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

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...

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Detalles Bibliográficos
Autores principales: Nii, Takenobu, Kohara, Hiroshi, Marumoto, Tomotoshi, Sakuma, Tetsushi, Yamamoto, Takashi, Tani, Kenzaburo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mary Ann Liebert, Inc. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876534/
https://www.ncbi.nlm.nih.gov/pubmed/27257519
http://dx.doi.org/10.1089/biores.2016.0009
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author Nii, Takenobu
Kohara, Hiroshi
Marumoto, Tomotoshi
Sakuma, Tetsushi
Yamamoto, Takashi
Tani, Kenzaburo
author_facet Nii, Takenobu
Kohara, Hiroshi
Marumoto, Tomotoshi
Sakuma, Tetsushi
Yamamoto, Takashi
Tani, Kenzaburo
author_sort Nii, Takenobu
collection PubMed
description 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.
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spelling pubmed-48765342016-06-02 Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency Nii, Takenobu Kohara, Hiroshi Marumoto, Tomotoshi Sakuma, Tetsushi Yamamoto, Takashi Tani, Kenzaburo Biores Open Access Original Research Article 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. Mary Ann Liebert, Inc. 2016-05-01 /pmc/articles/PMC4876534/ /pubmed/27257519 http://dx.doi.org/10.1089/biores.2016.0009 Text en © Takenobu Nii et al. 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.
spellingShingle Original Research Article
Nii, Takenobu
Kohara, Hiroshi
Marumoto, Tomotoshi
Sakuma, Tetsushi
Yamamoto, Takashi
Tani, Kenzaburo
Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
title Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
title_full Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
title_fullStr Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
title_full_unstemmed Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
title_short Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency
title_sort single-cell-state culture of human pluripotent stem cells increases transfection efficiency
topic Original Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876534/
https://www.ncbi.nlm.nih.gov/pubmed/27257519
http://dx.doi.org/10.1089/biores.2016.0009
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