Comparative Study Of The Transfection Of MSCs With Lentiviral Vector And Cationic Liposomes

Stem cell therapy for tissue repair and regenerative medicine has opened up entirely new possibilities. Mesenchymal stem cells (MSCs) are important adult stem cells, have been widelyutilized and studied. MSCs are capable of self-renewal properties, and the ability to differentiate into a variety of cell types, such as adipocytes, chondrocytes and osteoblasts. Because of these intrinsicadvantages, MSCs are considered an ideal cell source for regenerative medicine. Although MSCbased therapies have many advantages, there are still many factors influence the success of treatment. The in vitro expansion conditions and cell culture period affects themultipotency and phenotypes of MSCs.In order to solve these problems and enhancethe therapeutic efficacy, the genetically modified MSCs became a promising approach. Various of viral and non-viral transfection methods have been developed, the goal is to make the ideal target gene expression. Viral-vector transfection method has high transfection efficiency andlong-term gene expression, but the disadvantages are also obvious, such as cell toxicity, immunogenicity, carcinogenicity, poor target cell specificity, high costs and inability to transfer large size genes.Non-viral transfection method shows low transfectionefficiency, transient gene expression, however, safety, ability to transfer large size genes, low toxicity, easiness for preparation offers a promising potential for transfection. In addition, non-viral vectors may be manipulated by modification of the target gene transport tissue or cell specific ligands. MSCs genetically modified to make the advantages of stem cell therapy increase the clinical benefits will be further enhanced. Through the gene transfectio, MSCs can be guided to differentiate into desired cell lines.After genetic modification, MSCs can also become a carrier of the gene or drug. Furthermore,MSCs could be traced easily both in vitro or in vivo after fenetic modification with fluorescent proteins.Thus, geneticmodification has become a powerful tool for the molecular and biological mechanism studies, thereforeaccelerate the use ofhuman bone marrow mesenchymal stem cells in clinical applications.Objective:1.To in vitro harvest bone marrow mesenchymal stem cells through the whole bone marrow adherence method. Master cell culture techniques.2.With cationic lipid rat bone marrow mesenchymal stem cells were infected.Master non-viral transfection technology for optimal cationic liposome/DNA ratio to be applied in the future in other studies.3.With lentiviral vector rat bone marrow mesenchymal stem cells were infected. Master lentiviral transfection techniques, optimal MOI, in order to be applied in the future in other studies.Methods:MSCs were isolated and purified by the whole bone.Expression of MSCs surface antigens was detected by flow cytometer.At the third passage,MSCs were incubated in osteogenous for7d. Osteogenous potential was assessed by alkaline phosphatase staining.E. coli DH5a was used for the preparation of competent cells.Plasmid DNA extraction from the competent cells using a plasmid miniprep system. Set different cationic liposome/DNA ratio and non-viral transfection of MSCs, target gene expression and cellular morphology were observed byfluorescence microscopy to obtain the optimal cationic liposome/DNA ratio.Lentiviral vectors using different MOI to transfect MSCs,target gene expression and cellular morphology were observed by fluorescence microscopy to obtain the optimal MOI. ResultsThe whole bone marrow adherence method is an ideal methord to separateand purify bone marrow mesenchymal stem cells.Advantages including simple operation, small impact on cell viability and the high purity of bone marrow mesenchymal stem cells.When appropriate inducer presented, mesenchymal stem cells in vitro could be induced into osteogenesis cells, are considered an ideal cell source for regenerative medicine. Cationic liposomes, as a vector transfecting bone marrow mesenchymal stem cells, have lower transduction effciency, but lower toxicity is one of its advantages. Viral transfection has high transfection efficiency, but itscytotoxicity can not be ignored. Genetic modification of marrow mesenchymal stem cells offers a promising potential for stem cell based therapies and numerous clinical benefits could be achieved.