title: The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells.
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).
authors.affiliation: Mitochondrial Reproductive Genetics Group, Division of Medical Sciences, The Medical School, University of Birmingham, UK.
authors.forename: Justin C
authors.initials: JC
authors.lastname: St John
authors.forename: Alexandra
authors.initials: A
authors.lastname: Amaral
authors.forename: Emma
authors.initials: E
authors.lastname: Bowles
authors.forename: João Facucho
authors.initials: JF
authors.lastname: Oliveira
authors.forename: Rhiannon
authors.initials: R
authors.lastname: Lloyd
authors.forename: Mariana
authors.initials: M
authors.lastname: Freitas
authors.forename: Heather L
authors.initials: HL
authors.lastname: Gray
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Navara
authors.forename: Gisela
authors.initials: G
authors.lastname: Oliveira
authors.forename: Gerald P
authors.initials: GP
authors.lastname: Schatten
authors.forename: Emma
authors.initials: E
authors.lastname: Spikings
authors.forename: João
authors.initials: J
authors.lastname: Ramalho-Santos
mesh_terms: D001562:Benzimidazoles; D002232:Carbocyanines; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000302:isolation & purification; D005456:Fluorescent Dyes; D018628:Gene Dosage; D015894:Genome, Human; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000502:physiology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D000077278:RNA, Mitochondrial; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
chemical_list: D001562:Benzimidazoles; D002232:Carbocyanines; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D000077278:RNA, Mitochondrial; D034741:RNA, Small Interfering; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D012313:RNA