Studies On The Differentiation Of Insulin-Producing Cells Induced From Placenta-Derived Mesenchymal Stem Cells And It's Glucose-Reducing Effect On Diabetic Mice

Objective:Placenta mesenchymal stem cellc(PMSCs) were isolated from placenta tissue in use of the characteristic of mesenchymal stem cell that grows adherently and cultured and passaged in vitro.PMSCs have the potential to differentiate to nerve cell, adipocyte and osteoblast which the characteristics of which are similar to marrow mesenchymal stem cells.And they have been established as seed cells which are used to research the potention to differentiate into all kinde of cells including ectoderm,mesoderm,and endoderm.PMSCs are the source of new stem cells that will be used in future clinical substitution treatment and the gene therapy.This has aroused the widespread interest.This research attempts to discuss the function of placenta mesenchyma stem cells,including isolation,cultivation,differentiation and transplantation into diabetic mice.Diabetes mellitus(DM) is a serious disease that had affected about 200 million persons in the world can be classified toⅠandⅡ.The mechanism of DM is the damage ofβ-cells of pancrea.β-cells cannot produce and secrete sufficient amounts of active insulin in response to an increased demand for insulin which causes blood glucose elevation and sendary complications associated with diabetes.This kind of patients only could depend upon the life-long injection of exogenous insulin,this is not convenient and ofen failing to match insulin with prevailing blood gloucose concentration and mostly not sufficient for preventing the secondary complications. The ideal treatment is pancreas transplantation.But the lack of cadaveric islets for transplantatirs on determines that researchers must explore alternative source of graft material.So recently many approaches have been taken in the world,such as beta-cell replacement therapy,which is a permanent replacement for the lack of endogenous insulin production.The use of stem cells for generating healthy tissues has the potential to change therapies for diabetes mellius resulting from the destruction of beta-cells.Cell engeering of non-beta cells and selective expansion of stem cells are key potential sources,then transplantation of insulin-producing cells becomes an ideal method.So recently many scholars are seeking the source of stem cells which can differentiate to IPCs.Dudng the past years progress has been made in the definition of new strategies to produce mature pancreatic beta cells.The most significant advance is the use of pluripotent embryonic stem cells(ESCs),serving as a kind of regeneration therapy,because they are not only renewable but also constitute a limitless source of various cells.But they are easy to cause the tumor occurrence and initiate the discussion of ethics problem.Marrow mesenchymal stem cell is also the hot spot and the widest applied at present,but MSCs decreases with aging and are dificult to get,so finding a new source of stem cells is imminent.Kaviani confirmed that placenta tissue contains cells which have the ability to differentiate firstly in 2002, then Fukuchi et al.obtained adherent cells from placenta lobule which can express several kinds of symbolic genes of stem cells.Bailo and Yen et al.isolated fibroblast-like cells that grow swirlly similar to MSCs from placenta tissue,this surveyed its morphology feature furtherly.Recent years,many internal laboratories have isolated and cultured PMSCs successfully and induced them differentiate to nerve cell,adipocyte and osteoblast.These progress suggested that placenta stem cells would be manipulated to differentiate into IPCs which express and secrete insulin.Such information is crucial to assess the feasibility of using PMSCs as a potential source for cell replacement therapy.In our study we would focus on PMSCs and demonstrate that PMSCs can be differentiate into IPCs in vitro which can secret active insulin,and the induced IPCs transplantation may play glucose-reducing effect on diabetes mice.Our results may provide evidence that can be applied as a practical therapeutic strategies for cell transplantation against diabete mellitus using PMSCs.Methods:1.Isolated cultivation of PMSCs in vitro:First,we isolated the placenta tissue of Embryo side from full-term c-section and digested them with collagenaseⅣ,then we collected cell precipitation and cultured in medium which contains 10%fetal calf serum and high-glucose Dulbeccos modified eagle medium.The conditions were 37℃,5%CO2,95%saturated humidity,changing the fluid every 2-3 days and observing cell's growth situation. 