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研究目的（著者による）
The effect of exposure of different stem cells types to extremely low frequency magnetic fields on neuronal differentiation in relation to gene expression should be investigated.
詳細情報
The study was conducted in the view of a possible therapeutic use for transplantations to treat neurodegenerative diseases. Human bone marrow-derived mesenchymal stem cells and mouse neural stem cells were either exposed to different magnetic fields (0-200 Hz; exposure groups) or not exposed (control groups). Additionally, tests were conducted with Egr1-knockdown and Egr1-overexpressing human bone marrow-mesenchymal stem cells as Egr1 was supposed to play a crucial role in magnetic field mediated neuronal differentiation. Finally, tests were conducted with mice, in which the loss of dopaminergic neurons in Parkinson's disease was simulated by the administration of 6-hydroxy-dopamine (6-OHDA). The mice were divided into the following groups: 1) only administration of 6-OHDA, 2) 6-OHDA and sham transplantation, 3) 6-OHDA and transplantation of human bone marrow-mesenchymal stem cells, 4) 6-OHDA and transplantation of Egr1-overexpressing human bone marrow-mesenchymal stem cells, 5) 6-OHDA, transplantation of Egr1-overexpressing human bone marrow-mesenchymal stem cells and subsequent exposure to the magnetic field, 6) 6-OHDA and transplantation of embryonic midbrain cells (positive control). Stem cell transplantations were conducted 4 weeks after injection of 6-OHDA and mice were exposed afterwards.
影響評価項目
neuronal differentiation of two different stem cell lines
cells were exposed in 35 mm cell culture plates in a system formed by two Helmholtz coils (15 cm inner diameter), which produced a vertical magnetic field in a cell culture incubator with 5% CO2 at 37°C
Additional information
control cultures were grown in a separate incubator without Helmholtz coils
分子生合成: protein expression in the pretest: TuJ1 and DAPI staining; protein expression in human and mouse stem cells: neuronal markers TuJ1, TH, vMAT2, NeuroD1 and MAP2; protein expression in mice (only after 4 weeks): TH (tyrosine hydroxylase, marker for dopaminergic neurons) in brain sections (immunofluorescence, fluorescence microscopy); gene expression in human and mouse stem cells: global gene expression (microarray) and gene expression of neuronal differentiation related genes (real-time PCR)
細胞機能: electrophysiology recordings of human stem cells (inward sodium currents and outward potassium currents, patch-clamp technique)
形態学/組織学的変化: morphological appearance of human and mouse stem cells (observations during immunofluorescence; see "protein expression in human and mouse stem cells" under "molecular biosynthesis")
神経系への影響: see "cognitive/behavioral endpoints" and "protein expression in mice" under "molecular biosynthesis"
認知的／行動学的影響評価項目: apomorphine-induced turning behavior of mice (after 2 and 4 weeks; deletion of dopaminergic neurons and injection of human stem cells in the midbrain): counting of turns/time
In the pretest (field 1), a significant increase of TuJ1 was observed in cells exposed to the 50 Hz magnetic field compared to cells exposed to the other magnetic fields. Human stem cells (field 2) showed a significant increase of TuJ1 and NeuroD1 compared to the control group and moreover, these cells showed a neuronal morphology. Additionally, these cells showed electrophysiological properties after exposure comparable to those of primary neurons. In exposed mouse stem cells (field 3), a significant increase of the TuJ1 and TH protein expression as well as a neuronal morphology were observed compared to the control. A total of 57 genes were up-regulated in exposed human and mouse stem cells (fields 2+3) compared to the control groups with Egr1 showing the highest up-regulation in both cell types. Egr1-overexpressing cells showed a high degree of neuronal differentiation after exposure to the magnetic field (field 2) with significant increases of the expression of neuronal marker genes compared to the control group, whereas the Egr1-knockdown cells did not show neuronal differentiation and no significant increase of the expression of neuronal marker genes compared to the control. The apomorphine-induced turning behavior of mice was significantly reduced in group 5 after exposure to the magnetic field (field 4) compared to mice without transplantation (group 1+2) or without exposure (groups 3+4), indicating a replacement of destroyed neurons and neuronal differentiation of injected cells in vivo, which was confirmed by immunofluorescence. The authors conclude that Egr1 could induce a 50 Hz magnetic field mediated neuronal differentiation of stem cells.
