Abstract

Transcription factor (TF) networks are thought to regulate embryonic stem cell (ESC) pluripotency. However, TF expression dynamics and regulatory mechanisms are poorly understood. We use reporter mouse ESC lines allowing non-invasive quantification of Nanog or Oct4 protein levels and continuous long-term single-cell tracking and quantification over many generations to reveal diverse TF protein expression dynamics. For cells with low Nanog expression, we identified two distinct colony types: one re-expressed Nanog in a mosaic pattern, and the other did not re-express Nanog over many generations. Although both expressed pluripotency markers, they exhibited differences in their TF protein correlation networks and differentiation propensities. Sister cell analysis revealed that differences in Nanog levels are not necessarily accompanied by differences in the expression of other pluripotency factors. Thus, regulatory interactions of pluripotency TFs are less stringently implemented in individual self-renewing ESCs than assumed at present.

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Acknowledgements

We thank C. Raithel, A. Ziegler, S. Ammersdörfer and B. Vogel for technical support, M. Strasser and F. Buggenthin for helpful discussions and T. Hilger for statistical advice. This work was supported by the German Federal Ministry of Education and Research (BMBF), the European Research Council starting grant (Latent Causes), the BioSysNet (Bavarian Research Network for Molecular Biosystems), the International Human Frontier Science Program Organization, and by the German Research Foundation (DFG) within the SPPs 1395 and 1356. S.S. and O.H. acknowledge financial support for this project from SystemsX.ch.

Author information

Author notes

    • Adam Filipczyk
    • , Carsten Marr
    •  & Simon Hastreiter

    These authors contributed equally to this work.

Affiliations

  1. Research Unit Stem Cell Dynamics, Helmholtz Zentrum München—German Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany

    • Adam Filipczyk
    • , Simon Hastreiter
    • , Philipp S. Hoppe
    • , Dirk Loeffler
    • , Konstantinos D. Kokkaliaris
    • , Max Endele
    • , Bernhard Schauberger
    • , Oliver Hilsenbeck
    • , Stavroula Skylaki
    •  & Timm Schroeder
  2. Institute of Computational Biology, Helmholtz Zentrum München—German Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany

    • Carsten Marr
    • , Justin Feigelman
    • , Michael Schwarzfischer
    • , Bernhard Schauberger
    • , Oliver Hilsenbeck
    • , Jan Hasenauer
    •  & Fabian J. Theis
  3. Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland

    • Simon Hastreiter
    • , Philipp S. Hoppe
    • , Dirk Loeffler
    • , Konstantinos D. Kokkaliaris
    • , Max Endele
    • , Oliver Hilsenbeck
    • , Stavroula Skylaki
    •  & Timm Schroeder
  4. Technische Universität München, Center for Mathematics, Chair of Mathematical Modelling of Biological Systems, Boltzmannstraße 3, 85748 Garching, Germany

    • Jan Hasenauer
    •  & Fabian J. Theis
  5. Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany

    • Konstantinos Anastassiadis

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Contributions

A.F. and S.H. performed experiments and analysed data with C.M., J.F. and M.S. M.S. developed QTFy and performed protein number estimation with J.H. J.F. performed dynamical modelling and parameter estimation with J.H. P.S.H. established quantitative TF imaging and maintained and advised on FACS procedures with M.E. K.A. produced reporter ESC lines. F.J.T. designed and supervised data analysis, modelling and QTFy software development, and commented on the manuscript. T.S. designed and supervised the study, developed and maintained long-term bioimaging with D.L. and K.D.K., and single-cell tracking and analysis software with B.S., O.H. and S.S., and wrote the manuscript with A.F., C.M. and S.H.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Fabian J. Theis or Timm Schroeder.

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https://doi.org/10.1038/ncb3237

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