Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation

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Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation

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Jeong-Eun Park a, Young-Kwon Seo a, Hee-Hoon Yoon a, Chan-Wha Kim b, Jung-keug Park a, Songhee Jeon a,⇑ a b

Dongguk University Research Institute of Biotechnology, Dongguk University, 3-26, Pil Dong, Choong-Gu, Seoul 100-715, Republic of Korea School of Life Sciences and Biotechnology, Korea University, 1-5, Anam Dong, Seongbuk-Gu, Seoul 136-701, Republic of Korea

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Article history: Received 12 November 2012 Received in revised form 30 January 2013 Accepted 4 February 2013 Available online xxxx Keywords: Extremely low frequency electromagnetic fields Neural differentiation Bone-marrow mesenchymal stem cells Epidermal growth factor receptor Reactive oxygen species

a b s t r a c t Even though the inducing effect of electromagnetic fields (EMF) on the neural differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) is a distinctive, the underlying mechanism of differentiation remains unclear. To find out the signaling pathways involved in the neural differentiation of BM-MSCs by EMF, we examined the CREB phosphorylation and Akt or ERK activation as an upstream of CREB. In hBM-MSCs treated with ELF-EMF (50 Hz, 1 mT), the expression of neural markers such as NF-L, MAP2, and NeuroD1 increased at 6 days and phosphorylation of Akt and CREB but not ERK increased at 90 min in BM-MSCs. Moreover, EMF increased phosphorylation of epidermal growth factor receptor (EGFR) as an upstream receptor tyrosine kinase of PI3K/Akt at 90 min. It has been well documented that ELF-MF exposure may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentrations. Thus, we examined EMF-induced ROS production in BM-MSCs. Moreover, pretreatment with a ROS scavenger, N-acetylcystein, and an EGFR inhibitor, AG-1478, prevented the phosphorylation of EGFR and downstream molecules. These results suggest that EMF induce neural differentiation through activation of EGFR signaling and mild generation of ROS. Ó 2013 Published by Elsevier Ltd.

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1. Introduction

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There have been attempts to use stem cells for clinical translation of neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis, since it was discovered 130 years ago (Lindvall et al., 2012). In recent studies, it has been demonstrated that MSCs are capable of differentiation into neurons when they were exposed to either specific factors including growth factors, neurotrophins, cytokines (Sanchez-Ramos et al., 2000), or specific chemical compounds such as bmercaptoethanol, dimethyl sulfoxide (DMSO) and butylated hydroxyanizole (BHA) (Bertani et al., 2005; Jiang et al., 2002; Kabos et al., 2002). Chemical induction has at least two advantages over factor induction; (i) chemically induced hBM-MSCs acquire morphological features and express specific markers of mature neuronal cells in relatively short time; (ii) the process produces a larger amount of differentiated neuronal cells (Munoz-Elias et al., 2003). On the other hands, disadvantages of chemical induction are reduced viability of MSCs and difficulty of obtaining functional neurons. Indeed, induction with exogenous growth factors has

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⇑ Corresponding author. Tel.: +82 2 2290 1376; fax: +82 2 2271 3489.

limitations to use due to restricted sources, high cost, risk of rejection, and disease propagation (Burdon et al., 2011). Therefore, there have been many trials to find a more affordable and efficient way to differentiate MSCs to neuronal cells and electromagnetic fields has been proposed to be a good tool for promoting neural differentiation of them (Cho et al., 2012; Cuccurazzu et al., 2010). Although it is not lead directly to genotoxic effects, various cellular processes such as both internal and external of the cell membranes, signal transduction pathways, cell-cycle regulation and cell proliferation and/or differentiation can be affected by ELF-EMF exposure (Foster, 2003; Kavet et al., 2001). It has been well documented that ELF-MF exposure may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentrations. ROS production is not restricted to phagocytic cells, but also cellular proliferation and differentiation (Kanda et al., 2011; Sauer et al., 2000; Sundaresan et al., 1995). Moreover, ROS production has been found to be increased during differentiation of human MSCs into adipocytes (Tormos et al., 2011). Previously, we reported that ELF-EMF induces neural differentiation of hBM-MSCs (Cho et al., 2012). However, the mechanisms of ELF-EMF have not been defined. Therefore, in the present study, we investigated whether EMF-induced ROS production is involved in the MSCs differentiation and which signaling pathway may be activated in this system.

