Direct Contact With Mesenchymal Stem Cells Protects β-cells From Hypoxia-induced Cell Dysfunction And Upregulates E-cadherin During Islet Transplantation

BackgroudIslet transplantation is a promising cure for type-1diabetes.When islettransplantation is used to treat diabetes,>50%of transplanted β-cells undergo apoptosistriggered by non-immunological factors within a few days of transplantation. Hypoxiaplays an important role in this apoptosis, as islet cells are sensitive to low-oxygenconditions. Mesenchymal stem cells (MSCs) have been transplanted together with isletcells in animal models to reduce allograft rejection and to promote engraftment, but theeffects of direct interactions between the MSCs and β-cells are poorly understood.ObjectiveThe purpose of this study was to explore the underlying mechanisms by which MSCsprotect islet cells from hypoxic challenges both in vitro and in vivo.Methods1． The isolation of MSCs: The femur and tibia of F344rats were aseptically harvested.Bone marrowcells were obtained by flushing the bone marrow cavity with culturemedium. After48h, non-adherent cells were discarded, andthe medium was replacedevery2d thereafter. At passage three, cells were characterized usingfluorescence-activated cell sorting, and the ability to differentiate into adipocytes or osteoblasts under specific conditions was assessed.The isolation of islets: Rats were anesthetized with ether, and the proximal region ofthe common bile duct was cannulated. We then injected8–10ml of1mg/mlcollagenase V in medium. Islets were purified by using the histopaque1077in adiscontinuous density gradient.2． Islets were divided into four groups: control, islets alone, indirect contact, and directcontact. For the control group, islets were cultured at37°C in5%CO2for24h. For theislets-alone, indirect-contact, and direct-contact groups, the plates were placed intomodular incubator chambers, and the chambers were flushed with a hypoxicatmosphere (1%O2,5%CO2, and93%N2) at37°C. ELISA was used to assess thefunction of islets. TUNEL and FASC were used to analysis the apoptosis of islets ofdifferent groups.3． The co-transplantation of MSCs and islets. The direct contact group: MSCs and isletswere mixed before transplantation and then transplanted into the inferior pole of leftrenal capsule. The indirect contact group: MSCs were transplanted into the upper poleof left renal capsule and the islets were transplanted into the inferior pole of left renalcapsule. The islets alone group: islets were transplanted into the inferior pole of leftrenal capsule. And the control group: only MSCs were transplanted into the inferiorpole of left renal capsule. The blood glucose was measured in time.4． MSCs and INS-1cell line were co-cultured in the hypoxia for24h. Andimmunostaining, western blot and RT-PCR were used in the experiments.Results1. Mesenchymal stem cell of Passage3-6were used in the following experiments. FASCresults showed that the expression of CD29、CD105、CD90was high, and theexpression of CD34、 CD45、 CD80was very low. ELISA results indicated that thesecretion of IGF-1, HGF and VEGF was normal. Under appropriate conditions, theywere able to differentiate into adipocyte-like and osteoblast-like cells, as assessed byoil red O and Von Kossa staining, respectively. The islets were separated by theretrograde perfusion with collagenase V. 2. Mesenchymal stem cell could functionally protect the islets from hypoxia throughdirect contact manner. And mesenchymal stem cell could reduce the cell death rate ofislets under hypoxic condition.3. Mesenchymal stem cell could enhance the efficiency of islets transplantation in adirect contact manner. Mesenchymal stem cell could preserve the PDX-1expression inthe early graft.4. Mesenchymal stem cell could inhibit the activation of NF-κB and its downstream geneexpression of the INS-1cell, thus preserve the expression of PDX-1and Mafa. AndE-cadherin may participate in this process.ConclusionIn conclusion, MSCs modulate hypoxia-induced NF-κB signaling within islet cellsthrough direct cell contact, thereby improving the engraftment of transplanted islets.