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

Journal name:
Nature
Volume:
465,
Pages:
175–181
Date published:
DOI:
doi:10.1038/nature09017
Received
Accepted
Published online

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.

At a glance

Figures

  1. Aberrant silencing of the Dlk1-Dio3 gene cluster in mouse iPSCs.
    Figure 1: Aberrant silencing of the Dlk1–Dio3 gene cluster in mouse iPSCs.

    a, Strategy for comparing genetically matched ES cells and iPSCs generated with the doxycycline-controllable collagen-OKSM system. b, Morphology of collagen-OKSM ES cells and iPSCs. c, Unsupervised clustering of four ES cell and six derivative iPSC lines based on microarray expression data. d, Scatter plot of microarray data comparing iPSCs and ES cells with differentially expressed genes highlighted in green (twofold, P<0.05, t-test with Benjamini–Hochberg correction). e, Heat map showing relative expression levels of selected mRNAs in ES cells and iPSCs. f, Schematic representation of the Dlk1–Dio3 gene cluster with maternally and paternally expressed transcripts shown in red and blue, respectively. g, Heat map showing miRNAs that are differentially expressed between ES cells and iPSCs (twofold, P<0.01, t-test).

  2. Developmental consequences of Dlk1-Dio3 silencing.
    Figure 2: Developmental consequences of Dlk1–Dio3 silencing.

    a, Heat map showing relative expression levels of Gtl2, Rian and other select genes in ES cells and iPSCs derived from haematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP), granulocytes (Gran), peritoneal fibroblasts (PF) and tail-tip fibroblasts (TTF). Four iPSC clones expressing ES-cell-like levels of Gtl2 and Rian were identified (highlighted by asterisks) (iPSC clone number 18 was analysed only by qPCR; see Supplementary Fig. 1b). b, Strategy for assessing the developmental potential of iPSC clones by injection into diploid (2n) and tetraploid (4n) blastocysts to produce chimaeric or all-iPSC mice, respectively. c, Images of representative chimaeras with agouti coat colour indicating iPSC origin. d, Quantification of coat colour chimaerism in mice derived from indicated Gtl2off clones (green diamonds), Gtl2on iPSC clones (red diamonds) and ES cells (open diamonds). e, Statistical analysis of coat colour chimaerism in mice derived form Gtl2off and Gtl2on iPSC clones. Error bars indicate standard deviations, n = 38 for Gtl2off clones and n = 11 for Gtl2on clones, P<0.001. f, Images of two GFP+ all-iPSC neonates (left) and two agouti all-iPSC mice (right). g, Scatter plot showing intensity levels of all probe sets covered by microarray analysis; highlighted in green are those probe sets that were significantly different between 4n complementation-competent iPSCs (clones 19, 44, 47 and 49) and non-4n complementation-competent iPSCs (clones 18, 20, 45 and 48) (twofold, P<0.05, t-test with Benjamini–Hochberg correction).

  3. Epigenetic silencing of the Gtl2 locus in iPSCs.
    Figure 3: Epigenetic silencing of the Gtl2 locus in iPSCs.

    a, Structure of the Dlk1–Dio3 locus with the position of the genomic regions (I–VII) analysed by pyrosequencing indicated by black bars. b, Degree of DNA methylation at IG-DMR and Gtl2 DMR in three Gtl2off iPSC clones (green bars), three Gtl2on iPSC clones (red bars), three ES cell clones (red open bars), as well as the parental tail-tip fibroblasts (TTF, grey bars). Analysis of the other regions is shown in Supplementary Fig. 5. c, Prevalence of activation-associated (acH3, acH4 and H3K4me) and repression-associated (H3K27me) chromatin marks at the Gtl2 promoter in two Gtl2off iPSC clones, two Gtl2on iPSC clones and ES cells. d, Gtl2 expression levels as measured by qPCR in subclones derived from Gtl2off clone 45 and Gtl2on clone 49 in the absence (-) or presence (+) of doxycycline (Dox). e, Bright-field images of iPSC culture in the absence or presence of all-trans retinoic acid (RA). f, Expression levels of Gtl2, other imprinted genes (Igf2, Igf2r), and the pluripotency marker Pou5f1 in cells cultured with (+) or without (-) RA. All error bars indicate standard deviations with n = number of CpGs within the corresponding region in b and n = 3 in c and f.

