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研究目的（著者による）
The effects of exposure to a extremely low frequency magnetic field and of magnetic field deprivation on the cell reprogramming into a pluripotent state of mouse fibroblasts and the underlying mechanisms of action should be investigated. It was hypothesized that exposure to the magnetic field could influence reprogramming via epigenetic mechanisms. The study was conducted in view of a possible therapeutic use of magnetic fields in cell reprogramming.
詳細情報
Somatic cells can be reprogrammed into induced pluripotent stem cells by an overexpression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM). In a first step, the influence of the magnetic field on this type of reprogramming was examined and mouse fibroblasts were divided into the following groups: Transfection with OSKM and exposure to 1) 10 Hz, 2) 50 Hz, 3) 100 Hz, 4) OSKM transfection and no exposure (control group), 5) OSKM transfection and magnetic withdrawal and 6) fibroblasts without transfection. The effect of the magnetic field on the reprogramming success was compared with the addition of valproic acid and vitamin C, which are well-known stimulants of cell reprogramming. To investigate the mechanisms of action, inhibitors like e.g. of the histone lysine methyltransferase Mll2 and an experimentally-induced overexpression of Mll2 were examined. In a further step, it was examined whether the magnetic field exposure can replace individual transcription factors: 7) transfection with Oct4 only and 50 Hz exposure, 8) transfection with Oct4 and Sox2 and 50 Hz exposure. A control group was carried out without exposure for both groups, respectively.
the solenoid parameters were: axial symmetry, radius (R = 7.5 cm), current (I = 200 mA), and number of loops (N = 1000); the geometry of the system assured field uniformity for the exposed cultures; the surfaces of the culture plates were parallel to the force lines of the alternating magnetic field; temperature in the cultures was maintained at 37 ± 0.1°C
three-axis Helmholtz coils and the three-axis magnetic sensor, which measured the geomagnetic field accurately and made the system capable of canceling the earth's field inside the coil; cell cultures were located in the center of the three-axis Helmholtz coil in the incubator
mouse fibroblasts, transfected with lentivirus with OKSM gene constructs from HEK293 cells
方法
影響評価項目／測定パラメータ／方法
分子生合成: protein expression of reprogramming markers (alkaline phosphatase (commercial staining kit), Oct4, Nanog, Sox2 (immunofluorescence stain with Alexa Fluor and counterstain with DAPI, fluorescence microscopy and flow cytometry)); molecular mechanisms of action: global gene expression profile (unknown method) and mRNA expression (real-time RT-PCR) of histone lysine methyltransferase Mll2 and several genes associated with the cell cycle, amount of methylated histone H3K4me3 and Mll2 (Western Blot)
細胞機能: molecular mechanisms of action: protein-DNA-interactions of different histones like H3K4me3 with marker genes for reprogramming (e.g. Oct4, Nanog und Esrrb) (chromatin immuno-precipitation (ChIP) quantitative PCR), methylation state of Oct4 and Nanog (bisulfite sequencing, PCR); pluripotency proof: generation of teratomas (injection of induced pluripotent cells in mice, extraction of tumors after 4 weeks), generation of chimeras (injection of induced pluripotent cells into B6XDBA F2 host blastocysts)
Exposure to a magnetic field (groups 1, 2, 3) significantly improved reprogramming efficiency compared to the control group (group 4), with the most enhanced effect found in group 2 (50 Hz). It was found that exposure to a 50 Hz magnetic field (group 2) during reprogramming induced epigenetic changes via the increased expression of Mll2, a histone lysine methyltransferase, which is known to contribute to the methylation of histone 3 lysine 4 (H3K4me3). H3K4me3 was accumulated in the cells at the same time. Magnetic field deprivation (group 5) impaired reprogramming into a pluripotent state, indicating the necessity of a magnetic field in the generation of induced pluripotent stem cells. However, it was demonstrated that reprogramming could be induced in this group via experimental overexpression of Mll2. A transfection with Oct4 alone and exposure to the magnetic field (group 7) could create pluripotent cells, indicating that the magnetic field could replace the transcription factors Sox2, Klf4 and c-Myc and thus facilitate reprogramming. The authors conclude that exposure of mouse fibroblasts to a 50 Hz magnetic field could promote cell reprogramming into a pluripotent state by increasing the Mll2 expression. Mll2 seems to mediate the magnetic field induced reprogramming via epigenetical histone methylation. Magnetic field exposure might be an efficient tool for the acquisition of pluripotent cells.
