Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells

Nature volume 458, pages 766–770 (2009)Cite this article

Abstract

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state1,2,3,4. 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 retroviral1, lentiviral5, adenoviral6 and plasmid7 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 lines8,9,10,11. 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 events12. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition13. 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 excision12, 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 sequences14 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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cell lines generated by PB-mediated factor transposition are reprogrammed.
Figure 2: Seamless factor removal from iPS cells using transposase-stimulated PB excision.
Figure 3: Cell lines reprogrammed by PB-mediated factor transposition are pluripotent.
Figure 4: Properties of secondary fibroblast reprogramming.

Similar content being viewed by others

Article 01 December 2022

Zixuan Zhao, Xinyi Chen, … Hanry Yu

References

  1. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)

    Article  CAS  Google Scholar 

  2. Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55–70 (2007)

    Article  CAS  Google Scholar 

  3. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007)

    Article  ADS  CAS  Google Scholar 

  4. Meissner, A., Wernig, M. & Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nature Biotechnol. 25, 1177–1181 (2007)

    Article  CAS  Google Scholar 

  5. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008)

    Article  CAS  Google Scholar 

  6. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949 (2008)

    Article  ADS  CAS  Google Scholar 

  7. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Ding, S. et al. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122, 473–483 (2005)

    Article  CAS  Google Scholar 

  9. Wu, S. C. et al. piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells. Proc. Natl Acad. Sci. USA 103, 15008–15013 (2006)

    Article  ADS  CAS  Google Scholar 

  10. Cadinanos, J. & Bradley, A. Generation of an inducible and optimized piggyBac transposon system. Nucleic Acids Res. 35, e87 (2007)

    Article  Google Scholar 

  11. Wang, W. et al. Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc. Natl Acad. Sci. USA 105, 9290–9295 (2008)

    Article  ADS  CAS  Google Scholar 

  12. Fraser, M. J., Ciszczon, T., Elick, T. & Bauser, C. Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol. Biol. 5, 141–151 (1996)

    Article  CAS  Google Scholar 

  13. Cary, L. C. et al. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172, 156–169 (1989)

    Article  CAS  Google Scholar 

  14. Kaji, K. et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 10.1038/nature07864 (this issue)

  15. Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005)

    Article  CAS  Google Scholar 

  16. Agha-Mohammadi, S. et al. Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness. J. Gene Med. 6, 817–828 (2004)

    Article  CAS  Google Scholar 

  17. Belteki, G. et al. Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res. 33, e51 (2005)

    Article  Google Scholar 

  18. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl Acad. Sci. USA 90, 8424–8428 (1993)

    Article  ADS  CAS  Google Scholar 

  19. Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008)

    Article  CAS  Google Scholar 

  20. Kaji, K. et al. The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nature Cell Biol. 8, 285–292 (2006)

    Article  CAS  Google Scholar 

  21. Kim, J. B. et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454, 646–650 (2008)

    Article  ADS  CAS  Google Scholar 

  22. Mikkelsen, T. S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008)

    Article  ADS  CAS  Google Scholar 

  23. Fujiwara, Y. et al. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc. Natl Acad. Sci. USA 91, 12258–12262 (1994)

    Article  ADS  CAS  Google Scholar 

  24. Huangfu, D. et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature Biotechnol. 26, 1269–1275 (2008)

    Article  CAS  Google Scholar 

  25. Wernig, M. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nature Biotechnol. 26, 916–924 (2008)

    Article  CAS  Google Scholar 

  26. Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nature Protocols 2, 3081–3089 (2007)

    Article  CAS  Google Scholar 

  27. Dafa’alla, T. H. et al. Transposon-free insertions for insect genetic engineering. Nature Biotechnol. 24, 820–821 (2006)

    Article  Google Scholar 

  28. Wilson, M. H., Coates, C. J. & George, A. L. PiggyBac transposon-mediated gene transfer in human cells. Mol. Ther. 15, 139–145 (2007)

    Article  CAS  Google Scholar 

  29. Mitra, R., Fain-Thornton, J. & Craig, N. L. piggyBac can bypass DNA synthesis during cut and paste transposition. EMBO J. 27, 1097–1109 (2008)

    Article  CAS  Google Scholar 

  30. Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2003)

    Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. 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

  2. Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada

    Iacovos P. Michael, Paria Mohseni, Ridham Desai & Andras Nagy

  3. The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK ,

    Wei Wang & Pentao Liu

  4. MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK

    Keisuke Kaji

Authors
  1. Knut Woltjen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Iacovos P. Michael
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paria Mohseni
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ridham Desai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maria Mileikovsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Riikka Hämäläinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rebecca Cowling
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Pentao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Marina Gertsenstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Keisuke Kaji
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hoon-Ki Sung
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Andras Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andras Nagy.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S11 with Legends, a Supplementary Reference and Supplementary Tables 1-2 (PDF 2962 kb)

Supplementary Movie 1

This Movie shows 2o fibroblast with dox inducible reprogramming factors. (MOV 15149 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07863

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing