Analysis of the mitochondrial DNA and its replicative capacity in induced pluripotent stem cells

Gael Cagnone, Vijesh Vaghjiani, William Lee, Claire Sun, Jacqueline Johnson, Ka-Yu Yeung, Justin C. St.John

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

1 Citation (Scopus)

Abstract

The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.

Original languageEnglish
Title of host publicationInduced Pluripotent Stem (iPS) Cells
Subtitle of host publicationMethods and Protocols
EditorsKursad Turksen, Andras Nagy
Place of PublicationNew York NY USA
PublisherHumana Press
Pages231-267
Number of pages37
Volume1357
ISBN (Print)978-1-4939-3054-8, 978-1-4939-3055-5
DOIs
Publication statusPublished - 1 Jan 2016

Publication series

NameMethods in Molecular Biology
Volume1357
ISSN (Print)10643745

Keywords

  • ATP
  • Differentiation
  • Mitochondrial DNA
  • Next-generation sequencing
  • Oxygen consumption
  • Pluripotent cells
  • Transcription and replication
  • Variants

Cite this

Cagnone, G., Vaghjiani, V., Lee, W., Sun, C., Johnson, J., Yeung, K-Y., & St.John, J. C. (2016). Analysis of the mitochondrial DNA and its replicative capacity in induced pluripotent stem cells. In K. Turksen, & A. Nagy (Eds.), Induced Pluripotent Stem (iPS) Cells: Methods and Protocols (Vol. 1357, pp. 231-267). (Methods in Molecular Biology; Vol. 1357). Humana Press. https://doi.org/10.1007/7651_2014_156