Generating Human Induced Pluripotent Stem Cell Via Low-Dose Polyethylenimine-Mediated Transfection: An Optimized Protocol Publisher Pubmed



Shayestehfar M¹ ; Farahi S² ; Kheiri Yeganeh Azar B³ ; Memari A⁴ ; Baluchnejadmojarad T⁵ ; Faghihi F⁶ Show Affiliations
Source: DNA and Cell Biology Published:2022

Abstract

Human dermal fibroblasts (HDFs) can be reprogrammed through different strategies to generate human induced pluripotent stem cells (hiPSCs). However, most of these strategies require high-cost materials and specific equipment not readily accessible in most laboratories. Hence, liposomal and virus-based techniques can replace with polyethylenimine (PEI)-mediated transfection to overcome these challenges. However, few researchers have addressed the PEI's ability to transfect HDFs. This study used PEI reagent to transfer oriP/EBNA1-based vector into HDFs to produce hiPSC lines. We first described conditions allowing the efficient transfection of HDFs with low cytotoxicity and without specific types of equipment and optimized several parameters relevant to the transfection procedure. We then monitored the effect of different N/P ratios on transfection efficiency and cytotoxicity using flow cytometry and fluorescent microscopy. By the results, we found that transfection efficiency was greatly affected by plasmid DNA concentration, PEI concentration, order of combining reagents, serum presence in polyplexes, and the duration of serum starvations. Moreover, using the optimized condition, we found that the N/P ratio of 3 achieved the highest percentage of HDFs positive for green fluorescent protein plasmid (∼40%) with minimal cell toxicity. We finally generated hiPSCs using the optimized protocol and oriP/EBNA1-based vectors. We confirmed hiPSC formation by characterizing tests: alkaline phosphatase staining, immunocytochemistry assay, real-time PCR analysis, in vitro differentiation into three germ layers, and karyotyping test. In conclusion, our results indicated that 25 kDa branched PEI could efficiently transfect HDFs toward generating hiPSCs via a simple, cost-effective, and optimized condition. © 2022, Mary Ann Liebert, Inc.