piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells | Nature

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells | Nature
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Abstract
Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state
. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral
, lentiviral
, adenoviral
and plasmid
transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies.
piggyBac
(PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines
10
11
. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events
12
. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition
13
. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision
12
, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences
14
delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.
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Figure 1:
Cell lines generated by PB-mediated factor transposition are reprogrammed.
The alternative text for this image may have been generated using AI.
Figure 2:
Seamless factor removal from iPS cells using transposase-stimulated PB excision.
The alternative text for this image may have been generated using AI.
Figure 3:
Cell lines reprogrammed by PB-mediated factor transposition are pluripotent.
The alternative text for this image may have been generated using AI.
Figure 4:
Properties of secondary fibroblast reprogramming.
The alternative text for this image may have been generated using AI.
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Acknowledgements
We thank J. Moffat for time-lapse image acquisition, P.-A. Pentilla for cell sorting, M.-S. Eiymo for assisting with initial PB test vector construction, J. Ure and M. Kownacka for technical assistance, M. Kibschull for establishing human embryonic fibroblasts, A. Cheung for discussions, and K. Vintersten for critical reading of the manuscript. This work was supported by the Wellcome Trust to P.L., and grants awarded to A.N. from the Canadian Stem Cell Network and JDRF.
Author Contributions
K.W. designed the experiments, cloned the transposon vectors, isolated and transfected fibroblasts, cultured mouse PB-iPS lines, performed alkaline phosphatase, LacZ and immunostaining, FACS analysis, dissected embryos, prepared DNA and performed Southern blotting, collected, analysed and interpreted data, and wrote the manuscript. I.P.M. designed experiments and assisted with initial cloning. P.M. and R.D. isolated fibroblasts, and assisted with cell culture, immunostaining and embryo dissections. M.M. transfected human fibroblasts, cultured human PB-iPS lines, performed alkaline phosphatase staining, immunostaining and differentiation assays. R.H. and K.W. performed the single transposon reprogramming studies and the removal of factors from iPS cells. R.C. carried out RT–PCR reactions. W.W. and P.L. provided the
PB-PGK-neo-bpA
and pCyL43 transposase plasmids, and guidelines for their use. M.G. generated and coordinated the iPSC chimaera production. K.K. performed immunostaining on induced secondary fibroblasts. H.-K.S. and I.P.M. performed teratoma assays and collected microscopy images. A.N. was responsible for the project concept, supervised the experiment design and data interpretation, and wrote the manuscript. All authors were involved in initial project design, discussed the results and contributed to the manuscript preparation.
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Authors and Affiliations
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada ,
Knut Woltjen, Iacovos P. Michael, Paria Mohseni, Ridham Desai, Maria Mileikovsky, Riikka Hämäläinen, Rebecca Cowling, Marina Gertsenstein, Hoon-Ki Sung & Andras Nagy
Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada
Iacovos P. Michael, Paria Mohseni, Ridham Desai & Andras Nagy
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK ,
Wei Wang & Pentao Liu
MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK
Keisuke Kaji
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Supplementary information
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This file contains Supplementary Figures S1-S11 with Legends, a Supplementary Reference and Supplementary Tables 1-2 (PDF 2962 kb)
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Woltjen, K., Michael, I., Mohseni, P.
et al.
piggyBac
transposition reprograms fibroblasts to induced pluripotent stem cells.
Nature
458
, 766–770 (2009). https://doi.org/10.1038/nature07863
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Received
06 November 2008
Accepted
12 February 2009
Published
01 March 2009
Issue date
09 April 2009
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Editorial Summary
Virus-free iPS cells
The discovery that non-germline adult cells can be reprogrammed to become pluripotent, able to differentiate into any cell type, opened up exciting possibilities. Reprogrammed cells — called induced pluripotent stem (iPS) cells — should have great potential in regenerative medicine, but most current methods of producing them involve viral gene delivery that could cause abnormalities in the induced cells. Two groups in this issue report on a collaboration that has succeeded in producing pluripotency in human cells without using viral vectors. Stable iPS cells were produced in both human and mouse fibroblasts using virus-derived 2A peptide sequences to create a multicistronic vector incorporating the reprogramming factors, delivered to the cell by the
piggyBac
transposon vector. The 2A-linked reprogramming factors, not required in the established iPS cell lines, were then removed.
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