title: Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation.
abstract: OBJECTIVE
  The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with mitochondrial DNA (mtDNA) mutation.
  
  
  METHODS
  Skin biopsies were obtained from two diabetic patients with mtDNA A3243G mutation. The fibroblasts thus obtained were infected with retroviruses encoding OCT4 (also known as POU5F1), SOX2, c-MYC (also known as MYC) and KLF4. The stem cell characteristics were investigated and the mtDNA mutation frequencies evaluated by Invader assay.
  
  
  RESULTS
  From the two diabetic patients we isolated four and ten putative mitochondrial disease-specific iPS (Mt-iPS) clones, respectively. Mt-iPS cells were cytogenetically normal and positive for alkaline phosphatase activity, with the pluripotent stem cell markers being detectable by immunocytochemistry. The cytosine guanine dinucleotide islands in the promoter regions of OCT4 and NANOG were highly unmethylated, indicating epigenetic reprogramming to pluripotency. Mt-iPS clones were able to differentiate into derivatives of all three germ layers in vitro and in vivo. The Mt-iPS cells exhibited a bimodal degree of mutation heteroplasmy. The mutation frequencies decreased to an undetectable level in six of 14 clones, while the others showed several-fold increases in mutation frequencies (51-87%) compared with those in the original fibroblasts (18-24%). During serial cell culture passage and after differentiation, no recurrence of the mutation or no significant changes in the levels of heteroplasmy were seen.
  
  
  CONCLUSIONS
  iPS cells were successfully generated from patients with the mtDNA A3243G mutation. Mutation-rich, stable Mt-iPS cells may be a suitable source of cells for human mitochondrial disease modelling in vitro. Mutation-free iPS cells could provide an unlimited, disease-free supply of cells for autologous transplantation therapy.
authors.affiliation: Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. j-fuji@sannet.ne.jp
authors.forename: J
authors.initials: J
authors.lastname: Fujikura
authors.forename: K
authors.initials: K
authors.lastname: Nakao
authors.forename: M
authors.initials: M
authors.lastname: Sone
authors.forename: M
authors.initials: M
authors.lastname: Noguchi
authors.forename: E
authors.initials: E
authors.lastname: Mori
authors.forename: M
authors.initials: M
authors.lastname: Naito
authors.forename: D
authors.initials: D
authors.lastname: Taura
authors.forename: M
authors.initials: M
authors.lastname: Harada-Shiba
authors.forename: I
authors.initials: I
authors.lastname: Kishimoto
authors.forename: A
authors.initials: A
authors.lastname: Watanabe
authors.forename: I
authors.initials: I
authors.lastname: Asaka
authors.forename: K
authors.initials: K
authors.lastname: Hosoda
authors.forename: K
authors.initials: K
authors.lastname: Nakao
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D004272:DNA, Mitochondrial / Q000235:genetics*; D058732:Embryoid Bodies / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D059785:Karyotype; D000090062:Kruppel-Like Factor 4; D018895:Microsatellite Repeats / Q000235:genetics; D009154:Mutation
chemical_list: D004272:DNA, Mitochondrial; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D000469:Alkaline Phosphatase