| Popis: |
Enteric glial cells (EGC) are a prominent cell type of all layers of the gut wall, virtually controlling all gastrointestinal functions. While the development of transgenic mice has led to major advances in understanding EGC biology, in vitro models are still fairly limited and do not allow for the robust and reproducible establishment of primary cultures discriminating EGC from the inner versus outer layers of the gut wall. Here we report a novel method to separately grow EGC from the inner and outer layers of the intestinal wall from the same mouse with a high degree of purity and cell heterogeneity. Our results indicate that EGC from the inner layers of the gut wall exhibit higher calcium response to ATP when compared to EGC from the outer layers. We also show that inner EGC cultures express lower levels of the transcription factor Sox 10 as compared to outer EGC cultures, which mirrors in situ differential expression of Sox10 in submucosal (inner) versus myenteric (outer) plexus assessed using wholemounts. Confocal microscopy analyses of wholemounts further demonstrate that a majority of calretinin-expressing ganglionic cells of the submucosal plexus express the EGC marker S-100β, while this population is marginally represented in ganglia of the myenteric plexus. Altogether this study describes a novel method of EGC primary cultures permitting for the first time to compare inner versus outer EGC and provides in vitro and ex vivo evidence that inner EGC and outer EGC are phenotypically and functionally distinct. |
