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

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.

A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K-111 of Panax ginseng and the sequence amplified.

Panax ginseng is widely used as a Chinese medicine, but it takes a long time to reach harvest and to establish its qualified strains. In the course of searching high quality Panax ginseng, we found a useful random amplified polymorphic DNA (RAPD) primer, which showed a 725 base pair band for a selected elite strain Aizu K-111 (now called Kaishusan) including its cultured tissues, while the other strains did not necessarily show this band. We sequenced the DNA fragment amplified and designed primers to improve electrophoretic profiles, based on the sequence.

Mass Production of Ginseng (Panax ginseng) Embryoids on Media Containing High Concentrations of Sugar1.

A lot of ginseng embryoids were produced by culturing on high concentrations of sugar media from the embryogenic tissues obtained by moderately high temperature treatment. When the sucrose concentration was 100 g/l, the number of embryoids produced were over 100 pieces per g of inoculum weight. It was about ten times of that produced by culturing on 30 g/l of sucrose. Glucose showed an effect similar to sucrose on the basis of weight percentage. However, mannitol did not show this effect. The embryoids obtained by these processes redifferentiated to normal plantlets on culturing on the medium containing 30 g/l of sucrose. The saponin components of the tissue containing embryoids showed a similar pattern to those of natural GINSENG by HPLC.

Embryoid Formation by High Temperature Treatment from Multiple Shoots of Panax ginseng1.

We developed a new technology to induce embryoids by a moderate high temperature treatment from multiple shoots of PANAX GINSENG (Araliaceae). The number of formed embryoids was 10 times higher than that of untreated tissue. Normal plantlets were regenerated from the embryoids by transplanting them on a hormone-free medium. They contained ginsenosides Rb (1), Rg (1) and the other saponins as well as those of natural ginseng.