title: Analysis of Mitochondrial DNA in Induced Pluripotent and Embryonic Stem Cells.
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.
authors.affiliation: The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: William
authors.initials: W
authors.lastname: Lee
authors.affiliation: The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: Richard D W
authors.initials: RD
authors.lastname: Kelly
authors.affiliation: The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|Molecular Basis of Metabolic Disease, Division of Metabolic and Vascular Health, Warwick Medical School, The University of Warwick, Clifford Bridge Road, Coventry, CV2 2DX, UK.
authors.forename: Ka Yu
authors.initials: KY
authors.lastname: Yeung
authors.affiliation: The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: Gael
authors.initials: G
authors.lastname: Cagnone
authors.affiliation: The Molecular Basis of Mitochondrial Disease Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: Matthew
authors.initials: M
authors.lastname: McKenzie
authors.affiliation: The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia. justin.stjohn@monash.edu.
authors.forename: Justin C
authors.initials: JC
authors.lastname: St John
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D004272:DNA, Mitochondrial; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018628:Gene Dosage; D015870:Gene Expression; D015723:Gene Library; D050259:Genes, Mitochondrial; D059014:High-Throughput Nucleotide Sequencing; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D004272:DNA, Mitochondrial
keywords: Differentiation; Mitochondrial DNA,; OXPHOS,; POLGA,; Pluripotency,; mtDNA replication,