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

Pub Date : 2024-06-04 DOI:10.3103/s0095452724030022

Sumreen Begum, Sehrish Jabeen, Syed Adibul Hasan Rizvi

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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.

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间充质干细胞中肝细胞核因子 1α（Hnf-1α）电穿孔的优化方案

摘要本研究旨在探讨电穿孔肝细胞核因子1α（Hnf-1α）在小鼠间充质干细胞（mBM-MSCs）中的优化应用。通过 Neon 电穿孔系统将 Hnf-1α 质粒 DNA 转染到 mBM-MSCs 中。细胞保存在完全DMEM培养基中。单次 0.5 μg Hnf-1α 电穿孔后，mBM-间充质干细胞在 24 小时、72 小时和 21 天后的存活率差异在统计学上不显著。对转染效率进行了涡轮绿色荧光蛋白（tGFP）荧光成像检测。在脉冲电压（V）为 1000、脉冲宽度（ms）为 10 和脉冲数为 3 的参数下，mBM-间充质干细胞在 24 h 时的转染效率为（***p 值为 0.001，66.5 ± 12.2%）。转染 0.5 μg Hnf-1α 的效率在 72 小时（40.2 ± 10.9%）和 21 天（31.7 ± 5%）时有所下降。250 μg/mL 硫酸 G418 用于筛选 Hnf-1α 转染阳性细胞。独立实验的 TaqMan-qRT-PCR 结果显示，Hnf-1α 的表达与上述规定参数有显著的倍数差异。因此，将 0.5 μg Hnf-1α 质粒转染到 2.5 × 105 mBM-间充质干细胞中，脉冲电压为 1000 V，脉冲宽度为 10 ms，脉冲数为 3，这是之前没有报道过的。因此，这种具有高效转染率的优化程序可用于肝脏、胰腺、肾脏、肠道以及其他特殊壁龛组织的进一步基因功能和分化研究。

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