Human Embryonic Stem Cell Protocols pp 347-374

Part of the Methods In Molecular Biology book series (MIMB, volume 331) | Cite as

The Analysis of Mitochondria and Mitochondrial DNA in Human Embryonic Stem Cells

  • Justin C. St. John
  • Alexandra Amaral
  • Emma Bowles
  • João Facucho Oliveira
  • Rhiannon Lloyd
  • Mariana Freitas
  • Heather L. Gray
  • Christopher S. Navara
  • Gisela Oliveira
  • Gerald P. Schatten
  • Emma Spikings
  • João Ramalho-Santos

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).

Key Words

Mitochondria mitochondrial DNA human embryonic stem cells differentiation cardiomyocytes homoplasmy heteroplasmy transcription and replication 

References

  1. 1.
    Kehat I., Kenyagin-Karsenti D., Snir M., et al. (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108, 407–414.PubMedGoogle Scholar
  2. 2.
    St. John J. C., Lloyd R. E. I., Bowles E. J., Thomas E. C., and Shourbagy S. H. (2004) The consequences of nuclear transfer for mammalian foetal development and offspring survival. A mitochondrial DNA perspective. Reproduction 127, 631–641.PubMedCrossRefGoogle Scholar
  3. 3.
    Green D. R. and Reed J. C. (1998) Mitochondria and apoptosis. Science 281, 1309–1312.PubMedCrossRefGoogle Scholar
  4. 4.
    Wallace D. C. (1999) Mitochondrial diseases in man and mouse. Science 283, 1482–1488.PubMedCrossRefGoogle Scholar
  5. 5.
    Chinnery P. F., Andrews R. M., Turnbull D. M., and Howell N. N. (2001) Leber hereditary optic neuropathy: does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation Am. J. Med. Genet. 98, 235–243PubMedCrossRefGoogle Scholar
  6. 6.
    Howell N., Ghosh S. S., Fahy E., and Bindoff L. A. (2000) Howell Longitudinal analysis of the segregation of mtDNA mutations in heteroplasmic individuals. J. Neurolog. Sci. 172, 1–6.CrossRefGoogle Scholar
  7. 7.
    Barritt J. A., Brenner C. A., Malter H. E., and Cohen J. (2000) Interooplasmic transfers in humans. Reprod. Biomed. Online 3, 47–48.CrossRefGoogle Scholar
  8. 8.
    Steinborn R., Schinogl P., Zakhartchenko V., et al. (2000) Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nat. Genet. 25, 255–257.PubMedCrossRefGoogle Scholar
  9. 9.
    St. John J. C. and Schatten G. (2004) Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer. Genetics 167, 897–905.PubMedCrossRefGoogle Scholar
  10. 10.
    Moyes C. D., Battersby B. J., and Leary S. C. (1998) Regulation of muscle mitochondrial design. J. Exp. Biol. 201, 299–307.Google Scholar
  11. 11.
    Miller F. J., Rosenfeldt F. L., Zhang C., Linnane A. W., and Nagley P. (2003) Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by PCR-based assay: lack of change of copy number with age. Nucl. Acids Res. 31, 61.CrossRefGoogle Scholar
  12. 12.
    Gahan M. E., Miller F., Lewin S. R., et al. (2001) Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using realtime polymerase chain reaction. J. Clin. Virol. 22, 241–247.PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang H., Cooney D. A, Sreenath A., et al. (1994) Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content. Mol. Pharm. 46, 1063–1069.Google Scholar
  14. 14.
    Michaels G. S., Hauswirth W. W., and Laipis P. J. (1982) Mitochondrial DNA copy number in bovine oocytes and somatic cells. Dev. Biol. 94, 246–251.PubMedCrossRefGoogle Scholar
  15. 15.
    Fisher R. P. and Clayton D. A. (1988) Purification and characterization of human mitochondrial transcription factor 1. Mol. Cell. Biol. 8, 3496–3509.PubMedGoogle Scholar
