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piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells

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.

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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.

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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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Correspondence to Andras Nagy.

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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)

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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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