  4. Developmental defects in embryos derived from Gtl2off iPSCs.
    Figure 4: Developmental defects in embryos derived from Gtl2off iPSCs.

    a, Images of ‘all-iPSC’ E11.5 embryos obtained with Gtl2on clone 47 and Gtl2off clone 48, both of which express EGFP ubiquitously from the ROSA26 locus. b, Frequency of dead and live E11.5 all-iPSC embryos obtained with two Gtl2on (red bars) and two Gtl2off (green bars) iPSC clones upon 4n blastocyst injection. Number of blastocysts transferred per clone is indicated in brackets. c, Expression of Gtl2, Rian, Mirg and the paternally expressed gene Dlk1 in Gtl2off MEFs relative to Gtl2on MEFs (upper panel) as well as in Gtl2mKO MEFs relative to MEFs isolated from wild-type embryos (lower panel). d, In situ hybridization for Gtl2 mRNA in MEFs derived from all-iPSC embryos generated with either Gtl2on clone 44 or Gtl2off clone 48. e, Expression levels of Gtl2, Rian, Mirg and Dlk1 in the indicated tissues isolated from all-iPSC embryos produced with Gtl2off iPSCs relative to the levels seen in tissues derived from Gtl2on iPSCs. f, Degree of DNA methylation at the indicated Dlk1–Dio3 regions in Gtl2off, Gtl2on, Gtl2mKO and wild-type MEFs. g, Gtl2 expression levels in iPSC lines derived by subcloning Gtl2off clone 45 in the presence of valproic acid (VPA). h, Images of a neonatal stillborn pup (left) and a uterus filled with resorptions (right) derived after 4n blastocyst injections with either VPA-10 or the parental iPSC clone 45, respectively. All error bars indicate standard deviations with n = 3 in c, n = 5 in e and n = number of CpGs in f.

Accession codes

Primary accessions

Gene Expression Omnibus

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Author information

  1. These authors contributed equally to this work.

    • Matthias Stadtfeld &
    • Effie Apostolou

Affiliations

  1. Howard Hughes Medical Institute at Massachusetts General Hospital, Center for Regenerative Medicine; Harvard Stem Cell Institute, 185 Cambridge Street, Boston, Massachusetts 02114, USA

    • Matthias Stadtfeld,
    • Effie Apostolou,
    • Patricia Follett &
    • Konrad Hochedlinger
  2. Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA

    • Matthias Stadtfeld,
    • Effie Apostolou,
    • Toshi Shioda &
    • Konrad Hochedlinger
  3. Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 42 Church Street, Cambridge, Massachusetts 02138, USA

    • Matthias Stadtfeld,
    • Effie Apostolou &
    • Konrad Hochedlinger
  4. Department of Reproductive Biology, National Institute for Child Health and Development, Tokyo 157-8535, Japan

    • Hidenori Akutsu
  5. Department of BioScience, Tokyo University of Agriculture, Tokyo 156-8502, Japan

    • Atsushi Fukuda &
    • Tomohiro Kono
  6. Sanofi-Aventis, 270 Albany Street, Cambridge, Massachusetts 02139, USA

    • Sridaran Natesan

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.

Competing financial interests

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

Corresponding author

Correspondence to:

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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Supplementary information

PDF files

  1. Supplementary Information (4.9M)

    This file contains Supplementary Figures 1-10 with legends and Supplementary Tables 1, 4, 6 and 7.

Excel files

  1. Supplementary Table 2 (41K)

    This table shows expression levels of imprinted genes in ES cells and iPSCs.

  2. Supplementary Table 3 (224K)

    This table shows the global miRNA expression in ES cells and iPSCs.

  3. Supplementary Table 5 (188K)

    This table shows the global miRNA expression of 4n complementation-competent and non-competent iPSCs.

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

Additional data