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Biores Open Access. 2016 May 1;5(1):127-36. doi: 10.1089/biores.2016.0009. eCollection 2016.

Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.

Author information

1
Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan .
2
Project Division of ALA Advanced Medical Research, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan .
3
Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.; Department of Advanced Molecular and Cell Therapy, Kyushu University Hospital, Fukuoka, Japan.
4
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University , Hiroshima, Japan .
5
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.; Department of Advanced Molecular and Cell Therapy, Kyushu University Hospital, Fukuoka, Japan.

Abstract

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.

KEYWORDS:

gene expression; gene transfer; stem cells

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