The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells.
Authors
St John, Justin C.Amaral, Alexandra
Bowles, Emma J.
Oliveira, João Facucho
Lloyd, Rhiannon E.I.
Freitas, Mariana
Gray, Heather L.
Navara, Christopher S.
Oliveira, Gisela
Schatten, Gerald P.
Spikings, Emma
Ramalho-Santos, João
Affiliation
University of BirminghamIssue Date
2006Subjects
Mitochondriamitochondrial DNA
human embryonic stem cells
differentiation
cardiomyocytes
homoplasmy
heteroplasmy
transcription and replication
Metadata
Show full item recordAbstract
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).Citation
St John, J.C. et al (2006) 'The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells' Methods in molecular biology 331:347-74Publisher
Humana PressPubMed ID
16881526Additional Links
http://www.ncbi.nlm.nih.gov/pubmed/16881526http://link.springer.com/protocol/10.1385%2F1-59745-046-4%3A347
Type
Book chapterLanguage
enISSN
1064-3745ae974a485f413a2113503eed53cd6c53
10.1385/1-59745-046-4:347