Letter | Published:

Inhibition of mTOR induces a paused pluripotent state

Nature volume 540, pages 119123 (01 December 2016) | Download Citation

Abstract

Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2–3 days around the time of blastocyst implantation1. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions2. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth3, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent—able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.

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Acknowledgements

We are grateful to K. Shokat for the kind gift of RapaLink-1. We thank R. Blelloch, B. Bruneau, M. Conti, S. Fisher, D. Ruggero, and members of the Santos Laboratory for critical reading of the manuscript. This research was supported by grants NIH 5P30CA082103 to the UCSF Center for Advanced Technology, NIH P30DK063720 to the UCSF Flow Cytometry Core, NSF 1442504 and NIH R01CA163336 to J.S.S., and NIH R01OD012204 and R01GM113014 to M.R.-S.

Author information

Affiliations

  1. Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Diabetes Center, University of California, San Francisco, San Francisco, California 94143, USA

    • Aydan Bulut-Karslioglu
    • , Steffen Biechele
    • , Trisha A. Macrae
    •  & Miguel Ramalho-Santos
  2. Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA

    • Hu Jin
    • , Miroslav Hejna
    •  & Jun S. Song
  3. Departments of Bioengineering and Physics, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA

    • Hu Jin
    • , Miroslav Hejna
    •  & Jun S. Song
  4. The Centre for Phenogenomics (TCP), Toronto, Ontario M5T 3H7, Canada

    • Marina Gertsenstein

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Contributions

A.B.-K., S.B. and M.R.-S. conceived of the project. A.B.-K. and S.B. isolated embryos. A.B.-K. performed most paused embryo and ES cell cultures, embryo stainings and quantifications, and ES cell experiments with the following exceptions: T.A.M. performed HPG, EU and cell cycle analyses on ES cells. S.B. generated diapaused embryos, performed all NSET embryo transfers, and parturitions when necessary. H.J., and M.H. analysed RNA-seq data under the supervision of J.S.S. M.G. performed parallel embryo culture for surgical transfers and ES cell culture for aggregations, supported by staff of TCP Model Production Core. M.R.-S. supervised the project. A.B.-K. and M.R.-S. wrote the manuscript with feedback from all authors.

Corresponding author

Correspondence to Miguel Ramalho-Santos.

Reviewer Information

Nature thanks K. Kaji, T. Rodríguez and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the uncropped western blot images with size marker indications.

Excel files

  1. 1.

    Supplementary Table 1

    This file shows the survival data for Extended Data Figure 1c.

  2. 2.

    Supplementary Table 2

    This file shows the Gene expression values from RNA-seq of serum, 2i and paused ES cells (this study), Boroviak et al. samples and Scognamiglio et al. samples.

  3. 3.

    Supplementary Table 3

    This file contains the Gene ontology analysis of genes upregulated in serum, 2i or paused ES cells.

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DOI

https://doi.org/10.1038/nature20578

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