title: 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.
authors.affiliation: Center for the Study of Biological Complexity, Life Sciences Program, Virginia Commonwealth University, Richmond, VA 23284-3020, USA. siyer@vcu.edu
authors.forename: S
authors.initials: S
authors.lastname: Iyer
authors.forename: E
authors.initials: E
authors.lastname: Xiao
authors.forename: K
authors.initials: K
authors.lastname: Alsayegh
authors.forename: N
authors.initials: N
authors.lastname: Eroshenko
authors.forename: M J
authors.initials: MJ
authors.lastname: Riggs
authors.forename: J P
authors.initials: JP
authors.lastname: Bennett
authors.forename: R R
authors.initials: RR
authors.lastname: Rao
mesh_terms: D000328:Adult; D000963:Antimetabolites / Q000494:pharmacology; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D050259:Genes, Mitochondrial; D006801:Humans; D006822:Hybrid Cells; D008297:Male; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D009245:NADH Dehydrogenase / Q000235:genetics*; D058953:Neural Stem Cells; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D016047:Zalcitabine / Q000494:pharmacology
chemical_list: D000963:Antimetabolites; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C098483:NADH dehydrogenase subunit 4; C499631:TFAM protein, human; D014157:Transcription Factors; D016047:Zalcitabine; D009245:NADH Dehydrogenase