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Putting mammalian early embryonic cells into dormancy

Nature Protocols (2026)Cite this article

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Abstract

Mammalian development starts at fertilization and continually progresses until birth, except in cases in which an interruption is favorable to the embryo and the mother. Many mammals have the ability to pause development in case of suboptimal resources or routinely as part of their reproductive cycle—a phenomenon called ‘embryonic diapause’. Diapause can be mimicked in vivo in mice via surgical removal of the ovaries or hormone injections. This procedure is laborious and invasive, ruling out its use across species. We have developed in vitro protocols through which mouse blastocysts, human blastoids and pluripotent stem cells from both species can be induced to enter a diapause-like dormant state via pharmacological inhibition of mTOR. Here, we describe in detail how embryos, blastoids and stem cells can be transitioned into and out of dormancy under different culture conditions. We further explain critical parameters to ensure success and propose experimental readouts. These in vitro embryonic dormancy setups can be used to uncover molecular mechanisms of dormancy, to test environmental or pharmacological effectors and to further innovate culture systems for species in which in vitro reproductive technologies are limited. We anticipate that researchers with ~1 year of embryo- and stem cell-handling experience should be able to achieve consistent results and evaluate outcomes. Altogether, inducing dormancy in vitro offers the possibility to slow down embryonic development for exploratory investigations of molecular mechanisms and eventually to expand the time window before implantation for clinical assays.

Key points

  • This set of in vitro protocols details the steps through which mouse and human pluripotent stem cells, mouse blastocysts and human blastoids can be reversibly induced to enter a diapause-like dormant state via pharmacological inhibition of mTOR.

  • Compared to traditional, surgery-based approaches, these protocols are simple and noninvasive and enable higher throughput analysis.

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Fig. 1: Morphology and cell type composition of proliferative and dormant human blastoids.
Fig. 2: Schematic overview of the protocol.
Fig. 3: Mouse and human ESCs in proliferative culture.
Fig. 4: Suboptimal hPSC dormancy cultures with differentiating colonies.
Fig. 5: Reprogramming of primed hPSCs in RSeT culture.
Fig. 6: Overview of mESC colonies in serum/LIF culture during mTORi-mediated dormancy transition.
Fig. 7: Overview of hPSC colonies in PXGL and RSeT culture during mTORi-mediated dormancy transition.
Fig. 8: Morphological evaluation of normal and diapaused mouse blastocysts.
Fig. 9: Overview of human blastoid morphology over 8 d of mTORi treatment.

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Data availability

All datasets can be found in the primary publications cited in the legends. Statistical source data for Fig. 6 are provided with this paper.

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Acknowledgements

D.P.I. was supported by the German Academic Exchange Service (DAAD) PhD Fellowship (91730547). H.H.K. was supported by the Austrian Science Fund (FWF), Lise Meitner Programme M3131-B and Marie Sklodowska-Curie grant agreement no. 101026451. The Rivron Lab is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-Co grant agreement no. 101002317 ‘BLASTOID: a discovery platform for early human embryogenesis’) and the Institute for Molecular Biotechnology (IMBA), Vienna. The Bulut-Karslioglu Lab is supported by the ERC (ERC-StG grant agreement no. 101117421, ‘DOR CODE’) and the Max Planck Society (to A.B.-K.). We thank members of the Bulut-Karslioglu and Rivron labs for discussions and especially Henrik Chrzanowski for images and cell counts.

Author information

Authors and Affiliations

  1. Max Planck Institute for Molecular Genetics, Berlin, Germany

    Dhanur P. Iyer & Aydan Bulut-Karslioglu

  2. Institute of Zoology, University of Innsbruck, Innsbruck, Austria

    Dhanur P. Iyer

  3. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria

    Heidar Heidari Khoei & Nicolas Rivron

Authors
  1. Dhanur P. Iyer
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  2. Heidar Heidari Khoei
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Contributions

D.P.I., H.H.K., N.R. and A.B.-K. conceived the study and designed the experiments. D.P.I. and H.H.K. performed the experiments. N.R. and A.B.-K. supervised the project. D.P.I., H.H.K., N.R. and A.B.-K. wrote and revised the manuscript.

Corresponding author

Correspondence to Aydan Bulut-Karslioglu.

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Competing interests

The Institute for Molecular Biotechnology, Austrian Academy of Sciences holds a patent application (EP21151455.9) describing the protocols for human blastoid formation and for the blastoid–endometrium interaction assay. H.H.K. and N.R. are the inventors on this patent. Dawn-Bio, a biotechnology company co-founded by N.R., has licensed the technologies described in this patent. The other authors declare no competing interests.

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Nature Protocols thanks Robert Froemke, Ann Sutherland and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Iyer, D. P. et al. Cell 187, 6566–6583.e22 (2024): https://doi.org/10.1016/j.cell.2024.08.048

van der Weijden et al. Nat. Cell Biol. 26, 181–193 (2024): https://doi.org/10.1038/s41556-023-01325-3

Bulut-Karslioglu, A. et al. Nature 540, 119–123 (2016): https://doi.org/10.1038/nature20578

Supplementary information

Source Data Fig. 6

Statistical source data

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Iyer, D.P., Heidari Khoei, H., Rivron, N. et al. Putting mammalian early embryonic cells into dormancy. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01303-z

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