Hypoxia Induced Bone Marrow Derived Mesenchymal Stem Cells Protect The Function Of Islet Graft

Background:Diabetes mellitus (DM) is a serious threat to human health. With the continuous rise of its morbidity, the number of patients in china will come the first in the world by 2025,reaching 37.6 million. Type 1 DM is associated with self immunity disorder, which result in the destroy of beta-cells as well as severe complications. Since type 1 DM patients rely on the insulin productions that help them to control the blood glucose, however, they also risk the insulin resistance and fatal hypoglycemia. Compared with nature insulin secreting, the use of insulin productions is convinced less timeliness and accuracy. Therefore, islet transplanta- tion seems the best treatment for type 1 DM patients to restore the normoglycemia, regardless of primary non-function(PNF). The aim of this study is to investigate a new strategy to protect islet graft from PNF in order to improve the success of transplantation.Bone marrow derived mesenchymal stem cells (BM-MSCs) are self-renewable pluripotent stromal cells that have the potential to give rise to cells of diverse lineages. BM-MSCs seem to be the best candidates for cell therapy to regenerate injured tissue in biological engineering. They are reported to represent the precursor cell for stromal tissues that support hematopoiesis, and to promote angiogenesis. They are also admitted to protect other cells through paracrine actions which would be amplified by hypoxia.This study was designed to isolate the MSCs from BM, and stimulate them with hypoxia before co-transplantion with islets. After transplantation, blood glucose and body weight are detected every 3 days in order to investigate the efficiency of the BM-MSCs in protection for islet graft, which may result in a new strategy against PNF and shortage of islet supply.Part one:Isolation and cultivation of murine BM-MSCs and biological features of the derived cellsObjective:to establish a method of isolating BM-MSCs from bone marrow and to identify their biological features.Method:To put the whole cells derived from mouse bone marrow in the plastic cell culture flask with the presence of special murine BM-MSCs selecting culture medium and generate the fibroblast-like cells with aderence. To complete the growth curve with MTT. To identify some positive or negative surface markers with Flow Cytometry. To investigate their in vitro differentiation capacities into adipogenic progenitors with the presence of special stimulating medium.Result:Majority of derived cells adhere to the flask within 24 hours, and present fibroblast-like appearance in passage 3 or later passage. Cells receive a sharp growth in every 4 days, and there is a certain difference between passage 3 cells and passage 6 cells. Passage 1 cells are positive for CD29 and CD71, negative for CD34 and CD 90, and weak-positive for CD45. CD29 and CD71 become even more intense with passage 3 while CD34 remains negative.3 weeks after presence of special stimulating medium, cells become ground with vacuole in side, and fat droplet could be observed in red in the vacuole after dye with oil red O.Part two:The effect of hypoxia on BM-MSCsObjective:To compare the difference between the BM-MSCs cultured in normal condition and cells grow in low oxygen density.Method:BM-MSCs are derived from murine bone marrow, and cultured in the presence of 20%O2 or 5%O2. Growth curves are established for cells from different conditions. Surface markers and differentiation capacity are also detected by same protocol. RT-qPCR and ELISA are used to investigate the level of VEGF,IL-6,MCP-1 and MMP-9.Result:Cell growing in low oxygen density also adhere to the flask within 24 hours, and become fibroblast-like after passage 2. Cells grow more rapid in hypoxia than in normal condition. The markers and differentiation capacity of these two are almost the same, while the expression of genes VEGF,IL-6,MCP-1 and MMP-9 is much higher in cells cultured in hypoxia. However, the secretion of MCP-1 and MMP-9 is much lower in cells growing in low oxygen density, and VEGF and IL-6 show the opposite.Part three:Murine BM-MSCs protect Allogeneic islets in vitroObjective:To investigate in vitro protection of murine BM-MSCs for allogeneic islets.Method:To culture the BM-MSCs in general islet medium for 48 hours in presence of 20%O2 or 5%O2, in order to generate conditioned medium. To isolate murine islets with type IV collagenase and density gradient centrifugation. To detect the apoptosis of allogeneic islets growing with condition medium in presence of 20%O2 or 1%O2, with AO/PI dye and Flow cytometry. To evaluate the insulin releasing capacity of islets cultured in different condition medium by Radioimmunoassay.Result:Islets are obtained after density gradient centrifugation from layer between 20% and 23% Ficll. AO/PI dye and Flow cytometry prove that islets grow in condition medium derived from hypoxia induced BM-MSCs show better morphology, low apoptosis and good tolerance to low oxygen density, compared with other groups. Moreover, islets grow in condition medium derived from hypoxia induced BM-MSCs have stronger insulin releasing capacity than other test groups.Part four:Co-transplant with murine BM-MSCs and allogeneic islets in vivoObjective:To investigate in vivo protection of murine BM-MSCs for allogeneic islets.Method:To establish type 1 DM models of mouse with peritoneal injection of STZ. To transplant 250.300,350 and 400 IEQ respectively to DM models under renal subcapsule, and observe the body weight, random blood glucose and fasting blood glucose in next 30 days. Allogeneic 1×106 BM-MSCs either cultured in normal condition or hypoxia are co-transplanted with 250 and 300 IEQ respectively under kidney subcapsule, and same parameters are detected. OGTT are performed in all recipients after one month.Result:The success rate of type 1 DM model reach 70%. Mice received 400 IEQ restore their blood glucose within 10 days after transplantation, while 250.300 and 350 IEQ recipients also get a certain but not a thorough alleviation in hyperglycemia. Hypoxia induced allogeneic BM-MSCs co-transplanted with 250IEQ or normal BM-MSCs co-transplanted with 300 IEQ will achieve the same efficiency as 400 IEQ alone.Conclusion:1. Whole BM cells adhering plus selecting medium is helpful in generating murine BM-MSCs. 2. BM-MSCs cultured in vitro maintain their biological features.3. Hypoxia induced BM-MSCs show the same biological features as cells growing in normal condition, but have more rapid growth speed and higher expression of gene VEGF.4. Type IV collagenase plus density gradient centrifugation is helpful in generating murine islets.5. Allogeneic BM-MSCs especially those induced with hypoxia exhibit efficient protection for islets in vitro against cell apoptosis, and improve their insulin releasing capacity through paracrine way6. Allogeneic BM-MSCs especially those induced with hypoxia are much helpful in restoring blood glucose disorder when co-transplanted with islets in vivo.7. Allogeneic BM-MSCs especially those induced with hypoxia would need less islets in co-transplantation and thus alleviate the donor shortage.