The Study On Human Mesenchymal Stem Cells Differentiating Into Insulin-producing Cells

Now diabetes mellitus is a common and frequently-occurring disease of threatening human health. But forthcoming therapeutic measures can not solve the physiological requiremen of diabetic to insulin and prevent the happening and proceeding of acute and chronic complications. Islet cell transplantation will effectively regain the diabetic's secretion of endogenous insulin and cure diabetes. However, the questions of availability of human pancreas islets and allograft rejection have hampered the application of islet cell transplantion. The studies about stem cells have provided a new resource of pancreas islets. It has been expected to produce lots of functional pancreatic islet cells by differentiation of stem cells and become the focus of clinical research. Due to the low yield, oncogenicity and ethical matters of embryonic stem cells, adult stem cells, especially mesenchymal stem cells (MSCs) have been paid more attention.MSCs are multipotent stem cells derived from mesoderm, possessing potent proliferative capacity and self-renewal and multilineage differentiation. MSCs can differentiate into cells derived from the same germ-layer, for example, osteocytes, adipocytes and muscle cells under the control of different environment and cytokines. The new works show that they can also transdifferentiate into liver cells, neuron-like cells and neuroglial cells derived from other germ-layer. MSCs have some advantages, including drawing the materials easily and slightly injury, multiple amplification easy in vitro, no ethical matters and immune rejection and differentiation easy. For this reason, MSCs have been the optimum and main source of seed cells of islet cell transplantion.In this respect, this experiment focused on human mesenchymal stem cells (hMSCs), induced hMSCs differentiating into insulin-producing cells by various kinds of factors in vitro, verified secreting function and proliferative activity of Ins-hMSCs by the techniques about morphology. enzymology, molecular biology and electrophysiology, investigated their differentiating mechanisms, implanted Ins-hMSCs into nude mice and assessed their function. Aim to grope the fit methods of inducing hMSCs differentiation, evaluate the effect of hMSCs in the diabetes treatment by islet transplantiation and provide theoretical and practical basis for application of hMSCs.一,In the procedure of primary culture of hMSCs, the most important problem is how to obtain pure cells. HMSCs were obtained by the orthodox density gradient fractionation combination with adherence screening and monocloning screening methods and then cultured and proliferated. The purity of hMSCs was higher and it was confirmed that they had typical biological characteristics of stem cells by flow cytometry, immunofluorescence, assay of cell cycle and transmission electron microscope and staining of telomerase. So they may be cell sources for extended experiments. We also found there were L-type voltage-gated Ca2+ channels (CaV) and delayed rectifier K+ channels (KDR) in part of undifferentiated hMSCs by patch clamp technique. It is well known that ion channels are close correlation with differentiation, proliferation and function of cells, therefore, the results of ion channels were help to approaching the mechanism of differentiation and function.二,HMSCs were induced for 30 days by high glucose combination with nicotinomide and Exendix-4, the clusters like pancreatic island formed and shape of cells became polygon. 14.5 percent of hMSCs could express insulin protein and 5.5 percent secrete insulin by DTZ staining, RT-PCR, immunofluorescence staining, Western Blot and flow cytometry. Ins-hMSCs had the characteristic of ultrastructure like endocrine cells and secretory vesicles like beta cell of islet by transmission electron microscope. It was testified there were insulin-granules in cytoplasm of Ins-hMSCs by immuno-electron microscopy. We also found that the quantity and magnitude of L-type CaV current and KDR current in Ins-hMSCs were more than that in hMSCs and this results indicated Ins-hMSCs had more L-type CaV and KDR channels and characteristic of ion channels like beta cell of islet and became more mature. From those results we comfirmed that hMSCs had differentiated into insulin-producing cells.三,Our expriements utilized matrigel, glucose, nicotinomide and Exendix-4 to set up the 3D-induced conditions. HMSCs differentiated into expressing insulin cells in the 3D-induced conditions for 16 days. This illustrated the 3D-induced conditions made for the formation of the clusters like islets and differentiation into insulin-producing cells. Thus the works provided a new idea and method for hMSCs differentiation into insulin-producing cells.四,To evaluate the insulin secreting function of Ins-hMSCs, we used RT-PCR and ELISA to detect the mRNA of GK and GLUT2 and secretory volume of insulin. The results were that Ins-hMSCs expressed mRNA of GK and GLUT2, 23.3mmol/L glucose stimulated Ins-hMSCs secreting more insulin than 5.5mmol/L glucose and secretory volume were increased 1.5 times. This showed Ins-hMSCs were sensitive to glucose and their secretory activity of insulin was regulated by glucose in external environment. In addition, by patch clamp technique and Fluo-3/AM staining, we also found glucose stimulated L-type CaV channels in the cytomembrane opening and Ca2+ was released from calcium storerooms in cytoplasm and this activity leaded the density of calcium in cytoplasm increasing rapidly. The response to glucose in Ins-hMSCs was similar to beta cell of islet.This findings demonstrated that Ins-hMSCs possessed physiological process of insulin secretion like mature beta cell of islet and relatively consummate function of secreting insulin.五,As the seeding cells of islet transplantation, it was more important of having proliferating activity and no potential oncogenicity. We used PCNA immunofluorescence staining and Annexin V-EGFP apoptosis kit to detect Ins-hMSCs and confirmed that few Ins-hMSCs got into apoptosis and maintained better proliferating activity. Telomerase and cystoskeleton staining showed that Ins-hMSCs possessed normal cystoskeleton structure, expressed telomerase protein and the expressing intensity was lower than that of spongioblastoma cells. The results suggested Ins-hMSCs had no tumorigenic potential and was safe for islet transplantation.六,To investigate differentiated mechanism of hMSCs, we observed not only PDX-1expression of the different induction period by immunofluorescence stain and the assay of RT-PCR and Western blot, but also the change of calcium channel after inducted for 10 days in high glucose condition. The results were that high glucose made Ins-hMSCs express PDX-1, nicotinamide and Exendix-4 promoted this expression, and then activated insulin gene and others genes correlated with differentiation into pancreatic islet. In addition, high glucose condition might promote hMSCs differentianting into insulin-producing cells by enhancing the open of calium channel and calium density in cytoplasm.七,To observe the roles of Ins-hMSCs in regulating blood suger in vivo, we transplanted Ins-hMSCs labelled by BrdU into the renal capsule of nude mice with diabetes, monitored blood sugar of nude mice and done immunofluorescence stain. It was observed that Ins-hMSCs could survive, keep proliferating, last expressing PDX-1, synthetize and secrete insulin, and then make nude mice with diabetes recovering in one week and keeping normal for 20 days.In short, hMSCs were induced differentiating into insulin-producing cells by high glucose combination with nicotinomide and Exendix-4 in this experiment. It was verificated systematically that Ins-hMSCs had consummate secreting function and the effect of turning down blood suger. Moreover the mechanisms of hMSCs differentiation were investigated. It will provide not only a new technological route and substantial method about hMSCs differentiation into pancreatic islet, but also important theoretic and experimental basis for better utilizing hMSCs to treat diabetes.