Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all–iPS cell mice from terminally differentiated B cells

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  • A Corrigendum to this article was published on 27 June 2012

Abstract

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor–mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs.

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Figure 1: Meg3 hypermethylation occurs late during reprogramming and requires Dnmt3a.
Figure 2: Ascorbic acid treatment prevents Meg3 silencing.
Figure 3: Epigenetic changes induced by ascorbic acid during reprogramming.
Figure 4: Molecular characterization of B-iPSCs generated in the presence of ascorbic acid.
Figure 5: Functional characterization of B-iPSC clones generated in the presence of ascorbic acid.

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Change history

  • 18 March 2012

    In the version of this article initially published online, the names of the authors Steen S.K. Ooi and Timothy H. Bestor were given incorrectly as Steen Oi and Tim Bestor. Timothy Bestor was also incorrectly affiliated with the Howard Hughes Medical Institute at Massachusetts General Hospital. His correct affiliation is with the Department of Genetics and Development at the College of Physicians and Surgeons of Columbia University. These errors have been corrected for the print, PDF and HTML versions of this article.

  • 15 May 2012

    In the version of this article initially published, there were errors in the units shown on the x axis of Figure 2e. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We are grateful to B. Payer (Harvard University) for providing M. musculus × M. castaneus fibroblasts, D. Wesemann for advice on B-cell isolation and immunoglobulin rearrangements, A. Foudi for genomic DNA from B cells, H. Su for technical assistance and A. Riley for help with blastocyst injections. We thank past and present members of the Hochedlinger laboratory for suggestions. M.S. was supported by a postdoctoral fellowship from the Howard Hughes Medical Institute (HHMI), E.A. was supported by a Jane Coffin Childs postdoctoral fellowship, and support to K.H. was from the US National Institutes of Health (DP2OD003266 and R01HD058013).

Author information

M.S., E.A. and K.H. conceived the experiments, analyzed the data and wrote the manuscript. M.S. and E.A. performed reprogramming experiments, qPCR analyses and genomic DNA preparation and isolated intermediates. E.A. performed chromatin immunoprecipitation. F.F. and P.J.P. analyzed genome-wide histone modification data. T.S. analyzed gene expression data. J.C. and S.Y.K. performed tetraploid embryo complementation experiments. R.M.W. generated Southern blot data. T.C., S.S.K.O. and T.H.B. provided Dnmt3a- and Dnmt3l-null fibroblasts.

Correspondence to Konrad Hochedlinger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18 (PDF 9180 kb)

Supplementary Table 1

Differential H3K4me3 enrichment in B-iPSCs and ESCs (XLS 94 kb)

Supplementary Table 2

Coordinates of genomic regions analysed by pyrosequencing (XLS 38 kb)

Supplementary Table 3

Primer sequences used for SSLP and ChIP analyses (XLS 27 kb)

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Stadtfeld, M., Apostolou, E., Ferrari, F. et al. Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all–iPS cell mice from terminally differentiated B cells. Nat Genet 44, 398–405 (2012) doi:10.1038/ng.1110

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