BACKGROUND: Now, it is still difficult to obtain the distribution of pancreatic stem cells in pancreatic tissue, and to isolate them effectively and in vitro optimal culture.
OBJECTIVE: To isolate pancreatic stem cells from pancreatic tissue in newborn Kunming mice and to culture them in vitro conditions for the morphological and biological characteristics observation and preliminary identification.
METHODS: The newborn SPF Kunming mouse pancreatic tissue were digested by V collagenase. Percoll discontinuous density gradient centrifugation was used for the separation of pancreatic tissue cells and exocrine cells and distribution at three different density interfaces. Different interface cells were collected to culture in Dulbecco's modified eagle medium containing serum-free, basic fibroblast growth factor and epidermal growth factor.
RESULTS AND CONCLUSION: Observation of the cell morphology and cell growth characteristics combined with dithizone staining confirmed that, pancreatic endocrine cells were located in the first and second density interface, whereas cells of pancreatic exocrine located in the third density interface after Percoll discontinuous density gradient centrifugation. A kind of large, round, mononuclear, colony-like grown cells were found in both exocrine and endocrine cells from pancreas, which showed active proliferation, positive for alkaline phosphatase staining and expressed specific markers of pancreatic stem cells-Nestin, these cells just were the pancreatic stem cells. With the extension of time in vitro, pancreatic stem cells from exocrine and endocrine part of pancreas expressed PDX-1 and CK-19 respectively, which showed the differentiation potential to pancreatic endocrine cells and exocrine cell respectively.