H. A. Khalil1, N. Y. Lei1, P. Rana1, W. Nie1, J. Wang6, J. Yoo3, F. Wang2, L. Li2, M. G. Stelzner1,4, M. G. Martín6, J. C. Dunn1, M. Lewis5 1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA 2Stowers Institute For Medical Research,Kansas City, MO, USA 3Tufts Medical Center,Surgery,Boston, MA, USA 4VA Greater Los Angeles Medical Center,Surgery,Los Angeles, CA, USA 5VA Greater Los Angeles Medical Center,Pathology,Los Angeles, CA, USA 6University Of California – Los Angeles,Pediatrics,Los Angeles, CA, USA
Introduction: The intestinal subepithelial myofibroblast (ISEMF) is an important component of the intestinal stem cell niche and a potential source of the Wnt-agonistic R-spondins. ISEMFs are traditionally identified by positive staining for α-smooth muscle actin (α-SMA) and vimentin and negative staining for desmin, but isolation is limited by lack of unique surface markers. We developed a surface marker-based approach for isolating ISEMFs.
Methods: ISEMFs were isolated from adult C57BL/6J mouse small intestine (n=4) and human duodenum (n=4) through digestion with collagenase D and dispase. Isolates were cultured in DMEM with 10% FBS, penicillin/streptomycin, epidermal growth factor, insulin, and transferrin, passaged weekly, and lysed for RNA. Deep sequencing was performed to assess the RNA expression of cell surface markers. Top surface markers were identified and paraffin-embedded mouse and human intestines were co-stained with antibodies against these surface markers as well as α-SMA. CD90 colocalized with α-SMA in the pericryptal region in mouse and human intestine and was used in the ensuing isolation algorithm. ISEMFs were isolated from digested whole mouse intestine (n=3) by fluorescence activated cell sorting using negative selection for annexin V and 7-AAD (apoptotic and dead cells), EpCAM (epithelial cells), CD31 (endothelial cells), and CD45 (hematopoietic cells). The resulting cells were sorted into CD90+ and CD90− groups. We analyzed unsorted and sorted cells for expression of α-SMA, vimentin, desmin, and R-spondin2 by qPCR.
Results: In culture, murine and human ISEMFs retained the characteristic positive staining for α-SMA and vimentin and negative staining for desmin. Deep sequencing of cultured mouse and human ISEMFs showed elevated expression of CD63, CD90, CD44, and CD248. This was confirmed by immunofluorescent staining of cultured ISEMFs, but staining mouse and human intestine for these markers showed α-SMA colocalization only with CD90. Isolation and sorting of digested mouse intestine by the negative and positive selection scheme described above resulted in α-SMA and vimentin enrichment in the CD90− group by 39.4±9.7 and 12.8±5.6 fold, respectively, relative to unsorted cells; the CD90+ group was enriched by 6.5±2.7 and 9.5±6.2 fold, respectively. Both CD90− and CD90+ populations were enriched with desmin (11.9±2.8 and 6.3±2.2, respectively, relative to unsorted cells). Expression of R-spondin2 was high in the CD90− and CD90+ populations (42.9±27.6 and 10.0±7.3, respectively, relative to unsorted cells).
Conclusion: Negative selection for epithelial, endothelial, and hematopoietic cells, with or without positive selection for CD90, yields cells that are enriched in the classical markers of ISEMFs and that highly express R-spondin2. Further work is needed to exclude desmin-positive smooth muscle cells.