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The Analysis of Mitochondria and Mitochondrial DNA in Human Embryonic Stem Cells

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Human Embryonic Stem Cell Protocols

Part of the book series: Methods In Molecular Biology ((MIMB,volume 331))

Abstract

As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated individual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell’s requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).

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Author information

Authors and Affiliations

  1. The Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical School, University of Birmingham, Birmingham, UK

    Justin C. St. John, Rhiannon Lloyd, Mariana Freitas & Gisela Oliveira

  2. Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal

    Alexandra Amaral, Emma Bowles & João Ramalho-Santos

  3. Pittsburgh Development Center, Magee-Women’s Research Institute, Pittsburgh, PA

    Heather L. Gray & Christopher S. Navara

  4. Pittsburgh Development Center, Magee-Women’s Research Institute, Departments of Obstetrics-Gynecology-Reproductive Sciences and Cell Biology-Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA

    Gerald P. Schatten

  5. School of Medicine, The University of Birmingham, Birmingham, UK

    Emma Spikings

Authors
  1. Justin C. St. John
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  2. Alexandra Amaral
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  3. Emma Bowles
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  4. João Facucho Oliveira
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  5. Rhiannon Lloyd
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  6. Mariana Freitas
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  7. Heather L. Gray
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  8. Christopher S. Navara
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  9. Gisela Oliveira
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  10. Gerald P. Schatten
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  11. Emma Spikings
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  12. João Ramalho-Santos
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Editor information

Editors and Affiliations

  1. Ottawa Health Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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© 2006 Humana Press Inc.

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St. John, J.C. et al. (2006). The Analysis of Mitochondria and Mitochondrial DNA in Human Embryonic Stem Cells. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods In Molecular Biology, vol 331. Humana Press. https://doi.org/10.1385/1-59745-046-4:347

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  • DOI: https://doi.org/10.1385/1-59745-046-4:347

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-497-5

  • Online ISBN: 978-1-59745-046-1

  • eBook Packages: Springer Protocols

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