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.

  • Enabling Technologies
  • Published:

Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors

Abstract

Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Iyer S, Alsayegh K, Abraham S, Rao RR . Stem cell-based models and therapies for neurodegenerative diseases. Crit Rev Biomed Eng 2009; 37: 321–353.

    Article  PubMed Central  Google Scholar 

  2. Wallace DC, Singh G, Lott MY, Hodge JA, Schurr TG, Lezza AM et al. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 1988; 242: 1427–1430.

    Article  CAS  PubMed Central  Google Scholar 

  3. Yen MY, Wang AG, Wei YH . Leber's hereditary optic neuropathy: a multifactorial disease. Prog Retin Eye Res 2006; 25: 381–396.

    Article  PubMed Central  Google Scholar 

  4. DiMauro S, Schon EA . Mitochondrial disorders in the nervous system. Annu Rev Neurosci 2008; 31: 91–123.

    Article  CAS  PubMed Central  Google Scholar 

  5. Wallace DC . A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 2005; 39: 359–407.

    Article  CAS  PubMed Central  Google Scholar 

  6. Debray FG, Lambert M, Mitchell GA . Disorders of mitochondrial function. Curr Opin Pediatr 2008; 20: 471–482.

    Article  PubMed Central  Google Scholar 

  7. Borland MK, Mohanakumar KP, Rubinstein JD, Keeney PM, Xie J, Capaldi R et al. Relationships among molecular genetic and respiratory properties of Parkinson's disease cybrid cells show similarities to Parkinson's brain tissues. Biochim Biophys Acta 2009; 1792: 68–74.

    Article  CAS  PubMed Central  Google Scholar 

  8. Ghosh SS, Swerdlow RH, Miller SW, Sheeman B, Parker WD, Davis RE . Use of cytoplasmic hybrid cell lines for elucidating the role of mitochondrial dysfunction in Alzheimer's disease and Parkinson's disease. Ann NY Acad Sci 1999; 893: 176–191.

    Article  CAS  PubMed Central  Google Scholar 

  9. Trimmer PA, Bennett Jr JP . The cybrid model of sporadic Parkinson's disease. Exp Neurol 2009; 218: 320–325.

    Article  CAS  PubMed Central  Google Scholar 

  10. Dhara SK, Hasneen K, Machacek DW, Boyd NL, Rao RR, Stice SL . Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures. Differentiation 2008; 76: 454–464.

    Article  CAS  PubMed Central  Google Scholar 

  11. Dhara SK, Stice SL . Neural differentiation of human embryonic stem cells. J Cell Biochem 2008; 105: 633–640.

    Article  CAS  PubMed Central  Google Scholar 

  12. Shin S et al. Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions. Stem Cells 2006; 24: 125–138.

    Article  PubMed Central  Google Scholar 

  13. Young A, Assey KS, Sturkie CD, West FD, Machacek DW, Stice SL . Glial cell line-derived neurotrophic factor enhances in vitro differentiation of mid-/hindbrain neural progenitor cells to dopaminergic-like neurons. J Neurosci Res 2010; 88: 3222–3232.

    Article  CAS  PubMed Central  Google Scholar 

  14. Dhara SK, Gerwe BA, Majumder A, Dodla MC, Boyd NL, Machacek DW et al. Genetic manipulation of neural progenitors derived from human embryonic stem cells. Tissue Eng Part A 2009; 15: 3621–3634.

    Article  CAS  PubMed Central  Google Scholar 

  15. Wilson PG, Cherry JJ, Schwamberger S, Adams AM, Zhou J, Shin S et al. An SMA project report: neural cell-based assays derived from human embryonic stem cells. Stem Cells Dev 2007; 16: 1027–1041.

    Article  CAS  Google Scholar 

  16. Jin K, Mao X, Xie L, Galvan V, Lai B, Wang Y et al. Transplantation of human neural precursor cells in matrigel scaffolding improves outcome from focal cerebral ischemia after delayed postischemic treatment in rats. J Cereb Blood Flow Metab 2010; 30: 534–544.

    Article  Google Scholar 

  17. Kang D, Kim SH, Hamasaki N . Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions. Mitochondrion 2007; 7: 39–44.

    Article  CAS  Google Scholar 

  18. Scarpulla RC . Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 2008; 88: 611–638.

    Article  CAS  Google Scholar 

  19. Wallace DC . Animal models for mitochondrial disease. Methods Mol Biol 2002; 197: 3–54.

    CAS  PubMed  Google Scholar 

  20. Iyer S, Thomas RR, Portell FR, Dunham LD, Quigley CK, Bennett JP . Recombinant mitochondrial transcription factor A with N-terminal mitochondrial transduction domain increases respiration and mitochondrial gene expression. Mitochondrion 2009; 9: 196–203.

    Article  CAS  PubMed Central  Google Scholar 

  21. Bonawitz ND, Clayton DA, Shadel GS . Initiation and beyond: multiple functions of the human mitochondrial transcription machinery. Mol Cell 2006; 24: 813–825.

    Article  CAS  PubMed Central  Google Scholar 

  22. Brown TA, Clayton DA . Release of replication termination controls mitochondrial DNA copy number after depletion with 2′,3′-dideoxycytidine. Nucleic Acids Res 2002; 30: 2004–2010.

    Article  CAS  PubMed Central  Google Scholar 

  23. Keeney PM, Quigley CK, Dunham LD, Papageorge CM, Iyer S, Thomas RR et al. Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model. Hum Gene Ther 2009; 20: 897–907.

    Article  CAS  PubMed Central  Google Scholar 

  24. Kirby DM, Rennie KJ, Smulders-Srinivasan TK, Acin-Perez R, Whittington M, Enriquez JA et al. Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation. Cell Prolif 2009; 42: 413–424.

    Article  CAS  PubMed Central  Google Scholar 

  25. Tachibana M, Sparman M, Sritanaudomchai H, Ma H, Clepper L, Woodward J et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 2009; 461: 367–372.

    Article  CAS  PubMed Central  Google Scholar 

  26. Facucho-Oliveira JM, St John JC . The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation. Stem Cell Rev 2009; 5: 140–158.

    Article  CAS  PubMed Central  Google Scholar 

  27. Sundaresan P, Kumar SM, Thompson S, Fingert JH . Reduced frequency of known mutations in a cohort of LHON patients from India. Ophthalmic Genet 2010; 31: 196–199.

    Article  CAS  PubMed Central  Google Scholar 

  28. McEachern MJ, Iyer S, Fulton TB, Blackburn EH . Telomere fusions caused by mutating the terminal region of telomeric DNA. Proc Natl Acad Sci USA 2000; 97: 11409–11414.

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

Partial funding for this work has been provided by NIH-1K18DC009121 (Bennett/Rao), NIH- 5P50NS039788 (Bennett), Qimonda Endowment from the VCU School of Engineering (Rao) and Fellowship from the American Parkinson's Disease Association (Iyer). Recombinant MTD–TFAM was obtained by Bennett through an MTA with Gencia Corporation, Charlottesville, VA. LHON cybrid cells were a kind gift from Dr Russell Swederlow.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to S Iyer or R R Rao.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Iyer, S., Xiao, E., Alsayegh, K. et al. Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors. Gene Ther 19, 469–475 (2012). https://doi.org/10.1038/gt.2011.134

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.134

Keywords

This article is cited by

Search

Quick links