  16. 16.
    Clayton D. A. (1998) Nuclear-mitochondrial intergenomic communication. Biofactors 7, 203–205.PubMedGoogle Scholar
  17. 17.
    Poulton J., Morten K., Freeman-Emmerson C., et al. (1994) Deficiency of the human mitochondrial transcription factor h-mtTFA in infantile mitochondrial myopathy is associated with mtDNA depletion. Hum. Mol. Genet. 3, 1763–1769.PubMedCrossRefGoogle Scholar
  18. 18.
    Hansson A., Hance N., Dufour E., et al. (2004) A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts. Proc. Natl. Acad. Sci. USA 101, 3136–3141.PubMedCrossRefGoogle Scholar
  19. 19.
    Larsson N. G., Wang J., Wilhelmssohn H., et al. (1998) Mitochondrial transcription factor A is necessary for mitochondrial DNA maintenance and embryogenesis in mice. Nat. Genet. 18, 231–236.PubMedCrossRefGoogle Scholar
  20. 20.
    Li H., Wang J., Wilhelmsson H., et al. (2000) Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy. Proc. Natl. Acad. Sci. USA 97, 3467–3472.PubMedCrossRefGoogle Scholar
  21. 21.
    Piko L. and Matsumoto L. (1976) Number of mitochondria and some properties of mitochondrial DNA in the mouse egg. Dev. Biol. 49, 1–10.PubMedCrossRefGoogle Scholar
  22. 22.
    Ebert K. M., Liem H., and Hecht N. B. (1988) Mitochondrial DNA in the mouse preimplantation embryo. J. Reprod. Fertil. 82, 145–149.PubMedCrossRefGoogle Scholar
  23. 23.
    Piko L. and Taylor K. D. (1987) Amount of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos. Dev. Biol. 123,364–374.PubMedCrossRefGoogle Scholar
  24. 24.
    Jansen R. and de Boer K. (1998) The bottleneck: mitochondrial imperatives in oogenesis and ovarian follicular fate. Mol. Cell. Endocrinol. 145, 81–88.PubMedCrossRefGoogle Scholar
  25. 25.
    Chen Y., He Z. X., Liu A., et al. (2003) Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Res. 13, 251–263.PubMedCrossRefGoogle Scholar
  26. 26.
    McKenzie M. and Trounce I. (2000) Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects. J. Biol. Chem. 275, 31,514–31,519.PubMedCrossRefGoogle Scholar
  27. 27.
    Dey R., Barrientos A., and Moraes C. T. (2000) Functional constraints of muclear-mitochondrial DNA interactions in xenomitochondrial rodent cell lines. J. Biol. Chem. 275, 31,520–31,527.PubMedCrossRefGoogle Scholar
  28. 28.
    Moraes C. T., Kenyon L., and Hao H. (1999) Mechanisms of human mitochondrial DNA maintenance: the determining role of primary sequence and length over function. Mol. Biol. Cell 10, 3345–3356.PubMedGoogle Scholar
  29. 29.
    Falkenberg M., Gaspari M., Rantanen A., Trifunovic A., Larsson N. G., and Gustafsson C. M. (2002) Mitochondrial transcription factors B1 and B2 activate transcription of human mtTFA. Nat. Genet. 31, 289–294.PubMedCrossRefGoogle Scholar
  30. 30.
    Nelson I., Hanna M. G., Wood N. W., and Harding A. E. (1997) Depletion of mitochondrial DNA by ddC in untransformed human cell lines. Som. Cell. Mol. Genet. 23, 287–290.CrossRefGoogle Scholar
  31. 31.
    White D. J., Mital D., Taylor S., and St. John J. C. (2001) Sperm mitochondrial DNA deletions as a consequence of long term highly active antiretroviral therapy. AIDS 15, 1061–1062.PubMedCrossRefGoogle Scholar
  32. 32.
    Dalakas M. C. (2001) Peripheral neuropathy and antiretroviral drugs. J. Periph. Nerv. Syst. 6, 14–20.CrossRefGoogle Scholar
  33. 33.
    Jazayeri M., Andreyev A., Will Y., Ward M., Anderson C. M., and Clevenger W. (2003) Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype. J. Biol. Chem. 278, 9823–9830.PubMedCrossRefGoogle Scholar
