Isolation, culture and identification of stem cells from the pancreas of newborn rats

Objective The aim of this study is to isolate and identify the pancreatic stem cells from the pancreas of neonatal rats. Methods The pancreatic tissue was collected from neonatal rats in aseptic environment, and then digested with 5.5 mg/ml collagenase IV. The organoid cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS). Differential centrifugation method was carried out to isolate the epithelial cell and fibroblast. The epithelial cells were purified for several times. The 4th-generation cells were then induced to differentiate into β islet cells, and stained with immunofluorescent signal. High concentration of glucose was applied to induce insulin secretion. Results The pancreatic stem cells in the pancreas of the neonatal rats possessed high proliferative rate and can be applied for continuous passage. Immunofluorescence staining of the4th-generation cells showed positive Pancreatic duodenal homeobox-1 (PDX-1), Nestin, neurogenin3 (NGN3), Vimentin, insulin and C-peptide which were pancreatic stem/progenitor cell specific proteins; after inducing to differentiation, the cells displayed positive with Dithizone staining, while immunohistochemistry test showed cells were PDX-1 and C-peptide positive; for glucose stimulation assay, insulin secretion of induced ICCs [(30.0±11.5) pIU/ml] was significantly higher than that of non-induced ICCs [(5.3±1.5) pIU/ml, P=0.035). Conclusion The isolated pancreas cells were pancreas stem cells. Key words: Diabetes; Pancreatic stem cells; Islet like cell clusters; Induced differentiation in vitro; Insulin