{"title":"Generating Human Induced Pluripotent Stem Cell Via Low-Dose Polyethylenimine-Mediated Transfection: An Optimized Protocol.","authors":"Monir Shayestehfar, Sara Farahi, Behjat Kheiri Yeganeh Azar, Amirhossein Memari, Tourandokht Baluchnejadmojarad, Faezeh Faghihi","doi":"10.1089/dna.2022.0331","DOIUrl":null,"url":null,"abstract":"<p><p>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, <i>in vitro</i> 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.</p>","PeriodicalId":2,"journal":{"name":"ACS Applied Bio Materials","volume":" ","pages":"903-916"},"PeriodicalIF":4.6000,"publicationDate":"2022-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Bio Materials","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1089/dna.2022.0331","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/8/18 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"MATERIALS SCIENCE, BIOMATERIALS","Score":null,"Total":0}
引用次数: 1
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.
期刊介绍:
ACS Applied Bio Materials is an interdisciplinary journal publishing original research covering all aspects of biomaterials and biointerfaces including and beyond the traditional biosensing, biomedical and therapeutic applications.
The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrates knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important bio applications. The journal is specifically interested in work that addresses the relationship between structure and function and assesses the stability and degradation of materials under relevant environmental and biological conditions.