  34. 34.
    Virbasius J. V. and Scarpulla R. C. (1994) Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis. Proc. Natl. Acad. Sci. USA 91, 1309–1313.PubMedCrossRefGoogle Scholar
  35. 35.
    King M. P. and Attardi G. (1996) Isolation of human cell lines lacking mitochondrial DNA. Meth. Enzymol. 264, 304–313.PubMedCrossRefGoogle Scholar
  36. 36.
    Seidel-Rogol B. L., and Shadel G. S. (2002) Modulation of mitochondrial transcription in response to mtDNA depletion and repletion in HeLa cells. Nucl. Acids Res. 30, 1929–1934.PubMedCrossRefGoogle Scholar
  37. 37.
    Giles R. E., Blanc H., Cann H. M., and Wallace D. C. (1980) Maternal inheritance of human mitochondrial DNA. Proc. Natl. Acad. Sci. USA 77, 6715–6719.PubMedCrossRefGoogle Scholar
  38. 38.
    Birky C. W. (1995) Uniparental inheritance of mitochondrial and chloroplast genes: Mechanisms and evolution. Proc. Natl. Acad. Sci. USA 92, 11,331–11,338.PubMedCrossRefGoogle Scholar
  39. 39.
    Hecht N. B. and Liem H. (1984) Mitochondrial DNA is synthesized during meiosis and spermiogenesis in the mouse. Exp. Cell Res. 154, 293–298.PubMedCrossRefGoogle Scholar
  40. 40.
    Sutovsky P., Moreno R. D., Ramalho-Santos J., Dominko T., Simerly C., and Schatten, G. (1999) Ubiquitin tag for sperm mitochondria. Nature 402, 371–372.PubMedCrossRefGoogle Scholar
  41. 41.
    St. John J., Sakkas D., Dimitriadi K., et al. (2000) Abnormal human embryos show a failure to eliminate paternal mitochondrial DNA. Lancet 355, 200.PubMedCrossRefGoogle Scholar
  42. 42.
    Schwartz M. and Vissing J. (2002) Paternal inheritance of mitochondrial DNA. N. Engl. J. Med. 347, 576–580.PubMedCrossRefGoogle Scholar
  43. 43.
    Mulligan R. C. and Berg P. (1980) Expression of a bacterial gene in mammalian cells. Science 209, 1422–1427.PubMedCrossRefGoogle Scholar
  44. 44.
    Fire A., Xu S., Montgomery M. K., Kostas S. A., Driver S. E., and Mello C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 39, 806–811.CrossRefGoogle Scholar
  45. 45.
    Anderson S., Bankier A. T., Barrell B. G., et al. (1981) Sequence and organisation of the human mitochondrial genome. Nature 290, 457–465.PubMedCrossRefGoogle Scholar
  46. 46.
    St. John J. C., Ramalho-Santos J., Gray H. L., et al. The expression of mitochondnal DNA transcription factors during early cardiomyocyte differentiation from human embnyonic stem cells. Cloning and Stem Cells 7, 141–153.Google Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Justin C. St. John
    • 1
  • Alexandra Amaral
    • 2
  • Emma Bowles
    • 2
  • João Facucho Oliveira
  • Rhiannon Lloyd
    • 1
  • Mariana Freitas
    • 1
  • Heather L. Gray
    • 3
  • Christopher S. Navara
    • 3
  • Gisela Oliveira
    • 1
  • Gerald P. Schatten
    • 4
  • Emma Spikings
    • 5
  • João Ramalho-Santos
    • 2
  1. 1.The Mitochondrial Reproductive Genetics Group, The Division of Medical Sciences, The Medical SchoolUniversity of BirminghamBirmingham
  2. 2.Center for Neuroscience and Cell Biology, Department of ZoologyUniversity of CoimbraCoimbra
  3. 3.Pittsburgh Development CenterMagee-Women’s Research InstitutePittsburgh
  4. 4.Pittsburgh Development Center, Magee-Women’s Research Institute, Departments of Obstetrics-Gynecology-Reproductive Sciences and Cell Biology-PhysiologyUniversity of Pittsburgh School of MedicinePittsburgh
  5. 5.School of MedicineThe University of BirminghamBirmingham

Personalised recommendations