Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation

Abstract

Aims/hypothesis 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/interpretation 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, diseasefree supply of cells for autologous transplantation therapy.

Tissue- and cell-type–specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model

“…We found that a rapid bimodal shift toward wild-type or mutant homoplasmy occurred upon reprogramming in MELAS iPSCs, replicating findings of recent reports (40,41). Rapid shifts in mitochondrial heteroplasmy have been observed to occur in germ line (i.e., stem cells) in cows (42,43), in human patients (44,45), in mice with heteroplasmic neutral or potentially pathogenic mtDNA variants (18,25,34), and in primates (46).…”

The Pharmacology of Regenerative Medicine

“…Examples of specific conditions for which patient-specific iPS cells have been obtained and differentiated into disease-relevant cell types include Parkinson's disease (Byers et al, 2012;Jang et al, 2012) and other neurodegenerative disorders Jung et al, 2012), Wilson's disease (Yi et al, 2012), lysosomal storage disorders (Huang et al, 2012b), and diabetes (Fujikura et al, 2012).…”

Novel Insights into Disease Modeling Using Induced Pluripotent Stem Cells

“…Disease Modeling Using Patient-Specific iPSCs To date, several patient-specific iPSC lines have been generated from patients with a wide variety of mainly monogenetic, early-onset diseases such as neurologic disorders, 44,45) heart disease, 46,47) metabolic disease, 48) hematologic disorders, 49) mitochondrial disease, 50) chromosomal abnormalities, 51) telomere disease, 52,53) sensory organ disorder, 54) and storage disease.…”