E-mail address: [email protected] (S. Jeon). 0197-0186/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.neuint.2013.02.002

Please cite this article in press as: Park, J.-E., et al. Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation. Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.02.002

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30 min at 37 °C, and then exposed to ELF-EMF for 90 min. The treated cells were incubated with 20 lM H2DCF-DA for 30 min. The mean fluorescence intensity was measured at 480 nm excitation and 530 nm emission using a fluorescence microplate reader (TECAN, infinite 200 pro, Männedorf, Switzerland) in at least three independent experiments.

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2.6. Western blot assay

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The cells was lysed with sampling buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.1 mg/ml bromphenol blue in Tris– HCl, pH 6.8), heated at 100 °C for 10 min. Twenty lg of cell lysates were electrophoresed by 10% SDS–PAGE and transferred onto nitrocellulose membrane. The blotted membrane was incubated with anti-phospho-EGFR (Epitomics, Mitten Road, California), anti-EGFR (Santa Cruz Biothechnology, Inc., Santa Cruz, CA), anti-phosphoCREB, anti-CREB, anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt (Cell Signalling Technology, Beverly, MA, USA), diluted in TBS-T containing 0.05% BSA, for overnight at 4 °C. The bound antibodies were detected by horseradish peroxidase-conjugated antirabbit or anti-mouse IgG and the bands were visualized using the ECL system (Thermo Fisher Scienctific, USA). Band images were obtained by using Molecular Imager ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and band intensity was analyzed by Image Lab™ software version 2.0.1 (Bio-Rad).

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2.7. Immunocytochemistry

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Cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with PBS containing 0.3% Triton X-100 for 10 min. Cells were incubated overnight at 4 °C with an appropriate primary antibody, anti-NF-L, anti-MAP2, anti-NeuroD1, anti-phospho-CREB (1:100, Cell Signaling Technology) and anti-phosphoEGFR (1:100, Epitomics). The following day, cells were washed and incubated for 1 h at RT with species-specific secondary antibody conjugated with anti-rabbit IgG-Alexa Fluor 488 conjugate (Cell Signaling Technology). Nuclei were then counterstained for 2 min with 40 6-diamidino-2-phenylindole (DAPI: 0.5 lg/ml; Molecular Probes) and finally cover-slipped with ProLong Gold antifade reagent (Molecular Probes). Images were obtained with Laser-scanning confocal microscopy (Nikon C2+, Nikon Inc, Augusta, GA).

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2.8. Statistical analysis

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Results were expressed as the mean Sstandard error of measurements (SEM). Statistical comparisons were performed using one-way ANOVA followed by the Tukey’s post hoc test using SPSS statistical software (v21, IBM Corporation, Somers, NY), and a value of p < 0.05 was considered significant.

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3. Results

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2.5. Measurement of reactive oxygen species (ROS)

3.1. Biological effects of ELF-EMF exposure on hBM-MSCs

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Intracellular reactive oxygen sepsis (ROS) generation was measured using a fluorometer. The ROS-sensitive dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Sigma–Aldrich, St. Louis, MO, USA). H2DCF-DA, was passively entered, converted to dichlorofluorescin diacetate (DCFH), reacted with ROS and formed the fluorescent product, dichlorofluorescin (DCF). The cells were cultured in 35 mm dish (5  104) for 24 h at 37 °C. Next day, NH medium were changed with DM for 16 h, and the cells preincubated in the presence or absence of 10 lM AG1478 and 5 mM NAC for

Recently, we reported that ELF-EMF induces hBM-MSC into neural differentiation in growth media (Cho et al., 2012). However, the mechanisms of action of EMF were not defined yet. Thus, in order to examine it, we used a neuronal differentiation media (DM). DM consisted of DMEM/F12 including hydrocortisone, insulin, forskolin, valproic acid and KCl, without BHA. These components plus BHA could induce MSCs into neurogenic phenotype but the cell die within 14 days of culture (Safford et al., 2002; Safford et al., 2004). Thus, we excluded BHA in this study.

