Gastric Mesenchymal Myofibroblasts Maintain Stem Cell Activity and Proliferation of Murine Gastric Epithelium in Vitro

Stem cells are influenced by a microenvironmental niche that includes mesenchymal cells. We established a novel long-term method for primary mouse glandular stomach culture with mesenchymal myofibroblasts to investigate gastric epithelial-mesenchymal interactions. A gastric mesenchymal myofibroblast (GMF) cell line was established from mouse glandular stomach. Glandular stomach cells from neonatal mice and GMF cells were co-cultured in a collagen gel. Cultured stomach cells yielded expanding sphere-like structures. In the GMF co-culture system, the number and size of gastrospheres were increased compared with control cultures ( P  = 0.009 and 0.008, respectively). Immunohistochemistry showed cells positive for human gastric mucin, HIK1083, and chromogranin A, indicating differentiation into surface mucous cells, mucous neck cells, and enteroendocrine cells, respectively. RNA in situ hybridization for Lgr5 showed Lgr5 + stem cells in the cultured gastrospheres. Lgr5 + cells were observed persistently in the epithelium of gastrospheres in the GMF co-culture system for 2 months. GMFs allowed the cultured gastric epithelium to maintain active proliferation similar to that seen in vivo . Real-time quantitative RT-PCR showed that Gas1 expression was higher in GMFs ( P  = 0.0445), and Hoxc8, Notch1, and Sox10 expressions were higher in intestinal mesenchymal myofibroblasts ( P  = 0.0003, 0.0143, and 0.0488, respectively). We show the potential role of GMFs in sustaining Lgr5 + stem cell activity and affecting normal gastric epithelial differentiation and proliferation.