Journal
DIABETOLOGIA
Volume 55, Issue 6, Pages 1689-1698Publisher
SPRINGER
DOI: 10.1007/s00125-012-2508-2
Keywords
Gene therapy; Monogenic forms of diabetes; Stem cells
Categories
Funding
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Ministry of Health, Labour and Welfare of Japan
- Takeda Medical Research Foundation
- Smoking Research Foundation
- Suzuken Memorial Foundation
- Japan Foundation of Applied Enzymology
- Novo Nordisk
- Lilly Education and Research Grant Office
- Grants-in-Aid for Scientific Research [21390252, 23126513, 24659444, 23591305, 21229013, 23122525, 24390204, 22591004, 23790847] Funding Source: KAKEN
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The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with mitochondrial DNA (mtDNA) mutation. Skin biopsies were obtained from two diabetic patients with mtDNA A3243G mutation. The fibroblasts thus obtained were infected with retroviruses encoding (also known as ), , (also known as ) and . The stem cell characteristics were investigated and the mtDNA mutation frequencies evaluated by Invader assay. 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 and 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. 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.
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