Previous studies have reported that extremely low-frequency electromagnetic fields (ELF-EMF) can affect the processes of brain development, but the underlying mechanism is largely unknown. The proliferation and differentiation of embryonic neural stem cells (eNSCs) is essential for brain development during the gestation period. To date, there is no report about the effects of ELF-EMF on eNSCs. In this paper, we studied the effects of ELF-EMF on the proliferation and differentiation of eNSCs. Primary cultured eNSCs were treated with 50 Hz ELF-EMF; various magnetic intensities and exposure times were applied. Our data showed that there was no significant change in cell proliferation, which was evaluated by cell viability (CCK-8 assay), DNA synthesis (Edu incorporation), average diameter of neurospheres, cell cycle distribution (flow cytometry) and transcript levels of cell cycle related genes (P53, P21 and GADD45 detected by real-time PCR). When eNSCs were induced to differentiation, real-time PCR results showed a down-regulation of Sox2 and up-regulation of Math1, Math3, Ngn1 and Tuj1 mRNA levels after 50 Hz ELF-EMF exposure (2 mT for 3 days), but the percentages of neurons (Tuj1 positive cells) and astrocytes (GFAP positive cells) were not altered when detected by immunofluorescence assay. Although cell proliferation and the percentages of neurons and astrocytes differentiated from eNSCs were not affected by 50 Hz ELF-EMF, the expression of genes regulating neuronal differentiation was altered. In conclusion, our results support that 50 Hz ELF-EMF induce molecular changes during eNSCs differentiation, which might be compensated by post-transcriptional mechanisms to support cellular homeostasis.
References
[1]
Lacy-Hulbert A, Metcalfe JC, Hesketh R (1998) Biological responses to electromagnetic fields. FASEB J 12: 395–420.
[2]
Kheifets LI, Afifi AA, Buffler PA, Zhang ZW (1995) Occupational electric and magnetic field exposure and brain cancer: a meta-analysis. J Occup Environ Med 37: 1327–1341. doi: 10.1097/00043764-199512000-00002
[3]
Qiu C, Fratiglioni L, Karp A, Winblad B, Bellander T (2004) Occupational Exposure to Electromagnetic Fields and Risk of Alzheimer's Disease. Epidemiology 15: 687–694. doi: 10.1097/01.ede.0000142147.49297.9d
[4]
Vazquez-Garcia M, Elias-Vinas D, Reyes-Guerrero G, Dominguez-Gonzalez A, Verdugo-Diaz L, et al. (2004) Exposure to extremely low-frequency electromagnetic fields improves social recognition in male rats. Physiol Behav 82: 685–690. doi: 10.1016/j.physbeh.2004.06.004
[5]
Das S, Kumar S, Jain S, Avelev VD, Mathur R (2012) Exposure to ELF- magnetic field promotes restoration of sensori-motor functions in adult rats with hemisection of thoracic spinal cord. Electromagn Biol Med 31: 180–194. doi: 10.3109/15368378.2012.695706
[6]
Liu YHR, Yu YM, Weng EQ (2003) Effects of extremely low frequency electromagnetic fields on apoptosis and cell cycle of mouse brain and liver cells. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 21 (5) 339–341.
[7]
Elizabeth H, McFarlane , Dawe Gavin S, Marks M, Iain CC (2000) Changes in neurite outgrowth but not in cell division induced by low EMF exposure: influence of field strength and culture conditions on responses in rat PC12 pheochromocytoma cells. Bioelectrochemistry 52: 23–28. doi: 10.1016/s0302-4598(00)00078-7
[8]
Lisi A, Ciotti MT, Ledda M, Pieri M, Zona C, et al. (2005) Exposure to 50 Hz electromagnetic radiation promote early maturation and differentiation in newborn rat cerebellar granule neurons. J Cell Physiol 204: 532–538. doi: 10.1002/jcp.20322
[9]
Bodega G, Forcada I, Suarez I, Fernandez B (2005) Acute and chronic effects of exposure to a 1-mT magnetic field on the cytoskeleton, stress proteins, and proliferation of astroglial cells in culture. Environ Res 98: 355–362. doi: 10.1016/j.envres.2004.12.010
[10]
Yoshimatsu T, Kawaguchi D, Oishi K, Takeda K, Akira S, et al. (2006) Non-cell-autonomous action of STAT3 in maintenance of neural precursor cells in the mouse neocortex. Development 133: 2553–2563. doi: 10.1242/dev.02419
[11]
Grassi C, D'Ascenzo M, Torsello A, Martinotti G, Wolf F, et al. (2004) Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death. Cell Calcium 35: 307–315. doi: 10.1016/j.ceca.2003.09.001
[12]
Piacentini R, Ripoli C, Mezzogori D, Azzena GB, Grassi C (2008) Extremely low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Ca(v)1-channel activity. J Cell Physiol 215: 129–139. doi: 10.1002/jcp.21293
[13]
Cuccurazzu B, Leone L, Podda MV, Piacentini R, Riccardi E, et al. (2010) Exposure to extremely low-frequency (50 Hz) electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6 mice. Experimental Neurology 226: 173–182. doi: 10.1016/j.expneurol.2010.08.022
[14]
Chen C, Zhou Z, Zhong M, Li M, Yang X, et al. (2011) Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway. Neurotox Res 20: 15–25. doi: 10.1007/s12640-010-9214-y
[15]
Schuderer J, Oesch W, Felber N, Spat D, Kuster N (2004) In vitro exposure apparatus for ELF magnetic fields. Bioelectromagnetics 25: 582–591. doi: 10.1002/bem.20037
[16]
Yao L, Chen X, Tian Y, Lu H, Zhang P, et al. (2012) Selection of housekeeping genes for normalization of RT-PCR in hypoxic neural stem cells of rat in vitro. Mol Biol Rep 39: 569–576. doi: 10.1007/s11033-011-0772-8
[17]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. doi: 10.1006/meth.2001.1262
[18]
International Commission on Non-Ionizing Radiation Protection (1998) Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). Health Physics 74: 494–522.