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2. Materials and methods

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2.1. Cell culture

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Human BM-MSCs (hBM-MSCs) were purchased from Lonza (Basel, Switzerland) and maintained in culture in non-hematopoietic (NH) stem cell medium (Meltenyi Biotech, Beergisch Gladbach, Germany) supplemented with 100 U/ml of penicillin, 100 lg/ml of streptomycin (Invitrogen, Carlsbad, CA, USA) in 37 °C incubator at 5% CO2 humidified atmosphere. BM-MSCs were used from passages 4 to 7 with similar results obtained throughout. BM-MSCs were used from three different donors to abolish donor variation.

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2.2. Neuronal induction

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To induce neuronal differentiation of hBM-MSCs, we modified published neuronal induction protocols (Safford et al., 2002). One day before the initiation of differentiation, the BM-MSCs were seeded in NH medium at a density of 5  103 cells/cm2. To initiate neuronal differentiation, the cells were washed with PBS and replaced with neuronal differentiation medium (DM) composed of DMEM/F12 with KCl (5 mM), valproic acid (2 lM), forskolin (10 lM), hydrocortisone (1 lM), and insulin (5 g/ml). We moved the culture dishes to electromagnetic fields within 16 h after treatment of differentiation medium during the period of neuronal induction and medium changes were made twice a week.

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2.3. Electromagnetic fields exposure system

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BM-MSCs were exposed to continuous sinusoidal ELF-EMF (50 Hz or 100 Hz, 1 mT) in a system formed by two Helmholtz coils (15 cm inner diameter) oriented to produce a vertical magnetic field. The exposure system used that of our previous report (Cho et al., 2012). Samples were placed at the center of a uniform field area. The system was located in a cell culture incubator with 5% CO2 at 37 °C. Control cultures were grown in a separate incubator without an exposure system. A thermometric probe placed in inside and outside the ELF-EMF generator revealed no significant temperature difference between culture media of exposed or unexposed cells.

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2.4. Measurement of viability

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The mitochondria activity of the BM-MSCs after ELF-EMF exposure was determined by colorimetric assay, which detects the conversion of 3(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma–Aldrich) to formazan. For the assay, cells (5  104) were incubated in the presence or absence of ELF-EMF exposure described above. At the end of each time point, MTT solution (1 mg/ml) was added. Then the cells were incubated at 37 °C for 1 h and solubilized with DMSO. The dish was shaken and measured using a spectrophotometer (Versamax microplate reader, Molecular Device; Sunnyvale, CA, USA) at a wavelength of 570 nm.

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To induce neuronal differentiation of hBM-MSCs, we replaced the growth media (NH) with DM for 16 h and then exposed the cells onto EMF equipment. To determine the alteration of viability after consistent exposure of 50 or 100 Hz, MTT assay was performed on hBM-MSCs at 4 and 8 day. As shown in Fig. 1, cell viability under exposure to EMF did not show any differences at all-time points. Given these results that ELF-EMF does not affect any cytotoxic effect on hBM-MSCs. To examine the effect of EMF on neuronal differentiation of hBM-MSCs, at 6 days after EMF treatment, neurogenic markers were examined by immunoblotting and immunohistochemistry. As shown in Fig. 1B, expressions of neuronal markers such as neuroD1, mitogen activated protein (MAP) 2, and low-molecularweight neurofilament (NF-L) were significantly increased in EMF treated cells compared with that of control. To confirm the expression level of neuronal markers in each hBM-MSCs, cells were stained with indicated antibodies after ELF-EMF exposure (50 or 100 Hz; 1 mT) (Fig. 1C). Taken together, these suggest that ELF-EMF exposure could enhance neuronal differentiation of hBM-MSC.

