The Impact Of Different Microenvironments On The Rat Bone Marrow Mesenchymal Stem Cells Differentiating Into Insulin-producing Cells

Diabetes mellitus is a devastating chronic disease and over 6% of the population is affected worldwide. The diabetic are necessary to inject insulin in their lifetime, but it is not ideal to inject insulin since insulin can't prevent the development of diseases derived form diabetes, such as kidney diseases, coronary heart disease, eye diseases,etc. The success achieved over last few years with islet transplantation suggests that diabetes can be cured by the replenishment of deficientβcells. However, the lack of donor organs and transplant rejection hinder the application of pancreatic islet transplantation.Stem cells ,which have the capacity for both self-renewal and multilineage differentiation into any type of cell, becoming the new source of islet cell replacement. Bone marrow mesenchymal stem cells(MSCs) have great ability of multi-directional differentiation and reproductive activity that make them potentially useful for cell therapy. A number of protocols have been described to induce MSCs to neuronal cells,adipocytes,smooth muscle myocytes,skeletal muscle myocytes,cartilage cells and insulin-producing cells(IPCs).The IPCs induced from MSCs can secrete insulin,but their secretory volume was less than 1% that of a normalβcells. How to promote MSCs differentiating into IPCs become the critical issue should be solved. The recent study have shown that the differentiation mechanism is the result of key gene expression through a definite signal transmitting passway and inhibition or activation of transcription factor induced by microenviroment factors.In this study,we try to explore the impact of different microenvironment on differentiating rat bone marrow MSCs into IPCs in vitro,then to study the capacity of MSCs differentiating into IPCs in diabetic rat's microenviroment in vivo,and to provide suitable"seed"cells for replacement therapy of diabetes mellitus.Isolation, cultivation and biological features of rat MSCs in vitro:MSCs were isolated from rat bone marrow,purified by their adhesive process onto the culture dish,serial subcultivated on dulbecco's modified eagle medium with 10% fetal bovine serum;The changes of cell morphology and the ultrastructure were observed, cell surface markers CD34,CD44 were detected by immunocytochemistry. The results show both primary and passage MSCs have maintained highly proliferative capacity.They have homogeneous fibrocyte-like shap under light microscope. The ultrastructure showed the young cell nature of stem cell. The cells were negative for CD34, while positive for CD44 by immunocytochemistry staining.Conclusion: MSCs can be cultured stably,expanded quickly and have higher purity with our method.The Impact of different microenvironments on the differentiating rat MSCs into IPCs: Third descended MSCs cells were then cultivated in vitro mimicking different microenvironments,in which control group was cultivated in serum free DMEM/F12 medium including keratinocyte growth factor(KGF),ITS and Nicotinamide, while experimental group was cultivated in serum free DMEM/F12 medium in which the rat pancreatic extract was added. During the cultivation period the cells were taken for light and electron microscopy at different time points, identified by dithizone(DTZ) and immunocytochemistry. The glucose stimulation test was performed in vitro and the levels of insulin and C-peptide in response to the different glucose concentration was assayed. The results show MSCs could differentiat into IPCs both in the control group and the experimental group. The IPCs harvested from experiment group had greater number and better function than that from control group . Conclusion :The microenvironment in which the rat pancreatic extract added could promote the rat bone marrow MSCs to differentiate into better IPCs in vitro。The capacity of MSCs differentiating into IPCs in diabetic rat's microenviroment in vivo: The rat MSCs and IPCs induced from MSCs were transplanted under the renal capsule of STZ induced SD rat. Blood glucose levels, weight, urine and survival rate were monitored. when rat's blood glucose levels begin to descend,grafts were then removed by unilateral nephrectomy. The blood glucoses level was monitored to investgate whether euglycemia reversal happenned, and we have immunohistochemistry staining to observe wherether the MSCs of transplanted could differentiate into IPCs. The result show MSCs and IPCs by injection into the renal capsule of STZ induced diabetic SD rat could decrease its blood glucose level and prolonged survival time. Immunohistochemistry also confirmed that the MSCs of transplanted were strongly positive for insulin. Conclusion: MSCs can differentiating into IPCs in diabetic rat's microenviroment in vivo.In summary, the present study provides evidence that rat MSCs have the capacity to differentiate into IPCs under different microenvironments both in vitro and in vivo. The microenvironment in which the rat pancreatic extract added could promote the rat bone marrow MSCs to differentiate into better IPCs in vitro. MSCs and IPCs transplanted into the renal capsule of STZ induced diabetic SD rats could decrease their blood glucose level and prolong their survival time. Obviously, there are still a number of fundamental questions about MSCs need to be answered before they can be safely and effectively used in clinical setting, but it provides us a new solution to deal with the shortage of beta cells for therapy of type 1 diabetes.