2.Detection of the phenotype in surface of PMSCs:We got the cells of 3 passages digested by EDTA,flushed by PBS twice.Then we adjusted the number of cells to 1×10~6/100ul,added mouse anti-person monoclonal antibody CD19,CD44,CD45, CD34,CD29 to six centrifuge tubes respectively which are filled with the cells and detected the phenotype by flow cytometry.3.PMSCs were induced to differentiate into osteoblast:We got the cells of 3 passages which growed well,took trypsase as cell suspension,stopped cells with 10%fetus calf serum,flushed cells twice with PBS,cultured them in induction medium(10%fetus calf serum,high-glucose DMEM,20mmol/L sadiumβ-glycerophosphate,50mg/L vitamin C,10mmol/L dexamethasone),cultured in 5% CO2 hatched box at 37℃,changed the fluid one time every 3-5 days.After 10 days we detected the active change of alkalinity phosphatase,observed the shape of cells and dyeing situation.4.PMSCs were induced and differentiated into IPCs:We got the cells of 3 passages which growed vigorously digested by trypsas,flushed cells with PBS twice,took the suspension cultured in six-well culture plate in induction medium.The induction system contained high-glucose DMEM,10%fetus calf serum,1%nonessential amino acids,0.1 mmol/Lβ-ME,1mmol/Lglutamine,5ug/LbFGF.5.Examination of insulin producing cells:After inducted and cultured for 14 days, cells were examinated by these methods:DTZ staining;immunohistochemistrical assay,;insulin detection assay using ELISA;detected the expression of mRNA of PDX-1,Insulin1,Insulin2 and Glut2 by RT-PCR,;detection of activity of secreted insulin.6.IPCs transplantation on diabetic mice:Pure Balb/c mice were chosen to be injected with streptozotocin in abdominal cavity with the dose of 200mg/kg.14 days later,mouse with the level of blood glucose＞16.7mmol/L were judged as DM.The model of DM mice were established.Cells of induced for 14 days were implanted subcutaneously into the shoulder of diabetic mice with the dose of 1×10~8 per mice in the form of cluster suspension for the first time.Repeated the same operation after 5 days and observed effect of transplantation in blood glucose reducing through detecting blood glucose level and the change of body weight at different time.Results: 1.Isolated cultivation of PMSCs in vitro Mononuclear cells were harvested after placenta tissue was digested,adhered after about 7-14 days,growed like whirlpool,filled the bottom of the bottle after about 4 weeks.The shape of cells varied from irregularity to fusiform and the body of cell was long and thin like fibroblast.The cells passaged at 1 week for the first time and steadily passaged to the 10th generation.2.Detection of the phenotype in surface of PMSCs According to the detection of the flow cytometry,cells were lowly positive for CD29(1.1%),high positive for CD44(98.8%) and CD105(67.4%),but negative for CD34(0.1%),CD45 (0.7%) and CD19(0.7%).3.PMSCs were induced to differentiate into osteoblast After 10 days we found the activity of alkalinity phosphatase in induction group was higher than that of control group,and the disparity had statistical significance.4.PMSCs were induced and differentiated into IPCs The aerosol cells assumed clump shape to grow gradually and took the form of the bird nest after 7 days induction.5.Examination of insulin producing cells(1) DTZ staining:the differentiated cells of day 14 after induction were harvested and stained by DTZ,the induced cells were found to be stained crimson red.(2)Immunohistoochemiistrical assay for insulin:we examined insulin immunoreactivity within the cluster on the day of 14 after induction. Immunoreactivity was observed in some induced cell clusters.(3) The results of ELISA showed that inducted IPCs could secrete insulin under the stimulation of high glucose.(4) Detection of activity of secreted insulin:The supematant of day 14 could reduce blood glucose significantly when injected into normal mouse and the level of blood glucose decreased from 7.0±0.9 mmol/Lto4.7±0.6 mmol/L 30 min after tail intravenous injection.(5) Gene expression of IPCs cluster:to clarify the characteristical features of the differentiated clusters,we analyzed the gene expression of avariety of endocrine pancreatic markers using RT-PCR analysis.RNA samples were obtained from the induced cells on day 7 and 14.Samples of day 7 expressed PDX-1 mRNA of pancreas specific gene.Samples of day 14 expressed Insulin1,Insulin2,PDX-1mRNA.No expression of Glut2 was observed in both of the two sample.6.IPCs transplantation on diabetic mice G1 represents blood glucose level before STZ injection.G2 is blood glucose level before IPCs transplantation.G3 is blood glucose level on 5 after the first transplantation.G4and G5 are blood glucose levels of day 5 and day 15 after the second IPCs tiansplantation respectivedly.The results showed that:Blood glucose level of IPCs transplantation group and DM model group before transplantation was increased significantly comparing with that before STZ induction.Blood glucose level of IPCs transplantation group reduced significantly on day 5 after the second transplantation comparing with that before IPCs transplantation,from 20.3±0.7mmol/Lto 17.3±0.39mmol/L,which implied that IPCs transplantation in diabetic mice was effective.Blood glucose level of DM model control and normal control were not changed during the period of experiment.The level of blood glucose had not obvious difference on the day 15 after the second transplantation.Conclusion:1.Cells that had the biological characteristics of mesenchymal stem cells exist in placenta tissue and can grow adherently and passage in the common medium.2.PMSCs could be differentiated into osteoblast.3.PMSCs could be differentiated into IPCs in proper induction system.4.The transplantation of insulin-producing cells inducted from PMSCs to DM mice can make the level of blood glucose reduced in a short period and improve general condition of the mice---playing the effect of substituted treatment.