Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.
Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.
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DOI：
10.1089/biores.2016.0009
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发表时间：
2016
影响因子：
--
通讯作者：
Tani K
中科院分区：
文献类型：
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作者：
Nii T;Kohara H;Marumoto T;Sakuma T;Yamamoto T;Tani K
高效的基因转移到人类多能干细胞（hPSC）中为再生医学和药物开发带来了巨大的希望。在过去的十年中，开发了各种方法用于将基因转移到hPSC中;然而，hPSC形成紧密堆积的集落，使得基因转移困难。在这项研究中，我们建立了一种稳定的培养方法，在单细胞状态下的hPSC，以减少细胞密度和研究基因转染效率，然后基因编辑效率。使用具有编码增强型绿色荧光蛋白的质粒DNA或mRNA的非脂质体转染试剂转染以单细胞状态培养的hPSC。我们发现大多数细胞（DNA > 90%; mRNA > 99%）被转染而没有未分化PSC标志物表达或多能性的损失。此外，我们证明了一种有效的基因编辑方法，使用靶向腺瘤性结肠息肉病（APC）基因的转录激活因子样效应核酸酶（TALEN）。我们的新方法可以改善hPSC基因转移技术，从而促进其用于人类再生医学。
Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell state were transfected using nonliposomal transfection reagents with plasmid DNA or mRNA encoding enhanced green fluorescent protein. We found that most cells (DNA > 90%; mRNA > 99%) were transfected without the loss of undifferentiated PSC marker expression or pluripotency. Moreover, we demonstrated an efficient gene editing method using transcription activator-like effector nucleases (TALENs) targeting the adenomatous polyposis coli (APC) gene. Our new method may improve hPSC gene transfer techniques, thus facilitating their use for human regenerative medicine.