Transplantation Of Mesenchymal Stem Cells In Streptozotocin-induced Diabetic Rat

Background:Diabetes mellitus(DM)results when there is an inadequate functional mass of pancreaticβcell and decrement in insulin levels. Exogenous insulin administration or oral hypoglycemic medicine has traditionally been the mode of treatment for this disease.However,they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes,kidneys and cardiovascular system.So the regeneration and maintenance of pancreatic endocrine tissue after the onset of islet destruction would have significant therapeutic impact on DM.Recent advancements in regenerative therapy for DM may include transplantation of pancreas,islet and stem cells.Unfortunately,pancreas or islet transplantation has historically been hampered by immune rejection as well as the scarcity of donor islets.As stem cell research progresses,more attention has focused on the possible use of the bone marrow mesenchymal stem cells in treating DM.Mesenchymal stem cells(MSCs),which are adherent stromal cells of a nonhematopoietic origin,they play a role in providing the stromal support system for haematopoietic stem cells in the marrow,also they have the ability to give rise to various differentiated cell types Recent studies have certified that MSCs could differentiate into osteoblast,chondrocyte,neuron-like cells, adipocyte and myocardial cell,et al,which suggested that they can differentiate into not only mesodermal lineage cells but also ectodermal and endodermal lineage cells.On the other hand,several reports have shown that intravenous delivery of MSCs results in their specific migration to a site of injury.This ability of implanted MSCs to seek out the site of tissue damage has been demonstrated in bone or cartilage fracture,myocardial infarction,and ischemic cerebral injury.And transplantation of MSCs will be helpful for the repair of damaged organs.Due to their potential for differentiation into different tissues,MSCs have emerged as a promising tool for clinical applications such as tissue engineering and cell and gene therapy.BM-MSCs are attractive candidates for clinical use because they are relatively easy to harvest and expand in culture.More importantly,these cells may be directly obtained from individual patients,thereby eliminating the complications associated with immune rejection of allogenic tissue.Currently,several studies have shown that bone marrow-derived MSCs can differentiate into insulin-producing cells(IPCs)under certain conditions. Studies of Tang et al.have shown that MSCs from mouse bone marrow could be in vitro induced to differentiate into cells which express certain genes(insulinâ… andâ…¡,Glut-2,glucose kinase,islet amyloid polypeptide and Pdx-1,et al.),and these differentiated cells were responsive to glucose challenge and capable of reversing hyperglycemia in diabetic mice.Recent studies by Banerjee demonstrate bone marrow stem cell remains its stemness under hyperglycemia status.All these studies suggest that using diabetic patient's own BM-MSCs as a source for the therapy of DM would be feasible.So,the study will focus on the following questions:first,In vitro culture and the biological characteristics and differentiation potential of the diabetic BM-MSCs were investigated;second,to explore whether this kind of transplanted diabetic BM-MSCs could contribute to the reversal of hyperglycemia and the possible mechanism.Objective:First,to study the method of separation and cultivation of MSCs from diabetic rat bone marrow.Establishing a stable system of MSCs' culture in vitro and to provide a cell model for the further study of stem cells.Second, MSCs cultivated in vitro were transplanted into the diabetic rats to explore the effect and possible mechanism of MSCs transplantation in treatment of diabetes mellitus.Methods:First,the classical whole bone marrow adhering method was used for the culture of MSCs.The BM-MSCs were cultured with DMEM-LG containing 10%fetal calf serum at 37℃in a humidified atmosphere containing 5%CO₂.The potential of multiple-differentiation of MSCs was identified through the induction into osteoblasts and lipoblasts,et al.The phenotype of MSCs(CD44,CD34,CD45,CD90,et al.)was identified by flow cytometry.And the MSCs of passage 3 wre used for the following experiment.Second,MSCs were labeled by BrdU.Diabetic rats was induced by STZ injection.The rats were divided into three groups:Normal control Group(no DM,rats treated with citrat buffer throught tail vein,n=10);DM control group(DM,no transplantation of MSCs,n=10);tested group(DM and transplantation of MSCs,n=10).Body weight,blood glucose and serum insulin of the rats were mornitored during the experiment after the transplantation of MSCs.The paraffin sections of pancreas were obtained from rats of each group.The technology of immunohistochemistry