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

  • Justin C. St. John
  • Alexandra Amaral
  • Emma Bowles
  • João Facucho Oliveira
  • Rhiannon Lloyd
  • Mariana Freitas
  • Heather L. Gray
  • Christopher S. Navara
  • Gisela Oliveira
  • Gerald P. Schatten
  • Emma Spikings
  • João Ramalho-Santos
Part of the Methods In Molecular Biology book series (MIMB, volume 331)


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).

Key Words

Mitochondria mitochondrial DNA human embryonic stem cells differentiation cardiomyocytes homoplasmy heteroplasmy transcription and replication 


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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Justin C. St. John
    • 1
  • Alexandra Amaral
    • 2
  • Emma Bowles
    • 2
  • João Facucho Oliveira
  • Rhiannon Lloyd
    • 1
  • Mariana Freitas
    • 1
  • Heather L. Gray
    • 3
  • Christopher S. Navara
    • 3
  • Gisela Oliveira
    • 1
  • Gerald P. Schatten
    • 4
  • Emma Spikings
    • 5
  • João Ramalho-Santos
    • 2
  1. 1.The Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical SchoolUniversity of BirminghamBirmingham
  2. 2.Center for Neuroscience and Cell Biology, Department of ZoologyUniversity of CoimbraCoimbra
  3. 3.Pittsburgh Development CenterMagee-Women’s Research InstitutePittsburgh
  4. 4.Pittsburgh Development Center, Magee-Women’s Research Institute, Departments of Obstetrics-Gynecology-Reproductive Sciences and Cell Biology-PhysiologyUniversity of Pittsburgh School of MedicinePittsburgh
  5. 5.School of MedicineThe University of BirminghamBirmingham

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