| Islet transplantation is an ideal treatment for type I diabetes mellitus(insulindependent diabetes mellitus),but the limited islet donor resources,the separation and purification procedures are cumbersome and immune rejection after transplantation greatly limit its clinical application.Therefore,the search for islet replacement cell transplantation in the treatment of type I diabetes mellitus has become a hot research topic.Adipose Mesenchymal Stem Cells(MSCs)have no ethical issue.They are easy to be isolated and cultivated and have strong proliferation and differentiation ability.After transplantation,they not only have immunomodulatory and immunosuppressive abilities,but also can promote tissue repair.Adipose MSCs can be induced into IPCs in vitro by simulating the development environment of islet ?-cells and changing the composition of medium,but the induction procedure is complex and harsh,which is easy to cause damage to cells,and the induction cost is high,requiring the cooperation of multiple small molecule compounds of cytokines with different concentrations and activation/inhibitor of signaling pathway,and the induction efficiency is not ideal.Therefore,it is an urgent problem to find a more convenient and efficient method to obtain high maturity IPCs in vitro.In this study,adipose MSCs were induced and differentiated into IPCs based on the optimal induction program obtained through screening.Absolute quantitative transcriptome sequencing was used to study the expression changes of related genes and signal pathways during the differentiation process,analyze and screen out new functional genes that may play an important role in the differentiation process,and verify their functions.Then,based on the screened cascades of regulatory genes,new functional genes were introduced to form multiple genes co-expression combinations,and efficient regulatory genes combination was analyzed and screened out to obtain Reprogramming MSCs-derived islet ?-cells(RM-?Cs),and the immunogenicity of RM-?Cs was detected.RM-?Cs and adipose MSCs were co-transplanted into model and clinical diabetic mellitus dogs to verify the feasibility of RM-?Cs.The test results are as follows:1.To determine the optimal scheme for inducing differentiation of adipose MSCs into IPCsAdipose MSCs were successfully isolated and identified by morphological observation,growth curve,three-line differentiation and flow cytometry.Five induction schemes were used to induce and differentiate canine adipose MSCs into IPCs,and the induction efficiency of these schemes was systematically compared to screen out the optimal induction scheme.2.Absolute quantitative transcriptome sequencing analysis of optimal schemeAbsolute quantitative transcriptome sequencing was performed on cells at each stage of the optimal induction scheme.A large amount of genetic data on IPCs induced differentiation of adipose MSCs were obtained.A total of 15,561 DEGs were obtained.In GO functional and KEGG pathway enrichment analysis,126,266 DEGs and multiple signaling pathways were obtained,respectively.These DEGs and signaling pathways may be involved in the induction of adipose MSCs into IPCs and insulin secretion.Eighteen genes were screened according to the significance of differential expression,gene expression level at each stage,gene function and protein interaction analysis results.3.Screening and functional verification of regulatory genes18 genes were identified and screened,and five genes(Dll1,Hnf1b,Foxa1,Pbx1,Rfx3)were found to stimulate the high expression of genes related to the regulation of pancreatic ?-cell development.After further functional verification,it was found that overexpression of these 5 genes could improve the efficiency of adipose MSCs differentiation into IPCs and the amount of insulin secreted by IPCs.After silencing each gene,the efficiency of induced differentiation and the amount of insulin secreted decreased,and these 5 genes could be used as regulatory genes for the differentiation of adipose MSCs into IPCs.22 cascaded regulatory genes of islet ?-cell development were verified,and four key cascaded regulatory genes(Pdx1,Ngn3,Pax4,MafA)were screened out for their great influence on the induction and differentiation of adipose MSCs into IPCs.4.Identify highly effective regulatory genes combinationBased on four key cascaded regulatory genes Pdx1,Ngn3,Pax4 and MafA,five genes Hnf1B,Dll1,Pbx1,Rfx3 and Foxal were introduced to form multiple co-expression combinations of multiple genes.Through comparing,analyzing and screening the reprogramming efficiency of each combination,six genes(Pbx1,Rfx3,Pdx1,Ngn3,Pax4 and MafA)were identified.The combination of 6 genes can efficiently reprogram adipose MSCs into RM-?Cs.The clusrter rate of adipocyte MSCs was 37.57%?44.40%,and the insulin secretion of RM??Cs was more than half of that of mature pancreatic ?-cells.These 6 gene combinations can be used as highly effective regulatory genes combination.5.Detection on immunogenicity changes of RM-?CsRM-?Cs was obtained through reprogramming mediated by a combination of six genes,the immunogenicity of RM-?Cs was detected.In vitro test results showed that,the RM-?Cs surface lacked DLA Class ? molecules and costimulatory molecules CD40 and CD80,can inhibit the proliferation of peripheral blood monocytes,have no binding site of anti-glutamate decarboxylase antibody(GADA)and anti-islet cell antibody(ICA).Presensitized spleen cells can cause RM-?Cs apoptosis.In vivo tests showed that,transplantation of RM-?Cs into the abdominal cavity can induce inflammatory cell infiltration and other immune responses.RM?Cs maintained low immunogenicity in vitro,but the immunogenicity was enhanced after transplantation in vivo,and the immunogenicity of adipose MSCs was significantly lower than that of RM-?Cs.6.Treatment of model diabetic dogs with RM-?Cs and adipose MSCs transplantationTo test the feasibility of RM-?Cs in the treatment of model diabetic dogs.The type I diabetes was successfully developed by 85%pancreatectomy and 5 mg/kg streptozotocin solution.Model diabetic dog was then treated with transplanted cells.The results indicate that,Transplanted RM-?Cs can be colonized in the body,respond to hyperglycemic stimulation,and reverse hyperglycemia in a certain period of time.Transplantation of RM-?Cs did not increase GADA and ICA levels in dogs.Co-transplantation of RM-?Cs with adipose MSCs can make RM-?Cs play a better role and prolong the time of RM-?Cs playing a role.Graft test showed that transplanted RM-?Cs could secrete insulin,but adipose MSCs did not secrete insulin,the transplanted cells may induce immune cell infiltration at a later stage.Cotransplantation of RM-?Cs and adipose MSCs is feasible for the treatment of model diabetic dogs.7.Treatment of clinical diabetic dogs with RM-?Cs and adipose MSCs transplantationTo test the feasibility of RM-?Cs in the treatment of clinical diabetic dogs,RM-?Cs and adipose MSCs were co-transplanted into 2 clinical diabetic dogs.The results showed that RM?Cs could secrete insulin,and reduce blood glucose in a certain period of time.After cell transplantation,the amount of exogenous insulin could be reduced by 75.00%and 85.71%,respectively.Transplantation of RM-?Cs did not increase GADA and ICA levels in dogs.Cotransplantation of RM-?Cs and adipose MSCs is effective in the treatment of clinical diabetic dogs.In conclusion,adipose MSCs can be efficiently reprogrammed into RM-?Cs by the combination of 6 genes(Pbx1-Pdx1-Ngn3-Pax4-Rfx3-MafA).RM-?Cs maintained low immunogenicity in vitro and enhanced immunogenicity after transplantation in vivo.RM-?Cs co-transplantation with adipose MSCs is feasible for the treatment of canine diabetes mellitus.This study provides a good reference and basis for islet cell replacement transplantation in the treatment of type I diabetes mellitus. |
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