The Experimental Research Of Inducing Human Umbilical Cord Wharton's Jelly-derived Mesenchymal StemCells(HUMSCs) Into Insulin-producing Cells

Objective: We co-cutured HUMSCs with pancreatic cells of neonatal rat and transplanted HUMSCs into the body of diabetic rat. To explore the possibility of inducing HUMSCs to differentiate into insulin-producing cells in microenviroment of pancreatic cells in neonatal rat and in body of diabetic rat.Methods:①Subjects: SD rats were bought from Traditional Chinese Medicine University Of Guangzhou. According to the animal ethical standard, animals were disposed in the experiment. The human umbilical cords were obtained from healthy caesarean delivered uterogestation foetus from The Second Affiliated Hospital of Shantou University. The fact of the experiment was known by the lying-in mother and her family member, and was be approved by hospital Ethics Committee.②Experiment Methods: The pancreatic cells of fetal rat were isolated by enzyme dispelling therapy and human umbilical cord mesenchymal stem cells were also isolated and cultured, and then HUMSCs were co-cultured with pancreatic cells. On the 3rd,7th and 14th day during co-culture, Insulin release after glucose challenge was tested with radio-immunity, Human pancreatic and duodenal homeobox 1(PDX-1) was detected with reverse transcription-polymerase chain reaction (RT-PCR) method, meanwhile Control Group lonely cultured was set up. Last Human umbilical cord mesenchymal stem cells were marked by Hoechst33258, which is a fluorescent dye. Then human umbilical cord mesenchymal stem cells were transplanted into the body of diabetic rats through vena caudalis. Under the fluorescence microscope, to observe whether stem cells field in the pancreas, and then human insulin gene were detected by RT-PCR.Results:①The morphology of human umbilical cord mesenchymal stem cells under co-culturing gradually changed from fibroblast to round and had the tend of forming clusters.②The results of radio-immunity manifested that the insulin quantity secreted by co-cultured HUMSCs could increase following the prolongation of co-culture. And the insulin quantity secreted by co-cultured HUMSCs could increase following different density of glucose when the cells have the tend of gathering to lumping. There was significant difference compared with control group(P<0.05).③RT-PCR analysis indicated that Un-cocultured HUMSCs didn't express Pdx-1; Co-cultured HUMSCs could express Pdx-1 on third day;Pdx-1 can also be detected on seventh day. But on fourteenth day, PDX-1 wasn't be expressed.④The cells marked with Hoechst33258 distribute in uniformity in the pancreas under the fluorescent microscope. The un-transplanted diabetic rat pancreas didn't express human insulin gene, but the human insulin gene could be detected in the rat pancreas after transplanted HUMSCs on sixty day.⑤The transplantation results showed that rats treated with HUMSCs significantly increased their lifespan, as compared with untreated rats.Conclusion: In this study, we successfully differentiated HUMSCs into mature islet-like cell clusters in microenviroment, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.