Skip to main content

Thank you for visiting 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.

Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells


Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals (‘all-iPSC mice’). In contrast, iPSC clones with normal expression of the Dlk1Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Aberrant silencing of the Dlk1–Dio3 gene cluster in mouse iPSCs.
Figure 2: Developmental consequences of Dlk1–Dio3 silencing.
Figure 3: Epigenetic silencing of the Gtl2 locus in iPSCs.
Figure 4: Developmental defects in embryos derived from Gtl2 off iPSCs.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The mRNA profiling data discussed in this paper have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE20576.


  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)

    CAS  Article  Google Scholar 

  2. Hochedlinger, K. & Plath, K. Epigenetic reprogramming and induced pluripotency. Development 136, 509–523 (2009)

    CAS  Article  Google Scholar 

  3. Maherali, N. et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  5. Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  7. Boland, M. J. et al. Adult mice generated from induced pluripotent stem cells. Nature 461, 91–94 (2009)

    ADS  CAS  Article  Google Scholar 

  8. Zhao, X. Y. et al. iPS cells produce viable mice through tetraploid complementation. Nature 461, 86–90 (2009)

    ADS  CAS  Article  Google Scholar 

  9. Kang, L., Wang, J., Zhang, Y., Kou, Z. & Gao, S. iPS cells can support full-term development of tetraploid blastocyst-complemented embryos. Cell Stem Cell 5, 135–138 (2009)

    CAS  Article  Google Scholar 

  10. Nagy, A. et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development 110, 815–821 (1990)

    CAS  PubMed  Google Scholar 

  11. Eggan, K. et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl Acad. Sci. USA 98, 6209–6214 (2001)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  13. Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  15. Chin, M. H. et al. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell 5, 111–123 (2009)

    CAS  Article  Google Scholar 

  16. Marchetto, M. C. et al. Transcriptional signature and memory retention of human-induced pluripotent stem cells. PLoS One 4, e7076 (2009)

    ADS  Article  Google Scholar 

  17. Wilson, K. D. et al. MicroRNA profiling of human-induced pluripotent stem cells. Stem Cells Dev. 18, 749–758 (2009)

    CAS  Article  Google Scholar 

  18. Pick, M. et al. Clone- and gene-specific aberrations of parental imprinting in human induced pluripotent stem cells. Stem Cells 27, 2686–2690 (2009)

    CAS  Article  Google Scholar 

  19. Doi, A. et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nature Genet. 41, 1350–1353 (2009)

    CAS  Article  Google Scholar 

  20. Deng, J. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature Biotechnol. 27, 353–360 (2009)

    CAS  Article  Google Scholar 

  21. Brambrink, T., Hochedlinger, K., Bell, G. & Jaenisch, R. ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. Proc. Natl Acad. Sci. USA 103, 933–938 (2006)

    ADS  CAS  Article  Google Scholar 

  22. Wakayama, S. et al. Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells 24, 2023–2033 (2006)

    CAS  Article  Google Scholar 

  23. Soldner, F. et al. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977 (2009)

    CAS  Article  Google Scholar 

  24. Stadtfeld, M., Maherali, N., Borkent, M. & Hochedlinger, K. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nature Methods 7, 53–55 (2009)

    Article  Google Scholar 

  25. Sommer, C. A. et al. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27, 543–549 (2009)

    CAS  Article  Google Scholar 

  26. Beard, C., Hochedlinger, K., Plath, K., Wutz, A. & Jaenisch, R. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, 23–28 (2006)

    CAS  Article  Google Scholar 

  27. da Rocha, S. T., Edwards, C. A., Ito, M., Ogata, T. & Ferguson-Smith, A. C. Genomic imprinting at the mammalian Dlk1-Dio3 domain. Trends Genet. 24, 306–316 (2008)

    Article  Google Scholar 

  28. Humpherys, D. et al. Epigenetic instability in ES cells and cloned mice. Science 293, 95–97 (2001)

    CAS  Article  Google Scholar 

  29. Seitz, H. et al. Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene. Nature Genet. 34, 261–262 (2003)

    ADS  CAS  Article  Google Scholar 

  30. Seitz, H. et al. A large imprinted microRNA gene cluster at the mouse Dlk1-Gtl2 domain. Genome Res. 14, 1741–1748 (2004)

    CAS  Article  Google Scholar 

  31. Takahashi, N. et al. Deletion of Gtl2, imprinted non-coding RNA, with its differentially methylated region induces lethal parent-origin-dependent defects in mice. Hum. Mol. Genet. 18, 1879–1888 (2009)

    CAS  Article  Google Scholar 

  32. Lin, S. P. et al. Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region. Development 134, 417–426 (2007)

    CAS  Article  Google Scholar 

  33. Steshina, E. Y. et al. Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5′ region. BMC Genet. 7, 44 (2006)

    Article  Google Scholar 

  34. da Rocha, S. T. et al. Gene dosage effects of the imprinted delta-like homologue 1 (dlk1/pref1) in development: implications for the evolution of imprinting. PLoS Genet. 5, e1000392 (2009)

    Article  Google Scholar 

  35. Lin, S. P. et al. Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12. Nature Genet. 35, 97–102 (2003)

    CAS  Article  Google Scholar 

  36. Carr, M. S., Yevtodiyenko, A., Schmidt, C. L. & Schmidt, J. V. Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain. Genomics 89, 280–290 (2007)

    CAS  Article  Google Scholar 

  37. Dean, W. et al. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125, 2273–2282 (1998)

    CAS  PubMed  Google Scholar 

  38. Tevendale, M., Watkins, M., Rasberry, C., Cattanach, B. & Ferguson-Smith, A. C. Analysis of mouse conceptuses with uniparental duplication/deficiency for distal chromosome 12: comparison with chromosome 12 uniparental disomy and implications for genomic imprinting. Cytogenet. Genome Res. 113, 215–222 (2006)

    CAS  Article  Google Scholar 

  39. Schmidt, J. V., Matteson, P. G., Jones, B. K., Guan, X. J. & Tilghman, S. M. The Dlk1 and Gtl2 genes are linked and reciprocally imprinted. Genes Dev. 14, 1997–2002 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Eggan, K. et al. Mice cloned from olfactory sensory neurons. Nature 428, 44–49 (2004)

    ADS  CAS  Article  Google Scholar 

  41. Li, J., Ishii, T., Feinstein, P. & Mombaerts, P. Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 428, 393–399 (2004)

    ADS  CAS  Article  Google Scholar 

  42. Ono, Y. & Kono, T. Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells. Biol. Reprod. 75, 210–216 (2006)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  44. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009)

    ADS  CAS  Article  Google Scholar 

  45. Navarro, P. et al. Molecular coupling of Xist regulation and pluripotency. Science 321, 1693–1695 (2008)

    ADS  CAS  Article  Google Scholar 

  46. Donohoe, M. E., Silva, S. S., Pinter, S. F., Xu, N. & Lee, J. T. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting. Nature 460, 128–132 (2009)

    ADS  CAS  Article  Google Scholar 

  47. Eminli, S. et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nature Genet. 41, 968–976 (2009)

    CAS  Article  Google Scholar 

  48. Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005)

    CAS  Article  Google Scholar 

  49. Coser, K. R. et al. Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray. Proc. Natl Acad. Sci. USA 100, 13994–13999 (2003)

    ADS  CAS  Article  Google Scholar 

  50. Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863–14868 (1998)

    ADS  CAS  Article  Google Scholar 

Download references


We are grateful to H. Arnold for assistance with GeneSifter analysis; K. Coser, K. Claycomb and P. August for technical support on Affymetrix expression profiling; S. Sato and M. Machida for technical assistance; V. Greco for advice on keratinocyte isolation; and S. Schubert for advice on miRNA isolation. We thank members of the Hochedlinger laboratory for helpful suggestions and A. Umezawa for discussions and support. M.S. was supported by a postdoctoral fellowship from the Schering Foundation, E.A. was supported by a Jane Coffin Childs postdoctoral fellowship and K.H. was supported by a NIH Director’s Innovator Award and by funds provided by the Harvard Stem Cell Institute, MGH and HHMI.

Author information

Authors and Affiliations



Author Contributions M.S., E.A. and K.H. conceived the ideas for this study, designed and analysed experiments and wrote the manuscript. M.S. derived iPSC lines, conducted in vitro differentiation assays and performed expression array analysis. E.A. conducted qPCR analyses, in situ hybridizations and chromatin immunoprecipitations. H.A. and A.F. performed nuclear transfer experiments. P.F. did blastocyst injections. T.S. performed microarray experiments and analyses. S.N. and T.K. provided important study materials.

Corresponding author

Correspondence to Konrad Hochedlinger.

Ethics declarations

Competing interests

K.H. is on the advisory board of iPierian.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 with legends and Supplementary Tables 1, 4, 6 and 7. (PDF 5027 kb)

Supplementary Table 2

This table shows expression levels of imprinted genes in ES cells and iPSCs. (XLS 41 kb)

Supplementary Table 3

This table shows the global miRNA expression in ES cells and iPSCs. (XLS 230 kb)

Supplementary Table 5

This table shows the global miRNA expression of 4n complementation-competent and non-competent iPSCs. (XLS 189 kb)

Pease note that the descriptions for Tables 2, 3 and 5 were updated on 2 May 2010

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Stadtfeld, M., Apostolou, E., Akutsu, H. et al. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465, 175–181 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


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.


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