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3.2. Effects of ELF-EMF exposure on CREB phosphorylation

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Cyclic AMP response element binding protein (CREB) is associated with neuronal survival, development and differentiation as well as axonal outgrowth and regeneration (Barco A, 2006; Carlezon et al., 2005; Dragunow, 2004) and it is involved in Ca2+-mediated action of ELF-EMF on neuronal differentiation of neuronal stem cells (Piacentini et al., 2008). Hence, we investigated whether

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CREB phosphorylation is modulated by ELF-EMF. Indeed, CREB phosphorylation was significantly increased at 90 min in response to ELF-EMF in hBM-MSCs (Fig. 2).

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3.3. ELF-EMF induced phosphorylation of CREB is mediated by EGFR

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Next, we were trying to find out upstream regulators of CREB. Protein kinase A (PKA) and Protein kinase C (PKC) was not activated by ELF-EMF (data not shown) whereas Akt (PKB) and phosphoinositide 3-kinase (PI3K) signaling pathway was activated (Fig. 2). However, ELF-EMF did not activate extracellular-regulated kinase (ERK) (Fig. 2). These data indicate that PI3K/Akt pathway may contribute to mediate the neuronal differentiation of hBMMSCs. Akt is activated through receptor tyrosine kinase pathways, such as those of platelet derived growth factor receptor (PDGF-R), insulin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor I (IGF-I) (Liao and Hung, 2010). Among those receptor tyrosine kinases, EGFR was known to be phosphorylated and clustered by ELF-EMF exposure in chinese hamster lung (CHL) fibroblast cells (Sun et al., 2004). Consistent with this previous report, phosphorylation of EGFR was induced following to exposure of cells to ELF-EMF for 90 min (Fig. 2). Moreover, we confirmed the EGFR clustering induced by ELF-EMF in hBM-MSCs by immunocytochemical staining (Fig. 3C). These data suggest that ELF-EMF exposure causes EGFR activation via phosphorylation and clustering, which may be lead the activation of the PI3K/Akt signaling pathway and an increase of the CREB phosphorylation.

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Fig. 1. Biological effects of ELF-EMF exposure on hBM-MSCs. (A) Viability of hBM-MSCs was examined by MTT assay with or without EMF stimulation for 4 or 8 days. Data represents mean ± SEM (n = 3, ⁄p < 0.05 vs. 100 Hz at 4 day) (B) At 6 days after ELF-EMF stimulation, cell lysates were immunoblotted with each neuronal marker antibody. The intensity of the each band was normalized to that of b-actin and presented in bar graphs (right panel). The data represent the means ± SEM of three independent experiments. ⁄p < 0.05 vs. NH treated, #p < 0.05 vs. DM treated sample. (C) Confocal fluorescence images of neuronal specific marker proteins were obtained from control and 50 Hz or 100 Hz of ELF-EMF exposure on hBM-MSCs for 5 days.

Please cite this article in press as: Park, J.-E., et al. Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation. Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.02.002

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Fig. 2. ELF-EMF induces EGFR, Akt and CREB phosphorylation in BM-MSCs. (A) BM-MSCs were stimulated with ELF-EMF for the indicated time, and whole cell lysates were immunoblotted with each antibody. (B) The intensity of the phosphorylated band was normalized to that of the total and presented in bar graphs. The data represent the means ± SEM of three independent experiments. ⁄p < 0.05 or ⁄⁄p < 0.001 vs. Control, #p < 0.05 vs. 180 min treated, àp < 0.05 vs. 45 min treated cells. C, Control, unstimulated cells.

Fig. 3. N-acetylcysteine and AG1478 prevents activation of EGFR and downstream signaling molecules induced by ELF-EMF in hBM-MSCs. BM-MSCs were pretreated with or without AG1478 (10 lM), or NAC (5 mM) for 30 min before ELF-EMF treatment. (A) Whole cell lysates were immunoblotted with each antibody. (B) The intensity of the phosphorylated band was normalized to that of the total and presented in bar graphs. The data represent the means ± SEM of three independent experiments (⁄p < 0.05 vs. 50 Hz-90 min). (C) Confocal fluorescence images of pEGFR localization (upper panel) and pCREB (lower panel) were obtained from control and ELF-EMF treated hBM-MSCs for 90 min with or without AG1478 and NAC pretreatment for 30 min.

