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

Takenobu Nii, Hiroshi Kohara, Tomotoshi Marumoto, Tetsushi Sakuma, Takashi Yamamoto, Kenzaburo Tani

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)127-136
Number of pages10
JournalBioResearch Open Access
Volume5
Issue number1
DOIs
Publication statusPublished - May 2016

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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