Protocol

Human Embryonic Stem Cell Protocols

Volume 331 of the series Methods In Molecular Biology pp 347-374

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

  • Justin C. St. JohnAffiliated withThe Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical School, University of Birmingham
  • , Alexandra AmaralAffiliated withCenter for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra
  • , Emma BowlesAffiliated withCenter for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra
  • , João Facucho Oliveira
  • , Rhiannon LloydAffiliated withThe Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical School, University of Birmingham
  • , Mariana FreitasAffiliated withThe Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical School, University of Birmingham
  • , Heather L. GrayAffiliated withPittsburgh Development Center, Magee-Women’s Research Institute
  • , Christopher S. NavaraAffiliated withPittsburgh Development Center, Magee-Women’s Research Institute
  • , Gisela OliveiraAffiliated withThe Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical School, University of Birmingham
    • , Gerald P. SchattenAffiliated withPittsburgh Development Center, Magee-Women’s Research Institute, Departments of Obstetrics-Gynecology-Reproductive Sciences and Cell Biology-Physiology, University of Pittsburgh School of Medicine
    • , Emma SpikingsAffiliated withSchool of Medicine, The University of Birmingham
    • , João Ramalho-SantosAffiliated withCenter for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra

* Final gross prices may vary according to local VAT.

Get Access

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).

Key Words

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