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3.4. ELF-EMF-induced CREB phosphorylation was abolished by treatment of EGFR inhibitor To confirm whether ELF-EMF-induced CREB phosphorylation may be through EGFR activation, we used the EGFR inhibitor, AG1478. Phosphorylation of EGFR induced by ELF-EMF was inhibited by treatment of AG1478 (Fig. 3A) and ELF-EMF-induced EGFR clustering and phosphorylation was also diminished by AG1478 treatment (Fig. 3C). Phosphorylation of Akt and CREB as EGFR downstream molecules was inhibited in the presence of AG1478. These data demonstrated that ELF-EMF triggers EGFR phosphorylation and subsequent activations of Akt and CREB.

3.5. ELF-EMF-mediated EGFR activation blocked by ROS scavenging

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To explore whether ELF-EMF-mediated EGFR activation is directly associated with reactive oxygen species (ROS), we pre-incubated hBM-MSCs with N-Acetyl-Cysteine (NAC), a scavenger of ROS, prior to ELF-EMF exposure. Scavenging of ROS with NAC treatment suppressed ELF-EMF-induced EGFR phosphorylation. In addition, ELF-EMF-induced Akt and CREB phosphorylation was blocked in the presence of NAC (Fig. 3A). Likewise western blotting results, immunocytochemistry analyses showed further diminished signals from phospho-EGFR and phospho-CREB in NAC treated cells. (Fig. 3C). Collectively, these

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Fig. 4. ROS production induced by ELF-EMF is required for neuronal differentiation of hBM-MSCs. The effects of ELF-EMF exposure on the generation of ROS was measured by incubation with DCF-DA. (A) Imaging of DCF-DA staining was performed at 90 min after ELF-EMF exposure. Scale bar; 100 lm (B) ROS generation was measured by the fluorescence intensity of DCF. Data are expressed as mean ± SEM (n = 3). ⁄⁄p < 0.001 vs. DM only, #p < 0.05 vs. AG only. (C) Neural differentiation of hBM-MSCs induced by ELFEMF was inhibited by AG1478 or NAC pretreatment. Western blot of NeuroD1 in hBM-MSCS grown in differentiation medium exposed ELF-EMF with AG-1478 or NAC for 5 days. The intensity of the each band was normalized to that of b-actin and presented in bar graphs (lower panel). The data represent the means ± SEM of three independent experiments. ⁄⁄p < 0.001 vs. 50 Hz.

Fig. 5. Schematic representation of the proposed mechanism that mediates the phosphorylation of CREB upon extremely low electromagnetic fields. This pathway is mediated by EMF-induced activation of NADH oxidase which generates ROS at the plasma membrane. And then, increased ROS phosphorylates EGFR which in turn activates CREB through PI3K/Akt pathway. EMF-induced CREB phosphorylation can promote neuronal differentiation of BM-MSCs.

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data showed that ELF-EMF-induced EGFR phosphorylation is mediated by ROS production and subsequently Akt and CREB phosphorylation is mediated by ROS-induced EGFR activation in hBM-MSCs.

3.6. ELF-EMF elevates ROS level

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Intracellular generation of ROS was measured via the fluorescent product dichlorofluorescein (DCF). ELF-EMF exposed cells

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showed an increase in DCF fluorescence intensity (Fig. 4B), indicating the ROS production by ELF-EMF, but the antioxidant NAC attenuated ROS generation (p < 0.05). The ROS levels were slightly reduced by preincubation with EGFR inhibitor, AG1478. These experiments support and extend previous observations that NAC attenuates EGF-dependent ROS generation (Paulsen et al., 2012). Images of ROS staining with H2DCF-DA showed correspondent results with fluorocytometry analysis. ELF-EMF exposed cells further highlighted fluorescent signals than DM only treated cells and H2O2 treated cells also exhibited an increase of signal likewise ELF-EMF exposed cells. But, when the NAC pretreated with DM, the fluorescent signal diminished in both ELF-EMF exposed cells and H2O2 treated cells (Fig. 4A).

