Nii Takenobu, Kohara Hiroshi, Marumoto Tomotoshi, Sakuma Tetsushi, Yamamoto Takashi, Tani Kenzaburo
Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan .
Project Division of ALA Advanced Medical Research, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan .
Biores Open Access. 2016 May 1;5(1):127-36. doi: 10.1089/biores.2016.0009. eCollection 2016.
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.
高效的基因导入人多能干细胞（hPSCs）对再生医学和药物开发具有巨大的前景。在过去十年中，人们开发了各种将基因导入hPSCs的方法；然而，hPSCs形成紧密堆积的集落，使得基因导入变得困难。在本研究中，我们建立了一种hPSCs单细胞状态的稳定培养方法以降低细胞密度，并研究了基因转染效率以及随后的基因编辑效率。使用非脂质体转染试剂将编码增强型绿色荧光蛋白的质粒DNA或mRNA转染到以单细胞状态培养的hPSCs中。我们发现大多数细胞（DNA转染率>90%；mRNA转染率>99%）被成功转染，且未分化的PSC标志物表达或多能性未丧失。此外，我们展示了一种使用靶向腺瘤性息肉病大肠杆菌（APC）基因的转录激活样效应核酸酶（TALENs）的高效基因编辑方法。我们的新方法可能会改进hPSC基因导入技术，从而促进其在人类再生医学中的应用。