analysis and double Immunofluorescence was used to detect the BM-MSCs in the pancreatic tissue and their differentiating state.Results:After 48 hours,several adherent cells could be found.And after 72 hours,the nonadherent cells were removed and only the adherent cells were cultered following 3～7days of culture.Fresh complete medium was replaced twice a week.At about 7～10 days,the isolated cells were developed to visible systematic colonies of adherent fibroblast-like cells.They arranged regularly with clear boundary and showed spindle-like or polygon morphology.Primary cultures were maintained for 10～14 days.Upon reaching near 80～90%confluence,cells were detached with a solution of 0.25%trypsin 3～5 minutes at 37℃.After centrifugation,cells were resuspended with DMEM-LG, replated at a ratio of 1:2 and referred to as first-passage cultures.And the morphology of MSCs was about uniform at passage 3.Flow cytometric analysis of the MSCs at passage 3 showed that these cells were negative for CD34,CD45,MHCâ… and they expressed high levels of CD44,CD90.In various induction differentiation conditions,BM-MSCs could differentiate into the osteoblast,chondrocyte,adipocyte and neuron-like cells.These results indicated that relatively purified BM-MSCs which also have multiple differentiation potential were isolated.The BM-MSCs were 89.5%labeled by BrdU and DAPI,which was green/blue double stained under fluorenscent microscope.Nearly 99%of the cells survived after labeling by BrdU.1%STZ was administered at a dose of 60mg/kg in a citrate buffer through tail vein of Wistar rat.During the experiment, the blood glucose of the normal control group maintained between 4.6±0.7 to 5.6±1.0mmol/L,for DM control group and DM test group,they are between 24.2±2.3 to 27.8±2.1mmol/L,17.7±3.9 to 26.1±3.2mmol/L,respectively.After transplantation of BM-MSCs,reduction in hyperglycemia as well as increased survival was found among experimental group whereas DM control group remained hyperglycemic throughout the experiment and the survival rate was low.Especially at the 45 days,the blood glucose of experimental group was obviously lower than that of DM control group(17.7 mmol/L±3.9 vs 27.8 mmol/L±2.1,p＜0.05).At each time point,blood glucose of experimental group or DM control group was higher than that of normal control group(p＜0.05).During the experiment,the body weight of normal control group increased gradually,whereas for DM control group,it deceased and no obvious change was found in experimental group.At the 45 days,the body weight of DM control group test group was obviously lower than that of normal control group(133.3g±13.1 vs.358.8g±21.6,p＜0.05);and the body weight of experimental group was significantly higher than that of DM control group(232.7 g±19.7 vs 133.3g±13.1,p＜0.05).And at the 45 days,there existed differences of serum insulin levels among three groups.Serum insulin levels of DM control group were obviously lower than that of normal control group(10.3mlU/ml±2.3 vs.33.2mlU/ml±3.8,p＜0.05);and the serum insulin levels of experimental group were significantly higher than that of DM control group(19.8 mlU/ml±4.8 vs 10.3mlU/ml±2.3,p＜0.05).The results of immunohistochemistry showed that the number of islets in experimental group was increased with smaller size and more ductual distribution which was suggestive of new islet formation.Image analysis of normal control as well as newly generated islets has been performed with paraffin sections of pancreas. The values for islet area as well as diameter of control islets were found to be significantly higher than those of newly generated islets(p＜0.05).On the other hand,double Immunofluorescence showed some BM-MSCs labeled by BrdU could be found in pancreas and most of these green-nucleated cells were distributed near pancreatic duct.These phenomenon supposed that transplanted BM-MSCs could homing at the pancreatic site.At the same time, we also could observe small amount cells with co-expression of BrdU(green nuclear)and insulin(red cytoplasm)which suggested that a few of transplanted BM-MSCs could transdifferentiate into IPCs.Conclusions:(1)The isolation and cultivation of MSCs from bone marrow are performed with the whole bone marrow adhering method.The cultured MSCs have uniform phenotype and multiple differentiation potential.This cell lineage can be used for further research.(2)Transplantation of MSCs in the STZ-induced diabetic can improve the hyperglycemia status in recipient rats. And a relatively small quantity of transplanted stem cells homing at the pancreatic site,with a few portion of which transdiferentiate into insulin-producing cells,therefore contribute to the islet regeneration.These observations imply that transplantation of BM-MSCs for the treatment of diabetic mellitus would be feasible and it is worth for further investigation.