Analysis of mitochondrial DNA in induced pluripotent and embryonic stem cells

William Lee, Richard David William Kelly, Ka Yu (Vanessa) Yeung, Gael Cagnone, Matthew McKenzie, Justin Charles St John

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

2 Citations (Scopus)

Abstract

The mitochondrial genome has a major role to play in establishing and maintaining pluripotency. Furthermore, mitochondrial DNA (mtDNA) copy is strictly regulated during differentiation. Undifferentiated, pluripotent cells possess fewer than 300 copies of mtDNA, which establishes the mtDNA set point and promotes cell proliferation and, as a result, these cells rely on glycolysis with some support from oxidative phosphorylation (OXPHOS) for the generation of ATP. The mtDNA set point provides the starting point from which cells increase their mtDNA copy number as they differentiate into mature functional cells. Dependent on cell types, mtDNA copy number ranges from 10 copies in sperm to several thousand in cardiomyocytes. Consequently, differentiating cell types can acquire the appropriate numbers of mtDNA copy to meet their specific requirements for ATP generated through OXPHOS. However, as reprogrammed somatic cells do not always achieve this, it is essential to analyze them for their OXPHOS potential and ability to regulate mtDNA copy number. Here, we describe how to assess mtDNA copy number in pluripotent and differentiating cells using real-time PCR protocols; assess expression of the mtDNA specific replication factors through real-time RT-PCR; identify mtDNA variants in embryonic and induced pluripotent stem cells; determine DNA methylation patterns of the mtDNA-specific replication factors; and assess mitochondrial OXPHOS capacity.
Original languageEnglish
Title of host publicationCell Reprogramming
Subtitle of host publicationMethods and Protocols
EditorsPaul J Verma, Huseyin Sumer
Place of PublicationTotowa NJ USA
PublisherHumana Press
Pages219 - 252
Number of pages34
Edition1st
ISBN (Print)9781493928477
DOIs
Publication statusPublished - 2015

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Mitochondrial DNA
  • OXPHOS
  • mtDNA replication
  • POLGA
  • Pluripotency
  • Differentiation

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