Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants

Abstract

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle–chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or β-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Efficient development and genomic integrity after nuclear genome exchange.
Figure 2: Spontaneous activation can be prevented through spindle cooling.
Figure 3: Low levels of mtDNA carryover.
Figure 4: swaPS cells support a normal metabolic profile.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Illumina array data have been deposited at the Gene Expression Omnibus (GEO) under accession number GSE42077; Affymetrix array data have been deposited at the GEO under accession number GSE42271.

References

  1. Jenuth, J. P., Peterson, A. C., Fu, K. & Shoubridge, E. A. Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA. Nature Genet. 14, 146–151 (1997)

    Article  Google Scholar 

  2. Bolhuis, P. A. et al. Rapid shift in genotype of human mitochondrial DNA in a family with Leber’s hereditary optic neuropathy. Biochem. Biophys. Res. Commun. 170, 994–997 (1990)

    Article  CAS  Google Scholar 

  3. Cree, L. M. et al. A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes. Nature Genet. 40, 249–254 (2008)

    Article  CAS  Google Scholar 

  4. Wai, T., Teoli, D. & Shoubridge, E. A. The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nature Genet. 40, 1484–1488 (2008)

    Article  CAS  Google Scholar 

  5. Steffann, J. et al. Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis. J. Med. Genet. 43, 244–247 (2006)

    Article  CAS  Google Scholar 

  6. Nuffield Council on Bioethics. Novel Techniques for the Prevention of Mitochondrial DNA Disorders: an Ethical Reviewhttp://www.nuffieldbioethics.org/news/discussion-event-novel-techniques-prevention-mitochondrial-dna-disorders-ethical-review (2012)

  7. Sato, A. et al. Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation. Proc. Natl Acad. Sci. USA 102, 16765–16770 (2005)

    Article  ADS  CAS  Google Scholar 

  8. Tachibana, M. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Craven, L. et al. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85 (2010)

    Article  ADS  CAS  Google Scholar 

  10. Egli, D. et al. Reprogramming within hours following nuclear transfer into mouse but not human zygotes. Nature Comm. 2, 488 (2011)

    Article  ADS  Google Scholar 

  11. Ganem, N. J., Godinho, S. A. & Pellman, D. A mechanism linking extra centrosomes to chromosomal instability. Nature 460, 278–282 (2009)

    Article  ADS  CAS  Google Scholar 

  12. Sathananthan, A. H. et al. Centrioles in the beginning of human development. Proc. Natl Acad. Sci. USA 88, 4806–4810 (1991)

    Article  ADS  CAS  Google Scholar 

  13. Kaufman, M. H., Robertson, E. J., Handyside, A. H. & Evans, M. J. Establishment of pluripotent cell lines from haploid mouse embryos. J. Embryol. Exp. Morphol. 73, 249–261 (1983)

    CAS  PubMed  Google Scholar 

  14. Draper, J. S. et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nature Biotechnol. 22, 53–54 (2004)

    Article  CAS  Google Scholar 

  15. Kim, K. et al. Histocompatible embryonic stem cells by parthenogenesis. Science 315, 482–486 (2007)

    Article  ADS  CAS  Google Scholar 

  16. Noggle, S. et al. Human oocytes reprogram somatic cells to a pluripotent state. Nature 478, 70–75 (2011)

    Article  ADS  CAS  Google Scholar 

  17. Hyun, C. S. et al. Optimal ICSI timing after the first polar body extrusion in in vitro matured human oocytes. Hum. Reprod. 22, 1991–1995 (2007)

    Article  Google Scholar 

  18. Brinkley, B. R. & Cartwright, J., Jr Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. Ann. NY Acad. Sci. 253, 428–439 (1975)

    Article  ADS  CAS  Google Scholar 

  19. van Oven, M. & Kayser, M. Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation. Hum. Mutat. 30, E386–E394 (2009)

    Article  Google Scholar 

  20. Mimaki, M. et al. Reversible infantile respiratory chain deficiency: a clinical and molecular study. Ann. Neurol. 68, 845–854 (2010)

    Article  CAS  Google Scholar 

  21. Wang, J., Venegas, V., Li, F. & Wong, L.-J. Analysis of mitochondrial DNA point mutation heteroplasmy by ARMS quantitative PCR. Curr. Protocols Human Genet. Chapter 19, Unit–19.16 (2011)

    Google Scholar 

  22. Bai, R.-K. & Wong, L.-J. C. Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach. Clinical Chem. 50, 996–1001 (2004)

    Article  CAS  Google Scholar 

  23. Lee, H. S. et al. Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck. Cell Rep. 1, 506–515 (2012)

    Article  CAS  Google Scholar 

  24. Meirelles, F. V. & Smith, L. C. Mitochondrial genotype segregation during preimplantation development in mouse heteroplasmic embryos. Genetics 148, 877–883 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Jenuth, J. P., Peterson, A. C. & Shoubridge, E. A. Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice. Nature Genet. 16, 93–95 (1997)

