Optimization and Comparison of the PolyFect Gene Delivery Method in Three Different Kinds of Mesenchymal Stem Cell Types

Abstract

Objective: The aim of this study was optimization of the PolyFect gene delivery methodof pcDNA3.1 expression vector transfected with the mouse pdx-1 gene in three differentkinds of mesenchymal stem cells and Hepa cells as well as comparison of transfectionefficiency leading to expression of the mentioned gene in the cell types used.Materials and Methods: Rat bone marrow-derived mesenchymal stem cells, C57 mousebone marrow-derived mesenchymal stem cells, human synovium derived mesenchymalstem 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 hourslater, the cells were treated with anti-mouse Pdx-1 antibody and immunocytochemicallyanalyzed using a fluorescent inverted microscope. Transfection conditions were optimizedin each of these cells by changing different lipofection parameters such as DNAconcentration, PolyFect reagent concentration and cell density.Results: The results demonstrated that for transfection of these cells, the best concentrationsof DNA and PolyFect reagent are 400 ng/μL and 6000 ng/μL respectively. For maximumtransfection efficiency, the best cell density in 12-well plates was 105 cells in Hepacells, 1.3×105 cells in rat bone marrow-derived mesenchymal stem cells, 1.5×105 cells inhuman synovium-derived mesenchymal stem cells and 105 cells in C57 mouse bone marrow-derived mesenchymal stem cells. Under the mentioned optimized conditions, the maximumefficiency of transfection was determined to be 50% for Hepa cells, 40% for rat bonemarrow-derived mesenchymal stem cells, 21% for human synovium-derived mesenchymalstem cells and 10% for C57 mouse bone marrow-derived mesenchymal stem cells.Conclusion: These findings implicate that the most important factor extremely influencingtransfection efficiency in mesenchymal stem cells is the cell derivation origin. Resultsof this study can be used in basic and clinical studies dealing with gene therapy in mesenchymalstem cells