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Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs)

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Mitochondrial Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2310))

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Abstract

Mouse embryonic stem cells (mESCs) can be grown in culture, recapitulating the different states of pluripotency of their in vivo counterparts, with notably different metabolic profiles. mESCs in a naïve pluripotent state present an ambivalent metabolism, using both glycolysis and oxidative phosphorylation as energy sources. Here, we describe a method to evaluate the oxidative function of naïve mESCs using the Seahorse Extracellular Flux Analyzer coupled to flow cytometry analysis of mitochondrial transmembrane potential using the TMRM fluorescence probe, thus assessing both oxygen consumption and mitochondrial membrane potential. This may be a useful protocol for understanding how mitochondrial oxidative function and potential of mESCs change in certain circumstances, and how is it related with various pluripotency/differentiation phenotypes.

Bibiana Correia and Maria Inês Sousa are the Co-first authors.

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Acknowledgments

The authors would like to acknowledge the members of the Biology of Reproduction and Stem Cells research group, at the Center from Neuroscience and Cell Biology, for the discussion and constructive feedback related to this work. We also thank Ana Sofia Rodrigues for optimizing the flow cytometry analysis of mitochondrial transmembrane potential using the TMRM probe.

Funding: This work was funded by Fundação para a Ciência e Tecnologia (FCT) Portugal for the Ph.D. scholarships attributed to M.I.S. (SFRH/BD/86260/2012) and B.S. (SFRH/BD/144150/2019). Funding was also provided by the STEM@REST Project (CENTRO-01-0145-FEDER-028871) and PAC CANCEL_STEM (POCI-01-0145-FEDER-016390. A.F.B. was hired through the STEM@REST Project (CENTRO-01-0145-FEDER-028871). Additional funding was provided by the European Regional Development Fund (ERDF) through the Centro 2020 Regional Operational Programme: project CENTRO-01-0145-FEDER-000012-HealthyAging2020, the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation, and the Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, I.P.: project POCI-01-0145-FEDER-007440 that attributed a fellowship to B. S. (BIM - IN0828).

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Authors and Affiliations

  1. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    Bibiana Correia, Maria Inês Sousa & João Ramalho-Santos

  2. CNC-Center for Neuroscience and Cell Biology, CIBB, Azinhaga de Santa Comba, Polo 3, University of Coimbra, Coimbra, Portugal

    Bibiana Correia, Maria Inês Sousa, Ana F. Branco & João Ramalho-Santos

Authors
  1. Bibiana Correia
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  2. Maria Inês Sousa
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  3. Ana F. Branco
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  4. João Ramalho-Santos
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Corresponding author

Correspondence to João Ramalho-Santos .

Editor information

Editors and Affiliations

  1. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    Carlos M. Palmeira

  2. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    Anabela P. Rolo

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Correia, B., Sousa, M.I., Branco, A.F., Ramalho-Santos, J. (2021). Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs). In: Palmeira, C.M., Rolo, A.P. (eds) Mitochondrial Regulation. Methods in Molecular Biology, vol 2310. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1433-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1433-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1432-7

  • Online ISBN: 978-1-0716-1433-4

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