Document Type : Original Article
Biochemistry Department, Pasteur Institute of Iran, Tehran, Iran
Objective: The aim of this study was optimization of the PolyFect gene delivery method of pcDNA3.1 expression vector transfected with the mouse pdx-1 gene in three different kinds of mesenchymal stem cells and Hepa cells as well as comparison of transfection efficiency leading to expression of the mentioned gene in the cell types used.
Materials and Methods: Rat bone marrow-derived mesenchymal stem cells, C57 mouse bone marrow-derived mesenchymal stem cells, human synovium derived mesenchymal stem cells and Hepa cells were used in this study. After culturing of the mentioned cells, mouse pdx-1 gene were transfected into them using the Qiagen PolyFect kit. 72 hours
later, the cells were treated with anti-mouse Pdx-1 antibody and immunocytochemically analyzed using a fluorescent inverted microscope. Transfection conditions were optimized in each of these cells by changing different lipofection parameters such as DNA concentration, PolyFect reagent concentration and cell density.
Results: The results demonstrated that for transfection of these cells, the best concentrations of DNA and PolyFect reagent are 400 ng/μL and 6000 ng/μL respectively. For maximum transfection efficiency, the best cell density in 12-well plates was 105 cells in Hepa cells, 1.3×105 cells in rat bone marrow-derived mesenchymal stem cells, 1.5×105 cells in human synovium-derived mesenchymal stem cells and 105 cells in C57 mouse bone marrow-derived mesenchymal stem cells. Under the mentioned optimized conditions, the maximum efficiency of transfection was determined to be 50% for Hepa cells, 40% for rat bone marrow-derived mesenchymal stem cells, 21% for human synovium-derived mesenchymal stem cells and 10% for C57 mouse bone marrow-derived mesenchymal stem cells.
Conclusion: These findings implicate that the most important factor extremely influencing transfection efficiency in mesenchymal stem cells is the cell derivation origin. Results of this study can be used in basic and clinical studies dealing with gene therapy in mesenchymal stem cells.