Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation.
Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation.
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DOI：
10.1007/s00125-012-2508-2
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发表时间：
2012-06-01
期刊：
影响因子：
8.2
通讯作者：
Nakao, K
中科院分区：
文献类型：
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作者：
Fujikura, J;Nakao, K;Nakao, K
目标/假设：本研究的目的是从线粒体DNA（mtDNA）突变的糖尿病患者中获得诱导多能干细胞（iPS）。用编码OCT 4（也称为POU 5 F1）、SOX 2、c-MYC（也称为MYC）和KLF 4的逆转录病毒感染由此获得的成纤维细胞。结果：从2例糖尿病患者中，我们分别分离出4个和10个线粒体疾病特异性iPS（Mt-iPS）克隆。Mt-iPS细胞的细胞遗传学正常，碱性磷酸酶活性阳性，多能干细胞标志物可通过免疫细胞化学检测。OCT 4和NANOG启动子区的胞嘧啶鸟嘌呤二核苷酸岛高度未甲基化，表明表观遗传重编程为多能性。Mt-iPS克隆能够在体外和体内分化为所有三个胚层的衍生物。Mt-iPS细胞表现出双峰程度的突变异质性。14个克隆中有6个的突变频率降低到检测不到的水平，而其他克隆的突变频率（51-87%）与原始成纤维细胞中的突变频率（18-24%）相比增加了数倍。在连续的细胞培养传代和分化后，没有复发的突变或异质性的水平没有显着的变化被seed.CONCLUSIONS/解释：iPS细胞成功地从患者产生的mtDNA A3243 G突变。突变丰富，稳定的Mt-iPS细胞可能是体外人类线粒体疾病建模的合适细胞来源。无突变的iPS细胞可以为自体移植治疗提供无限制的无疾病细胞供应。
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, disease-free supply of cells for autologous transplantation therapy.