• A Corrigendum to this article was published on 01 April 2015

Abstract

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

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Gene Expression Omnibus

Data deposits

Chromatin immunoprecipitation data are available at the National Center for Biotechnology Information Gene Expression Omnibus database under the series accession number GSE49766. Microarray data are available at the National Center for Biotechnology Information Gene Expression Omnibus database under the series accession number GSE45352.

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Acknowledgements

J.H.H. is supported by a generous gift from I. and P. Mantoux; and grants from the Leona M. and Harry B. Helmsley Charitable Trust, ERC (StG-281906) grant, BIRAX initiative, Israel Science Foundation (BIKURA, ICORE and Regular programs), ICRF, Fritz Thyssen Stiftung, The Benoziyo Endowment fund, Alon Scholar Program, and the Clore research prize. I.A is supported by the HFSP Career Development Award, an ISF-Bikura and the ERC (StG-309788). A.A.M. is supported by a Weizmann Dean fellowship. We thank N. Barkai and her group, K. Saha, B. Hendrich, J. Nichols and A. Surani, for reagents and advice. We thank the Weizmann Institute management for providing critical financial and infrastructural support.

Author information

Author notes

    • Yoach Rais
    • , Asaf Zviran
    •  & Shay Geula

    These authors contributed equally to this work.

Affiliations

  1. The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

    • Yoach Rais
    • , Asaf Zviran
    • , Shay Geula
    • , Ohad Gafni
    • , Elad Chomsky
    • , Sergey Viukov
    • , Abed AlFatah Mansour
    • , Inbal Caspi
    • , Vladislav Krupalnik
    • , Mirie Zerbib
    • , Itay Maza
    • , Nofar Mor
    • , Dror Baran
    • , Leehee Weinberger
    • , Noa Novershtern
    •  & Jacob H. Hanna
  2. The Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel

    • Diego A. Jaitin
    • , David Lara-Astiaso
    • , Ronnie Blecher-Gonen
    •  & Ido Amit
  3. The Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel

    • Zohar Shipony
    • , Zohar Mukamel
    •  & Amos Tanay
  4. The Department of Computer Science, Weizmann Institute of Science, Rehovot 76100, Israel

    • Zohar Shipony
    • , Zohar Mukamel
    •  & Amos Tanay
  5. The Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

    • Tzachi Hagai
  6. The Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 76100, Israel

    • Shlomit Gilad
    •  & Daniela Amann-Zalcenstein

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Contributions

Y.R., A.Z., S.Ge., N.N. and J.H.H. conceived the idea for this project, designed and conducted experiments and wrote the manuscript. S.Ge. conducted protein biochemical analysis. A.Z. conducted numerical modelling analysis. O.G., L.W. and N.M. assisted in chromatin immunoprecipitation experiments. N.N. and A.Z. conducted bioinformatics analysis. Y.R. and A.Z. conducted live imaging experiments and analysis. S.V. engineered human stem cell lines. I.A., D.A.J., D.L.-A., S.Gi., D.A.-Z. and R.B.-G. assisted with ChIP-seq experiments. E.C., Z.S., Z.M. and A.T. conducted RRBS analysis. Y.R. and M.Z. conducted microinjections. Y.R. and A.A.M. conduced embryo staining. Y.R., S.Ge. and J.H.H. conducted reprogramming experiments with help from I.C., I.M., V.K., T.H. and D.B.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Noa Novershtern or Jacob H. Hanna.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains supplementary discussions, a supplementary description for numerical modelling analysis and supplementary references.

Excel files

  1. 1.

    Supplementary Dataset 1

    This Supplementary Table shows Mbd3 localization following ChIP-Seq analysis. (i) Mbd3 bound regions in MEF cells and (ii) Mbd3 bound regions in MEF+OSKM samples, measured with ChIP-Seq and estimated with MACS software. Data appear in “bed” file format. (iii) Mbd3 bound target genes in MEF and (iv) Mbd3 bound target genes in MEF+OSKM cells. List generated by mapping of Mbd3 MACS peaks to an interval of 1Kb around the Transcription Start Sites of all mouse genes (Taken from USCS RefSeq known gene list).

Videos

  1. 1.

    Highly enhanced ES-like colony formation by OSKM upon Mbd3 depletion

    Live imaging of reprogramming in equivalent regions (5*6 mosaic) and phase contrast, after plating 150 cells per well. Video was prepared from time-lapse measurements taken every 8 hours for 6.5 days at 50X magnification (5X objective lens), time from DOX induction is indicated in the upper title. Note the accelerated ES-like colony formation in Mbd3flox/- cells in comparison to Mbd3+/+ donor MEFs. (n=4 independent experiments).

  2. 2.

    Time-lapse microscopic imaging of deterministic reprogramming

    Live imaging of Mbd3flox/- and control Mbd3+/+ full well mosaics with fluorescent mCherry and Oct4-GFP markers. Measurements were taken every 12 hours for 6 days at 50X magnification. In house automated segmentation protocol was run over time-lapse data tracking Oct4-GFP activation dynamic. Right upper image show Mbd3+/+ and left upper image show Mbd3flox/- full well mosaics. Time from DOX induction is given in the upper title. Lower left graph indicates cumulative Oct4-GFP+ colonies for Mbd3flox/- (red graph) and Mbd3+/+ (blue graph). Lower right graph indicates the average fraction of Oct4-GFP+ cells within single colonies. (n=4 independent experiments).

  3. 3.

    Time-lapse imaging of reprogramming dynamics of Mbd3flox/- cells with single colony view

    Live imaging of Mbd3flox/- full well mosaics with fluorescent mCherry and Oct4-GFP markers. Measurements were taken every 12 hours for 6 days at 50X magnification. For each time point full well mosaic (upper image) and up to 40 representative single colony images (two lower images) are shown. The two white rectangles on the full well mosaic represent the bounding box of the two single colonies that are shown in the lower images. In addition, lower left graph indicates cumulative Oct4-GFP+ colonies and lower right graph indicates the average fraction of Oct4-GFP+ cells within single colonies for Mbd3flox/-. (n=4 independent experiments).

  4. 4.

    Time-lapse imaging of reprogramming dynamics of Mbd3+/+ cells with single colony view

    As in supplementary Video 3, live imaging of Mbd3+/+ full well mosaics with fluorescent mCherry and Oct4-GFP markers, measurements were taken every 12h for 6 days at 50X magnification. (n=4 independent experiments).

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DOI

https://doi.org/10.1038/nature12587

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