Study On Differentiation Of Cynomolgus Macaque Bone Marrow Derieved Mesenchymal Stem Cells Into Insulin Producing Cells

Objective: Mesenchymal stem cells(MSCs)are a class of adult stem cells with selfrenewal ability,multi-differentiation potential and immunoregulatory function,which have shown strong therapeutic potential in more and more preclinical studies and clinical trials.Type 1 diabetes mellitus(T1DM)is a T cell-mediated organ-specific autoimmune disease characterized by destruction of islet β-cells and lack of insulin secretion.Based on the characteristics of MSCs and the pathogenesis of T1 DM,MSCs are usually transplanted directly into T1 DM models or patients,but due to the destruction of pancreatic microenvironment,MSCs are not able to cause endogenous islet β regeneration;in addition,very few MSCs spontaneously differentiate into insulin producing cells(IPCs)in vivo.Therefore,researchers are gradually trying to differentiate MSCs into IPCs in vitro by chemical induction or virus infection.Among them,chemical induction has developed to the "three-step method",but currently,there is still no uniform and standardized protocol,Therefore,in this paper,we will combine the different chemical induction protocols with 3D method to induce the differentiation of cynomolgus bone marrow mesenchymal stem cells(BMSCs)into IPCs in vitro to establish an effective and reproducible method.Methods: In combination with the 3D culture system,BMSCs were differentiated into IPCs in vitro by two different chemical induction protocols.The in vitro characteristics of the following aspects were compared: 1.Dithizone(DTZ)staining was used to identify whether insulin was expressed by different differentiation protocols.2.Immunofluorescence staining of Insulin and PDX-1(pancreatic duodenal homeobox factor-1)was used to identify the maturation of IPCs by different differentiation protocols.3.Glucose Stimulated Insulin Secretion(GSIS)experiment was used to identify the insulin secretion ability of IPCs by different differentiation protocols.Results: 1.The IPCs induced by two different in vitro chemical induction protocol showed DTZ staining,indicating that both protocol A and B showed insulin expression.2.The IPCs induced by two different in vitro chemical induction protocol showed specific fluorescent staining of islet development-related genes Insulin and PDX-1,indicating that both protocol A and B showed the development and function of IPCs.3.The IPCs induced by two different in vitro chemical induction protocol showed glucose secretion under insulin stimulation,indicating that both protocol A and B have insulin secretion function under glucose stimulation.Conclusion: In this study,both chemical induction protocols were able to effectively differentiate BMSCs into IPCs under 3D culture conditions.Discussion: 1.The expression of islet development and function-related genes under two protocol needs further study.2.The in vivo function of the transplanted cells under two protocol still needs further study.