Isolation And Identification Of Pancreatic Stem Cells Derived From Human Fetal Islet-Like Cell Clusters

Diabetes mellitus is one of the devastating disease that threaten the health of people in the world. At present, this disease could not cured completely by the therapy of traditional drugs administration or exogenous insulin injection. Along with the transplantation success of pancreatic islets and the improvement of immunosuppressive regimen, the possibilities of curing the type I and some of the type II diabetes become more and more promising. However, the widespread application of this transplant technology has been greatly blocked by the deficient human pancreatic islets from the cadavers.Pancreatic stem cells are adult stem cells in pancreas, have the ability for self-renewal and to differentiate into many types of cell lineages in the appropriate local environment. They have the advantage of being already primed to specifically produce pancreatic cell types. An effective way to overcome the deficient human pancreatic islets is to isolate and clone pancreatic stem cells as"seed cells"and to induce their differentiation into functional pancreatic islets abundant for transplant. This dissertation focuses on the isolation of islet-like cell clusters(ICCs), purification and expansion of stem cells from human fetal pancreas ICCs, identification of the cells by detection of the molecular markers it expressed, and observation it's potency by induction to differentiate intoβcells. The purpose of this research is to optimize the methords for isolation and identification of pancreatic stem cells, provide compatible seed cells for diabeitc clinical cure, and lay a foundation for clinical applications.Human fetal pancreas at 3-5 gestational months was obtained sterilely, and was cut into pieces, and then digested in collagenaseⅣ. After that, these cells were cultured in RPMI1640 medium supplemented with 10% newborn bovine serum(NBS) and islets-like cell clusters(ICCs) were presented. ICCs were transferred to petri dishes coated with gelatin and then cultured in DMED medium with low glucose supplemented with 10%NBS and 10ng/mL EGF. When ICCs were grew into monolayer, these cells were subcultured in RPMI1640 medium supplemented with 10% NBS. 4 days after subcultrue, many epithelial cell patches growing in cloning way could be observed. These cell patches were picked out to purify and were expanded in RPMI1640 medium supplemented with 10% NBS. Purified epithelial cells(HFIECs) had been passaged for 45 times, and 106-108 cells were cryopreserved per passage. The 13th passage HFIECs were randomly selected and were cultured on 96 well plates to mensurate the growth curve of the cells by MTT. The growth curve demonstrate that the capability of proliferation of these epithelial cells in 20% serum were extraordinary. These cells grew slowly at the first 3 days and proliferated rapidly during the 3rd to 5th day and then ceased growing. But these epithelial cells grow slower while cultured in 10% serum. The 6 th, 11 th and 21th passages HFIECs were analyzed immunohistochemically, and the results showed that PDX-1, CK-19, PCNA, CK-7, CK-3, E-adherin, Nestin, Glucagon, Glut2, Vimentin were expressed stably in the epithelial cells, but Insulin, Somatostatin, IAPP(Islet amyloid polypeptide)was not expressed. Total RNA of the 39th passage cells was extracted to perform RT-PCR analysis, and result showed that the PDX-1, CK-7, CK-19, Nestin, Glucagon, Acidβ-galactosidase, Glut2 and Vimentin were detected, but Ngn3, Insulin, Somatostatin, Pancreatic polypeptide and Glucokinase was undetected. FACS analysis of the 5th passage cells showed that CD29, CD44 and CD166 were positive, whereas CD11a, CD14, CD34, CD45, CD90, CD105 and CD117 were negative. CD29, CD44, CD166 were stablely expressed in the 39th passage cells but CD54, CD90, CD11a were unexpressed. The positive rates of CD14, CD34, CD45, CD105, CD117, CD 71, CD34 and CD45 were different from the 5th passage cells. The 39th HFIECs were induced for 16 days by 10mmol/NIC and 10μg/L HGF and DTZ positive clusters were obtained. The insulin secretion quantity reach the maximum at the 20d which proved that the cell reach the most mature stage. These results suggested theβcell differentiation. Renewable HFIECs from human fetal pancreas derived ICCs have been purified, expanded and identified in this research. HFIECs originate from ducts and have characteristics of pancreatic stem cells, and will provide as seed cells for research and clinical cure of diabetes.