Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent.
Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent.
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DOI：
10.3390/mps3030057
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发表时间：
2020-08-09
影响因子：
2.4
通讯作者：
Mosqueira D
中科院分区：
文献类型：
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作者：
Bodbin SE;Denning C;Mosqueira D
自首次获得人类多能干细胞衍生的心肌细胞（hPSC - CM）以来的20年里，它们在疾病建模研究中显示出了应用前景，同时其用于心脏修复的潜力也正在研究中。然而，低转染效率阻碍了这一模型系统潜力的更广泛实现。我们致力于制定一种使用普洛麦格（Promega）公司的ViafectTM试剂来提高hPSC - CM转染效率的实验方案。通过对四个关键参数的优化：（i）血清补充，（ii）重新接种和转染之间的时间，（iii）试剂与DNA的比例以及（iv）细胞密度，我们能够成功地将hPSC - CM的转染效率提高到约95%。转染后的hPSC - CM尽管存活率略有下降，但仍保持了较高的纯度和结构完整性，并且与肥大的表型分析实验保持兼容性。该实验方案以一种简单快速的方法克服了hPSC - CM转染效率有限的问题，确保转染基因至少持续表达14天，从而为心脏相关疾病的机制研究开辟了新的机遇，极大地增加了该领域的价值。
Twenty years since their first derivation, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown promise in disease modelling research, while their potential for cardiac repair is being investigated. However, low transfection efficiency is a barrier to wider realisation of the potential this model system has to offer. We endeavoured to produce a protocol for improved transfection of hPSC-CMs using the ViafectTM reagent by Promega. Through optimisation of four essential parameters: (i) serum supplementation, (ii) time between replating and transfection, (iii) reagent to DNA ratio and (iv) cell density, we were able to successfully transfect hPSC-CMs to ~95% efficiencies. Transfected hPSC-CMs retained high purity and structural integrity despite a mild reduction in viability, and preserved compatibility with phenotyping assays of hypertrophy. This protocol greatly adds value to the field by overcoming limited transfection efficiencies of hPSC-CMs in a simple and quick approach that ensures sustained expression of transfected genes for at least 14 days, opening new opportunities in mechanistic discovery for cardiac-related diseases.