[19]
Nikolova T, Czyz J, Rolletschek A, Blyszczuk P, Fuchs J, et al. (2005) Electromagnetic fields affect transcript levels of apoptosis-related genes in embryonic stem cell-derived neural progenitor cells. FASEB J 19 (12) 1686–1688. doi: 10.1096/fj.04-3549fje
[20]
Liu YX, Tai JL, Li GQ, Zhang ZW, Xue JH, et al. (2012) Exposure to 1950-MHz TD-SCDMA electromagnetic fields affects the apoptosis of astrocytes via caspase-3-dependent pathway. PLoS One 7: e42332. doi: 10.1371/journal.pone.0042332
[21]
Ivancsits Sabine, Diem Elisabeth, Pilger Alexander, Hugo W, Rüdiger , Jahn O (2002) Induction of DNA strand breaks by intermittent exposure to extremely-low-frequency electromagnetic fields in human diploid fibroblasts. Mutation Research 519: 1–13. doi: 10.1016/s1383-5718(02)00109-2
[22]
Burdak-Rothkamm S, Rothkamm K, Folkard M, Patel G, Hone P, et al. (2009) DNA and chromosomal damage in response to intermittent extremely low-frequency magnetic fields. Mutat Res 672: 82–89. doi: 10.1016/j.mrgentox.2008.10.016
[23]
Focke F, Schuermann D, Kuster N, Schar P (2010) DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure. Mutat Res 683: 74–83. doi: 10.1016/j.mrfmmm.2009.10.012
[24]
Nam C, Doi K, H N (2010) Etoposide induces G2/M arrest and apoptosis in neural progenitor cells via DNA damage and an ATM/p53-related pathway. Histol Histopathol 25 (4) 485–493.
[25]
Huang YY, Lu H, Liu S, Droz-Rosario R, Z S (2012) Requirement of Mouse BCCIP for Neural Development and Progenitor Proliferation. PLoS One 7 (1) e30638. doi: 10.1371/journal.pone.0030638
[26]
Matthew Hirsch L, B. Matthew Fagan , Dumitru Raluca, Jacquelyn Bower J, Yadav Swati, et al. (2011) Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells. PLoS One 6 (11) e27520. doi: 10.1371/journal.pone.0027520
[27]
Carlessi L, De Filippis L, Lecis D, Vescovi A, Delia D (2009) DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression. Cell Death Differ 16: 795–806. doi: 10.1038/cdd.2009.10
[28]
Ishiyama Munetaka, Miyazono Yoko, Sasamoto Kazumi, Ohkura Yosuke, Ueno K (1997) A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 44: 1299–1305. doi: 10.1016/s0039-9140(97)00017-9
[29]
Hashimoto D, Ohmuraya M, Hirota M, Yamamoto A, Suyama K, et al. (2008) Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells. J Cell Biol 181 (7) 1065–1072. doi: 10.1083/jcb.200712156
[30]
Nishino J, Kim I, Chada K, Morrison SJ (2008) Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell 135: 227–239. doi: 10.1016/j.cell.2008.09.017
[31]
Ruzhynsky VA, McClellan KA, Vanderluit JL, Jeong Y, Furimsky M, et al. (2007) Cell Cycle Regulator E2F4 Is Essential for the Development of the Ventral Telencephalon. Journal of Neuroscience 27: 5926–5935. doi: 10.1523/jneurosci.1538-07.2007
[32]
Vanderluit JL, Ferguson KL, Nikoletopoulou V, Parker M, Ruzhynsky V, et al. (2004) p107 regulates neural precursor cells in the mammalian brain. J Cell Biol 166: 853–863. doi: 10.1083/jcb.200403156
[33]
Bae I, Fan S, Bhatia K, Kohn KW, Fornace AJ Jr, et al. (1995) Relationships between G1 arrest and stability of the p53 and p21Cip1/Waf1 proteins following gamma-irradiation of human lymphoma cells. Cancer Res 55: 2387–2393.
[34]
Czyz J, Nikolova T, Schuderer J, Kuster N, Wobus AM (2004) Non-thermal effects of power-line magnetic fields (50 Hz) on gene expression levels of pluripotent embryonic stem cells-the role of tumour suppressor p53. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 557: 63–74. doi: 10.1016/j.mrgentox.2003.09.011
[35]
Louis SA, Mak CK, Reynolds BA (2013) Methods to culture, differentiate, and characterize neural stem cells from the adult and embryonic mouse central nervous system. Methods Mol Biol 946: 479–506. doi: 10.1007/978-1-62703-128-8_30
[36]
Graham V, Khudyakov J, Ellis P, Pevny L (2003) SOX2 Functions to Maintain Neural Progenitor Identity. Neuron 39: 749–765. doi: 10.1016/s0896-6273(03)00497-5
[37]
Pevny LH, Nicolis SK (2010) Sox2 roles in neural stem cells. Int J Biochem Cell Biol 42: 421–424. doi: 10.1016/j.biocel.2009.08.018
[38]
Favaro R, Valotta M, Ferri AL, Latorre E, Mariani J, et al. (2009) Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat Neurosci 12: 1248–1256. doi: 10.1038/nn.2397
[39]
Sikorska M, Sandhu JK, Deb-Rinker P, Jezierski A, Leblanc J, et al. (2008) Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation. J Neurosci Res 86: 1680–1693. doi: 10.1002/jnr.21635
[40]
Bertrand N, Castro DS, Guillemot F (2002) