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

Abstract

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.

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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.

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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.

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Acknowledgements

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.

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Authors

Contributions

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.

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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

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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). https://doi.org/10.1038/nature09017

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