Differentiation Of Bone Marrow-Derived Mesenchymal Stem Cells From Diabetic Patients Into Insulin-Producing Cells In Vitro

BACKGROUND AND OBJECTIVEDiabetes mellitus (DM), results when there is the progressive failure of functional B-cells. The destruction of insulin-producing β-cells is the main cause of type 1 diabetes and also can be seen in type 2 diabetes at the later stages.At present, a lot of ways were used in treating DM, for axample, which include injection exogenous insulin, or using oral antidiabetic drug. While traditional recombinant insulin therapy can control blood glucose levels, the therapeutic efficacy reduces with time.The success of Edmonton Protocol for pancreatic islet transplantation and pancreas teansplantation have sparked new interests in treating DM. Unfortunately, they has historically been hampered by immune rejection as well as the scarcity of donor islets, which have greatly diminished the benefits to patients and limited its wider clinical applications. Stem cells, which have self-renewal and pluripotecy, appeal to people for developing new source of organ, tiusse replacement and regeneration in present years.To date, scientists have turned their focus on mesenchymal stem cells(MSCs), which exist in bone marrow mainly and belong to one of comparatively primary marrow stroma cells. MSCs can secret a lot of cytokines, and several reports revealed that MSCs have selt-renewal and pluripotecy differentiation capacity, since under suitable condition in vitro, MSCs can be differentiated into many different typeies tissue-derived cells. The multilineage differentiation potential of MSCs populations derived from a variety of different species has been extensively studied in vitro since their first discovery in 1968 by Friedenstein et al. Bone marrow contains bone marrow-derived mesenchymal stem cells (BM-MSCs), which carry the more significant implications for possible clinical development because of their pluripotecy differentiation capacity. More and more reports revealed that that populations of BM-MSCs from human, canine, rabbit, rat, and mouse have the capacity to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, fat, tendon, muscle and ligament et al. At the same time, hepatic, biliary duct epithelium, endotheliocyte and neuroectodermal cells of donor origin can be found inrecipient animals upon transplantation of BM-MSCs. BM-MSCs carry the more significant implications for possible clinical development, because they are easily accessible for an autograft and routinely collected from adults without ethical concern inherent to fetal embryonic tissues. Accordingly, BM-MSCs has spurred considerable interests in better understanding the biology of BM-MSCs and their potential clinical applications in the therapy of organ or system specific diseases in the future. Therefore, the utilization of BM-MSCs is becoming the most promising therapy of DM.Under these premises, in this present study, we isolated BM-MSCs from both typel and type 2 DM patients marrow by first taking advantage of their preferential adherence to the plastic surface of the culture dish and uncovered culture conditions to keep BM-MSCs from DM patients at undifferentiation state. We then went to characterize these BM-MSCs celllines and identify clones by testing morphology feature, surface marker and differentiation capacity for further investigation. Finally, we induced BM-MSCs to transdifferentiate into insulin-producing cells under culture conditions containing high concentrations of glucose and the additions of 8-cell stimulating growth factors. Taken together, our results indicated that the bone marrow-derived MSCs from diabetic patients themselves could differentiate into insulin-producing cells in vitro and hinted that using a diabetic patient's own bone marrow-derived MSCs as a source of autologous insulin-producing cells for β-cell replacement would be feasible.Part 1 Isolation, culture-expanding and identification of bone marrow-derived mesenchymal stem cells from diabetic patients invitroOBJECTIVE1) Establish efficient methods for isolating, culture-expanding of BM-MSCs from diabetic patients;2) Investigate the chief biological properties of BM-MSCs from diabetic patients;3) Identify the differentiation capacity of BM-MSCs from diabetic patients into bone, cartilage, fat and neuroectodermal cells;MATERIALS AND METHODSUnder aseptic condition, 5 ml of heparinized bone marrow samples were obtained from the posterior superior iliac crest of DM patients. And then the diluted sample were isolated by Ficoll-Hypaque(1.077±0.001g/ml)density gradient centrifugation. Monuclear cells were culture-expanded in Dulbceco's Modifed Eagle's medium-low glucose (DMEM-LG) which contains 10% FCS. BM-MSCs from diabetic patients were characterized according to morphology by H.E and Giemsa staining. Representative cell surface antigen expression profiles of DM-MSCs analysed by flow cytometric analysis. Finally, the cells were differentiated into bone, cartilage, fat and neuroectodermal cells under certain conditionds in vitro.RESULTS1) BM-MSCs from diabetic patients exhibit a homonomous fibroblast-like morphology with spindle shape according to H.E and Giemsa staining;2) Primary cultures were maintained for 18～20 days. The cells were detached with a solution of trypsin-EDTA. BM-MSCs at passage 20 still can be kept in undifferentiation state; 3) Flow cytometric analysis of BM-MSCs showed that these cells expressed high levels of CD29, CD44 and CD106, and they negative for CD34, CD45 and CD14;4) BM-MSCs from diabetic patients have the multiple differentiation potentials, which can be differentiated into bone, cartilage, fat and neuroectodermal cells under certain conditionds in vitro;CONCLUSIONS1) BM-MSCs from diabetic patients can be successfully isolated, culture-expanded in vitro. By Ficoll-Hypaque(1.077±0.001g/mI)density gradient centrifugation, we can obtain relatively purified BM-MSCs in vitro.The results draw a conclusion that BM-MSCs posses powerful proliferation and amplification capacity in vitro, and BM-MSCs of passage 20 still can be kept in undifferentiation state, which will be more suitable for resesrch of organ or tiusse engineering, and cell/ molecular biology in the future;2) Flow cytometric analysis of the BM-MSCs from diabetic patients showed that these cells were negative for CD34, CD45 and CD14. They expressed high levels of CD29, CD44 and CD106. These results indicated that relatively purified BM-MSCs were isolated;3) BM-MSCs from diabetic patients were strong in the multiple differentiation potentials, which can be differentiated into bone, cartilage, fat and neuroectodermal cells under certain conditionds in vitro successfully;4) Culture-expandedh BM-MSCs were confirmed the presence of cells with adult multipotential stem cells-like characteristis and non-hematopoietic origin.Part 2 Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitroOBJECTIVE Study the differentiation capacity of the diabetic patient's BM-MSCs and test the feasibility of using the BM-MSCs for autologous B-cell replacement in treating DM.MATERIALS AND METHODSBM-MSCs were obtained from 10 DM patients and induced to IPCs under a three-stage protocol with high concentrations of glucose and the additions of -cell stimulating growth factors. RT-PCR was performed to detect multiple genes related to pancreatic B-cell development and function. The identity of the IPCs was illustrated by the analysis of morphology, ditizone staining and immunocytochemistry. Release of insulin by these cells was confirmed by immunoradioassay.RESULTS1) Typical islet-like cell clusters were observed at the end of protocol (18 days). Ditizone staining and immunohistochemistry for insulin are both positive;2) These differentiated cells at stage 2 (10 days) expressed nestin, PDX-1, Ngn3, Pax4, insulin, glucagon, but at stage 3 (18 days) we observed the high expression of PDX-1, insulin, glucagons;3) Insulin was secreted by these cells in response to different concentration of glucose stimulation in a regulated manner (P<0.05);CONCLUSIONS1) BM-MSCs from DM patients can be differentiated into functional IPCs under certain conditions in vitro;2) Using diabetic patient's own BM-MSCs as a source of autologous IPCs for β-cell replacement would be feasible;...