• A Corrigendum to this article was published on 17 June 2015

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Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

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  • 10 December 2014

    A minor addition was made to the Acknowledgements in the HTML and PDF versions.


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Gene Expression Omnibus

Data deposits

Microarray data have been deposited on Stemformatics ( and in the Gene Expression Omnibus database under accession number GSE49940.


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We are grateful for A. Bang’s expertise and assistance regarding flow cytometry. We thank M. Gertsenstein and M. Pereira for chimaera production, and K. Harpal for teratoma sectioning. We would also like to acknowledge the assistance and support of lab colleagues, collaborators and all the members of the Project Grandiose Consortium who are too numerous to name individually but who made a positive impact on this research. A.N. is Tier 1 Canada Research Chair in Stem Cells and Regeneration. This work was supported by grants awarded to A.N. and I.M.R. from the Ontario Research Fund Global Leadership Round in Genomics and Life Sciences grants (GL2), to A.N. from the Canadian stem cell network (9/5254 (TR3)) and Canadian Institutes of Health Research (CIHR MOP102575), to J.-S.S. by the South Korean Ministry of Knowledge Economy (no. 10037410), SNUCM research fund (grant no. 0411-20100074), and Macrogen Inc. (no. MGR03-11 and 12), to S.G. from the Australian Research Council (no. SR110001002), and to C.A.W. by a Queensland government Smart Futures Fellowship and an ARC by Stem Cells Australia and to T.P. grants from NHMRC and ARC. S.M.I.H. received a fellowship from the McEwen Centre of Regenerative Medicine.

Author information


  1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

    • Peter D. Tonge
    • , Andrew J. Corso
    • , Claudio Monetti
    • , Samer M. I. Hussein
    • , Mira C. Puri
    • , Iacovos P. Michael
    • , Mira Li
    • , Ian M. Rogers
    •  & Andras Nagy
  2. Institute of Medical Science, University of Toronto, Toronto, Ontario M5T 3H7, Canada

    • Andrew J. Corso
    •  & Andras Nagy
  3. Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5T 3H7, Canada

    • Mira C. Puri
  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5T 3H7, Canada

    • Iacovos P. Michael
  5. Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul 110-799, South Korea

    • Dong-Sung Lee
    • , Jong-Yeon Shin
    •  & Jeong-Sun Seo
  6. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799, South Korea

    • Dong-Sung Lee
    •  & Jeong-Sun Seo
  7. Department of Biochemistry, Seoul National University College of Medicine, Seoul 110-799, South Korea

    • Dong-Sung Lee
    •  & Jeong-Sun Seo
  8. Department of Systems & Computational Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA

    • Jessica C. Mar
  9. Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia

    • Nicole Cloonan
    • , David L. Wood
    • , Maely E. Gauthier
    •  & Sean M. Grimmond
  10. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia

    • Othmar Korn
    •  & Christine A. Wells
  11. Genome Biology Department, The John Curtin School of Medical Research, The Australian National University, Acton (Canberra), Australian Capital Territory 2601, Australia

    • Jennifer L. Clancy
    •  & Thomas Preiss
  12. Victor Chang Cardiac Research Institute, Darlinghurst (Sydney), New South Wales 2010, Australia

    • Thomas Preiss
  13. Life Science Institute, Macrogen Inc., Seoul 153-781, South Korea

    • Jong-Yeon Shin
    •  & Jeong-Sun Seo
  14. Department of Physiology, University of Toronto, Toronto, Ontario M5T 3H7, Canada

    • Ian M. Rogers
  15. Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario M5T 3H7, Canada

    • Ian M. Rogers
    •  & Andras Nagy
  16. QIMR Berghofer Medical Research Institute, Genomic Biology Lab, 300 Herston Road, Brisbane, Queensland 4006, Australia

    • Nicole Cloonan


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P.D.T. and A.N. conceived and designed the experiments, and wrote the manuscript. P.D.T. and A.J.C. derived all iPSC lines, performed real-time PCR analysis and bisulphite sequencing analysis of DNA methylation. P.D.T., C.M. and I.P.M. performed the in vivo characterization of the iPS lines (teratomas and chimaera formation). O.K., J.L.C. and T.P. assisted in data analysis. P.D.T., J.C.M. and C.A.W. performed microarray analysis. M.L., M.C.P., S.M.I.H. and I.M.R. performed pull-downs for genome-wide ChiP-seq. D.-S.L., J.-Y.S., and J.-S.S. performed genome-wide MethylC-seq and ChiP-seq. N.C., D.L.W., M.E.G. and S.M.G. performed RNA-seq.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andras Nagy.

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    Supplementary Information

    This file contains Supplementary Information sets 1-6. Supplementary Information 1 contains differentially expressed genes (two­tailed Welch t-test p<0.01, FDR<0.01) between 28 transgene-expressing reprogrammed lines and 3 ESC-like lines. Supplementary Information 2 contains differentially expressed genes (Welchs t-test p<0.01 FDR<0.05) between F-class and C-class cells. Supplementary Information 3 contains plurinet genes of statistically significant differential expression between F-class samples and ESC samples. Supplementary Information 4 contains differential gene expression upon inactivation of c-Myc expression in F-class cells. Supplementary Information 5 contains differential gene expression upon treatment of F-class cells with HDAC inhibitors. Supplementary Information 6 contains epigenetic status of loci that exhibited differential gene expression (>5 fold) between F-class state and ESC-like cell state. The status of three epigenetic marks are examined; H3K4 trimethylation (H3K4me3), H3K27 trimethylation (H3K27me3) and CpG methylation. Supplementary Information 7 contains primer sequences for quantitative RT-PCR analysis of gene expression.

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