in ISEMFS, regulates growth factor secretion from these cells. Epim−/− mice have increased crypt cell proliferation and crypt fission during weaning, and Epim deletion in ISEMFs further increases co‐cultured enteroid growth and budding. Although Epim is not expressed in epithelial cells, we showed that primary cultures of Epim−/− enteroids without ISEMFS have increased surface area and budding compared to wild type (WT) enteroids. Our aims are to understand the mechanisms for the Epim deletion‐induced increase in enteroid growth and epithelial proliferation, to examine epithelial differentiation, and to determine whether the observed increase in budding and area in Epim−/− compared to WT enteroids cultured without ISEMFs reflects stromal environmental effects on the stem cell niche that are lost with passaging.

Methods

Crypts were isolated from WT and Epim−/− mouse small intestines and cultured in vitro. Enteroids were imaged at 6 days post plating (dpp) and analyzed for surface area, budding and epithelial differentiation by H&E staining. Enteroids were passaged twice and reimaged each 6dpp, and harvested for RNA. RNAseq was performed to determine the effect of the Epim‐deleted stromal microenvironment on global epithelial gene expression.

Results

In primary crypt stem cell cultures, Epim−/− enteroids had significantly larger surface area and more buds vs. WTs (p<0.001) as expected. In addition, the percentage of goblet and Paneth cells per enteroid was significantly increased in Epim−/− enteroids (p=0.01). Stem cell marker Lgr5 and Olfm4 expression was significantly increased in Epim−/− vs WT enteroids by qRT‐PCR (p<0.05). RNAseq analysis revealed significant differences in expression of 86 epithelial genes. Gene ontology molecular function analysis of Epim−/− vs WT enteroids showed significant differences in G‐protein receptor coupled signaling, histone binding, and phosphatidylinositol phospholipase C pathways. These differences were retained after first passage (p<0.001 for surface area and buds). At the second passage, enteroid area and budding in Epim−/− vs. WT enteroids were no longer significantly different (p=NS).

Conclusions

Epim regulates mouse intestinal epithelial and stem cell proliferation, differentiation, and gene expression via stromal contributions to the niche microenvironment, affecting the rapidly dividing Lgr5 stem cell population and a subset of gut secretory cells. The stromal microenvironment is responsible for the differences in crypt cell proliferation and crypt fission in Epim−/− intestine.

Support or Funding Information

Supported by the American Physiological Society Undergraduate Summer Research Fellow program (CV) and NIH NIDDK 106382.