Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors (original) (raw)

Gene Therapy volume 19, pages 469–475 (2012)Cite this article

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 _Sfa_N1-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.

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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.

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Author notes

  1. E Xiao
    Present address: Current address: Neuroimaging Core; Genes, Cognition, and Psychosis Program, National Institute of Mental Health, Bethesda, MD, USA.,

Authors and Affiliations

  1. Center for the Study of Biological Complexity, Life Sciences Program, Virginia Commonwealth University, Richmond, VA, USA
    S Iyer & R R Rao
  2. Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA
    E Xiao, N Eroshenko & R R Rao
  3. Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA
    K Alsayegh & R R Rao
  4. School of Engineering and Applied Sciences, Harvard University, Boston, MA, USA
    N Eroshenko
  5. Integrated Life Sciences Program, Virginia Commonwealth University, Richmond, VA, USA
    M J Riggs & R R Rao
  6. Parkinson's Disease Center, Virginia Commonwealth University, Richmond, VA, USA
    J P Bennett Jr
  7. Department of Neurology, Virginia Commonwealth University, Richmond, VA, USA
    J P Bennett Jr

Authors

  1. S Iyer
  2. E Xiao
  3. K Alsayegh
  4. N Eroshenko
  5. M J Riggs
  6. J P Bennett Jr
  7. R R Rao

Corresponding authors

Correspondence toS Iyer or R R Rao.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on Gene Therapy website

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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

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