The Immune Protective Effects Of HAAT Gene On The Mouse IPSCs And The Induced Insulin-Producing Cells

Objective: The latest study found that the induced pluripotent stem cells (iPSCs) haveimmunogenicity, and the purpose of the research is to prevent immune rejection in thecell therapy with iPSCs and insulin-producing cells (IPCs) induced from iPSCs.Inthis research the mouse iPSCs-hAAT was established by transferring the human Alpha1-antitrypsin gene (AAT) on the basis of the mouse iPSCs construction. A preliminarystudy was taken to evaluate the immune protective effects of hAAT on the mouseiPSCs and the process of allograft transplantation for treatment of diabetes with IPCs.Methods:①The induction of mouse iPSCs: To obtain the mouse iPSCs, the mouseembryos fibroblasts (MEFs) isolated from C57mouse were transfected by theretrovirus containing Oct4, Sox2, c-Myc and Klf4genes using LipofectamineTM2000,and the MEFs were reprogrammed into the mouse iPSCs. Some identifications weretaken to the mouse iPSCs.②The immune protective effects of hAAT on the mouseiPSCs: hAAT gene was transferred into the mouse iPSCs using LipofectamineTM2000,and the mouse iPSCs-hAAT was selected by G418selection. After the CTLexperiment, inflammatory factors were detected by the ELISA and RT-PCR analysis,and apoptosis was detected by flow cytometry.③IPCs differentiation from the mouseiPSCs: The mouse iPSCs were induced by adding RA, Activin A, N2, and B27, bFGF,HGF and nicotinamide. The changes of cell morphology were observed during theprocess of differentiation. The differentiated cells were identificated byimmunofluorescence and RT-PCR analysis. The function of the differentiated cellswas evaluated by glucose-stimulated experiments.④The immune protective effectson allograft transplantation in diabetic mice with IPCs: The IPCs derived from twosources of iPSCs were transplanted under the left kidney capsule of BALB/c mouse.In order to evaluate the function of the insulin-secreting cells, the blood glucose,serum insulin level and glucose-stimulated experiments were detected. And the leftkidney was sliced and stained by hematoxylin and eosin to observe inflammatory cellinfiltration to explore the protective effects of hAAT gene on the insulin-secreting cells in this process.Results:①The cell morphology of the mouse iPSCs is very similar to mouse ESCs.And with the increasing of the induction time, the mouse iPSCs clones becamegradually obvious. The mouse iPSCs, which express the surface markers, such as AP,SSEA-1, Nanog and Oct4, can form a teratoma with all three germ layers.②Theresistant clones with stable expression of hAAT after G418selection were detected byWestern Blot. After7days of allograft spleen lymphocytes from the recipientmouseco-cultivation with mouse iPSCs and mouse iPSCs-hAAT, repectively, ELISAresults showed that IL-4secretion of iPSCs-hAAT group was significantly higher thanthat of iPSCs group (P<0.05), and the IFN-γ secretion of iPSCs-hAAT group wassignificantly lower compared with iPSCs group (P<0.05). And the proportion ofiPSCs-hAAT apoptosis was lower than that of iPSCsgroup detected by flow cytometry.The RT-PCR results showed that expression levels of IL-1β and IL-6of iPSCs-hAATwere lower and the levels of CRP was higher.③The differentiation induction processcould be roughly divided into three stages. And the growth state of mouse iPSCschanged from adherent state transferring into suspension culture, and typicalembryoid bodies were obtained. And then they were seeded on the dish coated with1%Matrigel. The cells spread to the surrounding and finally the cell mass appeared.The IPCs specifically expressed the proteins and genes including PDX1and Insulindetected by RT-PCR analysis, and Insulin expression gradually increased with theextension of the induction time. Immunofluorescence assay showed that at the finalstage of cell differentiation most cells were PDX1-positive (red), insulin-positive (red)and NKX6.1-positive (green) cells.The IPCs could secrete insulin and C-peptide,which were regulated by glucose concentration.④The IPCs derived from twosources of iPSCs both play biological effects, and the blood glucose levels reduced tothe normal level3days later after transplantation. The blood glucose decreasedsignificantly fast, the normal level kept longer and insulin secretion levels were higerin diabetic mice transplated with IPCs derived from iPSCs-hAAT. The glucosetolerance test showed that the IPCs derived from iPSCs-hAAT still played a certain role in regulating blood glucose on the14day after transplantation. The structure ofthe transplanted parts was normal on the14day after transplantation, theinflammatory response was weak, and there were a small amount of lymphocyte andmononuclear cell infiltration on the28day after transplantation with the IPCs derivedfrom iPSCs-hAAT. However, the inflammatory response was more obvious in indiabetic mice transplated with cells derived from iPSCs.Conclusion:①The mouse iPSCs were successfully obtained.②The mouseiPSCs-hAAT was established by G418selection. The hAAT gene could reduce thekilling effect of CTL by inhibiting the expression of inflammatory cytokines, and hasthe immune protective effects on the mouse iPSCs.③The IPCs were successfullyinduced from mouse iPSCs.④The IPCs induced from iPSCs-hAAT could playbiological effects in vivo. hAAT could reduce the immune attack by recipient mice tosome extent, and could effectively extend the survival time of IPCs to regulate bloodglucose levels.