Differentiation Of Adipose-derived Mesenchymal Stem Cells Into Insulin Producing Cells And Effects Of Over-expression MiR-375 In Differentiation Process

Diabetes is a common and serious metabolic disease,and islet transplantation is the best treatment at present,but the lack of islet source seriously restricted the use of this therapy.The non-pancreatic β-cells can gain the insulin secretion function after directed induction or genetic engineering;thereby bring the hope for replacing pancreatic beta cells for cell therapy of diabetes.In this study,we successfully established a system to induce the differentiation of human adipose-derived mesenchymal stem cells(haMSCs)into insulin-producing cells(IPCs)and investigated the effects of miR-375 overexpression on differentiation,which laid bases for diabetes treatment by haMSCs.Based on the key stps of differentiation embryonic stem cells into insulin-secreting cells and the differentiation exploratory experiments of haMSCs,five differentiation protocols was established and been tested in this study.Insulin expression was detected at the end of differentiation when the haMSCs were induced to differentiate by these protocols,and the best differentiation protocol is method E,which is differentiation haMSCs in medium added CYC,NOG,B27,RA for 3 days,then change to Nic,GLP1,B27,NOG containing medium for 5 days and use Nic,GLP1,IGF1,NOG containing medium culture for another 8-10 days.The induced cells were expressing Foxa2,Pdxl,Ngn3,Ins and Gcg,the immunofluorescence staining of insulin was positive,and we found that BMP signaling pathway has the regulatory role on the formation of insulin-secreting cells by addition of NOGGIN increasing insulin expression.The hucMSCs preserved in our laboratory was used as control cells in the haMSC differentiation process.MiR-375 plays an important role in the regulation of pancreatic islet cell development and maturation,and its expression may help stem cells to differentiate into insulin-secreting cells.In order to investigate the effects of miR-375 on the differentiation,we used the lentiviral vector to construct the haMSC cell line with stable expression of miR-375 and induce it to differentiate into IPCs.At the same time,a system which transiently overexpresses/inhibits miR-375 at third stage by liposome was established to explore the effects of overexpression/inhibition of miR-375 on haMSC differentiation.The results showed that miR-375 could decrease the expression of Gcg in the differentiated cells.The overexpression of miR-375 at the third stage of differentiation increased expression of Ngn3.The expression of miR-375 had little effect on Pdx1,Foxa2 gene expression.In conclusion,overexpression of miR-375 can promote cell differentiation into insulin-producing cells and reduce differentiation into glucagon-producing cells.