An Optimized Protocol of Electroporation of Hepatocyte Nuclear Factor 1 Alpha (Hnf-1α) in Mesenchymal Stem Cells - Cytology and Genetics

Abstract The objective of this study was to investigate the optimization of electroporation of hepatocyte nuclear factor 1 alpha (Hnf-1α) in murine mesenchymal stem cells (mBM-MSCs). mBM-MSCs were phenotypically observed and confirmed by positive expression of stemness markers with differentiation capacity into osteocytes. Hnf-1α plasmid DNA was transfected via Neon electroporation system into the mBM-MSCs. The cells were maintained in a complete DMEM medium. Following single 0.5 μg Hnf-1α electroporation the differences in viability of mBM-MSCs were statistically insignificant at 24 h, 72 h, and post-21 days. Fluorescence imaging of turbo green fluorescence protein (tGFP) was detected for the efficiency of transfection. The transfection efficiency was detected at parameters of 1000 pulse voltage (V), 10 pulse width (ms), and at 3 pulse number at 24 h (***p-value < 0.001, 66.5 ± 12.2%) in mBM-MSCs. The efficiency of transfected 0.5 μg Hnf-1α was decreased at 72 h (40.2 ± 10.9%) and 21 days (31.7 ± 5%). 250 μg/mL G418 Sulfate was used for the selection of Hnf-1α transfected positive cells. TaqMan-qRT-PCR results of independent experiments revealed significant fold differences in Hnf-1α expression with above mentioned defined parameters. Therefore, 0.5 μg Hnf-1α plasmid into 2.5 × 105 mBM-MSCs with a pulse voltage of 1000 V, pulse width of 10 ms, and pulse number of 3, was optimized, which was not reported before. These parameters can be considered for transfection with cell viability of 65–96% from 24 h to 21 days and 60–70% transfection efficiency after 24 h. Hence, this optimized procedure with efficient transfection rates can be applied for further gene functions and differentiation studies in the liver, pancreas, kidney, intestine, and for other tissues in specialized niches.