    Article  CAS  Google Scholar 

  26. Sharpley, M. S. et al. Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition. Cell 151, 333–343 (2012)

    Article  CAS  Google Scholar 

  27. Fujikura, J. et al. Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation. Diabetologia 55, 1689–1698 (2012)

    Article  CAS  Google Scholar 

  28. Birket, M. J. et al. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells. J. Cell Sci. 124, 348–358 (2011)

    Article  CAS  Google Scholar 

  29. Cowan, C. A. et al. Derivation of embryonic stem-cell lines from human blastocysts. New Engl. J. Med. 350, 1353–1356 (2004)

    Article  CAS  Google Scholar 

  30. Inoue, S., Fuseler, J., Salmon, E. D. & Ellis, G. W. Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system. Biophys. J. 15, 725–744 (1975)

    Article  ADS  CAS  Google Scholar 

  31. Bianchi, V., Coticchio, G., Fava, L., Flamigni, C. & Borini, A. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Human Reprod. 20, 1078–1083 (2005)

    Article  CAS  Google Scholar 

  32. Larman, M. G., Minasi, M. G., Rienzi, L. & Gardner, D. K. Maintenance of the meiotic spindle during vitrification in human and mouse oocytes. Reprod. Biomed. Online 15, 692–700 (2007)

    Article  CAS  Google Scholar 

  33. Forman, E. J. et al. Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection: a novel, paired randomized controlled trial using DNA fingerprinting. Fertil. Steril. 98, 644–649 (2012)

    Article  CAS  Google Scholar 

  34. Gook, D. A., Osborn, S. M., Bourne, H. & Johnston, W. I. Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum. Reprod. 9, 684–691 (1994)

    Article  CAS  Google Scholar 

  35. ASRM. Mature oocyte cryopreservation: a guideline. Fertil. Steril.http://dx.doi.org/10.1016/j.fertnstert.2012.09.028 (18 October 2012)

  36. Tachibana, M. et al. Towards germline gene therapy of inherited mitochondrial diseases. Naturehttp://dx.doi.org/10.1038/nature11647 (this issue)

  37. Tachibana, M., Sparman, M. & Mitalipov, S. Chromosome transfer in mature oocytes. Nature Protoc. 5, 1138–1147 (2010)

    Article  CAS  Google Scholar 

  38. Liu, D., Vader, G., Vromans, M. J., Lampson, M. A. & Lens, S. M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates. Science 323, 1350–1353 (2009)

    Article  ADS  CAS  Google Scholar 

  39. A mother’s gift, minus mitochondria. Nature Med. 16, 645 (2010)

  40. Society for Assisted Reproductive Technology, American Society for Reproductive Medicine. Assisted reproductive technology in the United States: 2000 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil. Steril. 81, 1207–1220 (2004)

  41. The Ethics Committee of the American Society for Reproductive Medicine. Financial compensation of oocyte donors. Fertil. Steril. 88, 305–309 (2007)

  42. Chen, A. E. et al. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell 4, 103–106 (2009)

    Article  CAS  Google Scholar 

  43. Lin, D. P.-C. et al. Comparison of mitochondrial DNA contents in human embryos with good or poor morphology at the 8-cell stage. Fertil. Steril. 81, 73–79 (2004)

    Article  CAS  Google Scholar 

  44. D’Amour, K. A. et al. Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells. Nature Biotechnol. 24, 1392–1401 (2006)

    Article  Google Scholar 

  45. Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnol. 27, 275–280 (2009)

    Article  CAS  Google Scholar 

  46. Burridge, P. W. et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PloS ONE 6, e18293 (2011)

    Article  ADS  CAS  Google Scholar 

  47. Lin, T. et al. A chemical platform for improved induction of human iPSCs. Nature Methods 6, 805–808 (2009)

    Article  CAS  Google Scholar 

  48. DiMauro, S. et al. Cytochrome c oxidase deficiency in Leigh syndrome. Ann. Neurol. 22, 498–506 (1987)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Chang and K. Eggan for discussions, Z. Hall for critical reading of the manuscript, and L. Yu and O. Nahum for SNP-array preparation. We thank anonymous oocyte donors for participating in research, and M. Spencer for a Lykos laser system. This work was supported by the New York Stem Cell Foundation, the New York State Stem Cell Science award C026184, and the Bernard and Anne Spitzer Fund.

Author information

Authors and Affiliations

Authors

Contributions

M.V.S. consented oocyte donors and retrieved oocytes. R.P. contributed IVF developmental data. R.S.G. and M.V.S. wrote institutional review board and consent documents. D.E., D.P. and S.N. designed and performed experiments with oocytes. D.P. and V.E. determined heteroplasmy. N.T. performed array analysis of single cells. D.E., D.P., V.E., L.S., K.A.W., H.H., M.Z. and D.J.K characterized stem-cell lines. D.E., D.P., V.E. and M.H. wrote the paper.

Corresponding authors

Correspondence to Mark V. Sauer or Dieter Egli.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9, Supplementary Tables 1-8 and Supplementary Karyotypes. (PDF 41490 kb)

SwaPS1 cardiomyocytes

This video shows a contracting embryoid body that had undergone directed differentiation toward a cardiac (mesodermal) lineage. (MOV 949 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Paull, D., Emmanuele, V., Weiss, K. et al. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632–637 (2013). https://doi.org/10.1038/nature11800

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11800

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing