Abstract
Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon–based approach to generate integration-free iPSCs. Transposons carrying 2A peptide–linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs.
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Acknowledgements
We thank R. Banerjee for karyotype analysis, M. Li, C. Kokubu and K. Horie for help, suggestions and comments, and members of Team 82 and the Research Support Facility of the Wellcome Trust Sanger Institute for their support. K.Y. is funded by postdoctoral fellowship of Japan Society for the Promotion of Science. This work is supported by the Wellcome Trust (WT077187).
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K.Y. designed and performed experiments, analyzed data and wrote the paper. R.R. and J.T. performed experiments and assisted in writing the paper. A.B. designed experiments, interpreted and assisted in writing the paper.
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Yusa, K., Rad, R., Takeda, J. et al. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods 6, 363–369 (2009). https://doi.org/10.1038/nmeth.1323
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DOI: https://doi.org/10.1038/nmeth.1323
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