| Islet cells replacement is considered as the optimal treatment for type I and parts of type II diabetes. In recent years some diabetes' patients received islet cell transplantation through hepatic portal vein. However, the availability of human pancreas islets for transplantation is limited, and immune rejection response is another difficulty for the transplantation treatment. Stem cells, which have self-renewal and pluripotecy, appeal to people for developing new source of islet cell replacement. More and more reports revealed that bone marrow stem cells have pleuripotent differentiation capacity, since myoblasts, cardiac myoblasts, endothelium, hepatic, biliary duct epithelium and neuroectodermal cells of donor origin can be found in recipient animals upon transplantation of bone marrow or enriched hematopoietic stem cells (HSCs). We are interested that if bone marrow stem cells can differentiate into islet-like cells and become the islet β cells replacement for transplantation treatment of diabetes. Firstly, we injected streptozotocin (STZ) into normal mice and obtained experimental diabetes model. Isolated bone marrow stem cells or mesenchymal stem cells were i.v. or i.p. injection into diabetic mice for 4 times. The symptom of hyperglycemia of diabetic mice was not improved after bone marrow cell transplantation. At 3, 4, 6, 9, 12 weeks after the first cell transplantation, pancreases were isolated and made into tissue sections. By using in situ hybridization for sry gene in Y chromosome we detected transplanted male cells which scattered in pancreas tissue. On the other hand we also detected DAPI-labeled bone marrow cells in pancreas. Most transplanted cells differentiated into acinus cells or pancreatic duct cells. No insulin positive transplanted cells were discovered. This result suggests that bone marrow stem cells can survive in pancreas and differentiated into pancreatic cells (less β cells) in pancreatic lesions. Hyperglycemia status of diabetic animals may be a bad environment for transplanted cells to differentiate into islet cells or long term hyperglycemia leads to functional failure of new differentiated islet cells.Since bone marrow stem cells transplantation can't improve symptom of diabetes, we sought to expand and induction of bone marrow mesenchymal stem cells differentiation islet-like cells and use as islet cell transplantation replacement. Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the human, bird or rodent bone marrow microenvironment defined by their ability, either in vitro or in vivo, to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, neural cells and myogenic lineages. The multipotential of these cells, the easy isolation and culture property, as well as their high ex vivo expansive potential makes these cells an attractive therapeutic tool. We designed two-step induction protocol based on the similarity of development of neuronal system and pancreas. Using the centrifugation of Percoll density, we separated MSCs from healthy people's bone marrow, and then we cultured them in the a-MEM added with 10% fetal bovine serum. The results of flow cytometry showed that the cells we separated and cultured have the surface markers of CD29 and CD71, and no expression of CD34, CD 11 a . We used bFGF and EGF to induce MSCs differentiation into nestin-postive islet-derived progeitor cells (NIPs), and used high glucose, betacellulin and nicotinamide to induce NIPs differentiation into insulin-secreting cells. The results showed that hMSCs can be induced to differentiation into islet-like cell cluster in vitro. After the first stage, hMSCs can become NIPs, and the cells changed into round shape cells after another 6 days, and clustered into islet-like cells structure. These cells were stained by dithizone. Insulin and glucagon protein could be detected in cytoplasm of the differentiated cells. The results of RIA assay showed that these cells could synthesize and secrete insulin. They had weak glucose response, which might be explained by not full maturation of them. Because the cytokines and nutrient involved in the development of pancreatic islets are so many and so far we don't know which one is the most suitable for this development process, we modified hMSCs by using gene and we got islet-like cells with good treatment function.Pancreatic duodenal homeobox-1 (PDX-1) is the key transcriptional factor for the development of pancreas. PDX-1 plays a role in the formation of early developmentof pancreas and in the maturation of pancreas islet cells. Because stem cells is a group of relative static cells and the rate of transfection with lipofectamine is very low, we choose virus vector to get high transfection rate. We constructed the adenovirus vector pAd-PDX-1. Firstly, human PDX-1 gene was ligated into shuttle vector pAdTrack-CMV. Then homologous recombination was performed in BJ5183 bacteria by cotransforming linearized shuttle plasmid with adenovirus backbone plasmid pAdEasy-1. The recombinant plasmid was packaged and amplified in 293 cells. In the present study, we used pAdEasy system to get the adenovirus vector with PDX-1, which is the base of the investigation of the PDX-1 expression and biological function of hMSCs.After being transfected with Ad-PDX-1 at MOM 50, hMSCs were successfully induced to differentiation into insulin-secreting cells. RT-PCR and Western blot analysis showed that the expression of PDX-1 in MSCs. MSCs began to aggregate into patch-like structures after 3 days of transfection with Ad-PDX-1 and gradually formed semi-spherical clusters at 7 days of culture. We used DMEM-LG medium containing GLP-1 and 5% fetal bovine serum to culture the transfected cells. These differentiated PDX-1+-hMSCs expressed multiple islet cell genes including neurogenin3, insulin, GK, Glut2 and glucagons and GLP-1 receptor genes. RT-PCR and RIA assay results showed that GLP-1 could increase mRNA and protein level of insulin. C-peptide production by the differentiated cells suggested that these cells could produce insulin indeed. These differentiated cells also expressed insulin and glucagon protein. They released insulin in a regulated manner in response to glucose challenge and different stimulators. When the voltage-clamp protocol applied to the cells, these differentiated PDX-1+-hMSCs showed the sensitivity of potassium channels to high glucose concentration and the islet cell specific excitability. These in vitro experiments suggest that the differentiated PDX-1+-hMSCs had the character of islet cells. Cell transplantation will be done in order to prove the function of these differentiated cells.Recent studies suggested that MSCs might exert immunoregulatory effects. Allogenic MSCs transplantation can prevent or treat Graft-Versus-Host-Disease (GVHD) caused... |
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