OC-065 Stem cell analysis by label retention within Barrett's oesophagus

Abstract

Introduction Stem cells are thought to be the only long lived cells in the gastrointestinal epithelium. It is thought they are the target for mutations that may lead to the premalignant Barrett9s Metaplasia (BM). These cells are thought to divide slowly allowing DNA dyes to be retained within the stem cells over a long period of time. Here, we have used this characteristic to identify, for the first time, the location of stem cells within the glands of Barrett9s oesophagus, an important premalignant condition. Methods We utilised iododeoxyuridine (IUdR) a thymidine analogue which is incorporated into the DNA of replicating cells, as a marker to identify the label retaining cells (LRC) as putative stem cells. Four patients with BM diagnosed with oesophageal adenocarcinoma undergoing oesophagectomy were infused with IudR prior to their surgery (7–69 days). After resection, tissue samples of normal oesophagus, BM, normal stomach and tumour were extracted for immunocytochemistry and FACS. Results Infusion 3 days or less in vitro in transformed cells and explants revealed appropriate abundant staining of the proliferative compartments. Results from FACS analysis showed the percentage of BRdU labelled cells was much greater in BM tissue (7.85%) compared with fundus (0.69%) or squamous (0.38%). However labelling at 7 days in both patients ex vivo showed positive discrete staining of LRC9s within various gastrointestinal tissue types including BM. LRCs were seen in the basal layer of the squamous epithelium, likewise LRCs were seen at the neck/foveolar regions of the gastric tissue conforming to the suggested gastric SC zone. LRC9s were seen at several locations in metaplastic tissue both at the base of the gland and in the neck region at 7, 11, 29 and 67 days, this ties in with preliminary data showing LGR5 (a stem cell marker) is also expressed at the base of the Barrett9s gland. Conclusion This is the first report of LRC9s within the human oesophagus and our findings correlate with the hypothesised location of SCs in the squamous epithelium and gastric mucosa. Furthermore LRCs were located in the base of the BM gland implying that this is the location of BM SC9s. Analysis and molecular characterisation of LRCs and BM SC9s and their niche are underway, involving the use of other GI SC markers.