3.7. Inhibition of EGFR and scavenging of ROS can suppress neural differentiation of hBM-MSCs induced by ELF-EMF So far, we confirmed that ELF-EMF can produce ROS in hBMMSCs, and this event can modify the EGFR signaling pathways. Eventually, ELF-EMF exposure can promote neural differentiation of hBM-MSCs. Next, in order to determine whether the increased ROS were responsible for the neural differentiation effects of ELFEMF, we used AG1478 and NAC. Significant increase in NeuroD1 was observed when cells were exposed to ELF-EMF for 5 days, which was attenuated by treatment of AG1478 and NAC, indicating that ELF-EMF promotes neural differentiation of hBM-MSCs via ROS-induced EGFR activation (Fig. 4C).

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In the present study, we demonstrated that ELF-EMF accelerates neural differentiation of BM-MSCs via ROS-induced EGFR activation and, subsequently, Akt and CREB phosphorylation. It has been well documented that ELF-MF exposure may alter cellular homeostasis by increasing intracellular ROS concentrations. In this study, we examined that ELF-EMF increased ROS levels in BM-MSCs and the produced ROS plays an important role in neural differentiation. Consistent with our observations, Kanda et al. demonstrated that ROS plays an important role during the early stage of adipocyte differentiation in MSCs. They showed that adipocyte differentiation is impaired in the presence of ROS scavengers or RNA interference against NOX4. Furthermore, they showed that CREB acts as a downstream of ROS in adipocyte differentiation (Kanda et al., 2011). However, signaling pathway of ROSmediated CREB phosphorylation was not elucidated yet. ROS can oxidize a conserved, essential cysteine residue in the catalytic domain of phosphatases, thereby disrupting the catalytic activity of the phosphatases (Barrett et al., 1999; Das and Vasudevan, 2007; Robinson et al., 1999). Moreover, mobile phone irradiation induces production of ROS that activates metalloproteinases and, consequently, the release of Hb-EGF (heparin-binding EGF) to activate EGFR and the MAPK cascade in HeLa cells (Friedman et al., 2007). Furthermore, synergistically, binding of EGF to the extracellular domain of the EGFR results in the assembly and activation of NADPH oxidase (NOX) complexes, which generate H2O2 (Friedman et al., 2007; Paulsen et al., 2012). However, in the present study, ELF-EMF did not activate the MAPK cascade in BM-MSCs. This discrepancy might be resulted from the differences in cell types and EMF frequency. Indeed, no short term activation of ERKs was detected in auditory hair cells treated for 15 min with high frequency EMFs (Halliwell and Gutteridge, 1997) and ERK phosphorylation is dependent on the radiation intensity (Consales et al., 2012). Thus, these suggest that ELF-EMF may stimulate increased EGFR signaling through ROS production.

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Some reports demonstrated that activated EGFR signaling in post-natal neural stem cells induces aggressive characteristics, including enhanced proliferation, survival and migration. In contrary, it has been reported that EGF but not PDGF stimulates differentiation of MSCs into bone forming cells (Kratchmarova et al., 2005) and EGF promotes the differentiation of umbilical cord blood derived MSCs into neuron-like cells (Jin et al., 2009). These support our observation that ELF-EMF-induced EGFR activation is required for the neuronal differentiation of hBM-MSCs. What remains to be clarified is that how ROS production is regulated by ELF-EMF and which oxidant/antioxidant system is implicated in this differentiation mechanism. We summarized our results as schematic representation and proposed that EMF-induced activation of NADH oxidase which generates ROS at the plasma membrane. And then, increased ROS phosphorylates EGFR which in turn activates CREB through PI3K/ Akt pathway. EMF-induced CREB phosphorylation can promote neuronal differentiation of BM-MSCs (Fig. 5). In conclusion, for the first time we demonstrated that ELF-EMF facilitates neuronal differentiation from hBM-MSCs and identified underlying mechanism.

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Acknowledgments

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This work was supported by the Pioneer Research Program of the National Research Foundation of Korea and was funded by the Ministry of Education, Science, and Technology (NRF-20090082941).

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Please cite this article in press as: Park, J.-E., et al. Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation. Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.02.002

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