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Virus-free induction of pluripotency and subsequent excision of reprogramming factors

Nature volume 458, pages 771–775 (2009)Cite this article

Abstract

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors1,2,3,4,5,6,7. However, such iPS cells contain a large number of viral vector integrations1,8, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming9,10, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia11,12. Two transient transfection-reprogramming methods have been published to address this issue13,14. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon15,16, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.

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Figure 1: Efficient reactivation of pluripotent markers in iPS cells generated by a non-viral multiprotein expression vector.
Figure 2: Non-viral iPS cells with a single-vector integration site.
Figure 3: Reprogramming cassette excision and pluripotency of the non-viral iPS cells.

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Acknowledgements

We thank A. Tsakiridis for advice on using the 2A peptide sequence and A. Nagy for providing data from his laboratory on generating human reprogrammed cell lines by combining the PB transposon system and the 2A-sequence-joined reprogramming factors (or MKOS single vector reprogramming system) as well as for his comments on the manuscript. We also thank I. Chambers for providing TNG MEFs and for discussion and comments on the manuscript, V. Wilson for advice on teratoma analysis, T. Kunath, S. Lowell, C. Blackburn and K. Vintersten for discussions and comments on the manuscript, and B. Hendrich for permission to start preliminary experiments of this work in his laboratory. We also thank J. Ure, L. Robertson, R. McLay and R. Wilkie for technical assistance, and Biomed unit staff for mouse husbandry. K.K. is the recipient of MRC career development fellowship in stem cell research. A.P. is the recipient of a BBSRC CASE PhD studentship. M.M., P.M. and K.W. were supported by grants from the Canadian Stem Cell Network and Juvenile Diabetes Research Foundation.

Author Contributions K.K. conceived the study, designed and executed the experiments, interpreted data and wrote the manuscript. K.N. performed immunoblotting and karyotype checking, and assisted with manuscript preparation. A.P. performed real-time PCR and in vitro differentiation experiments. M.M. generated human reprogrammed cells. P.M. performed immunostaining for human reprogrammed cells. K.W. constructed the PB/MKOS system, assisted human cell reprogramming experiments and analysis, and helped to prepare the manuscript.

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Authors and Affiliations

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

    Keisuke Kaji, Katherine Norrby & Agnieszka Paca

  2. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada ,

    Maria Mileikovsky, Paria Mohseni & Knut Woltjen

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

    Paria Mohseni

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  1. Keisuke Kaji
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  2. Katherine Norrby
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Correspondence to Keisuke Kaji.

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Kaji, K., Norrby, K., Paca, A. et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458, 771–775 (2009). https://doi.org/10.1038/nature07864

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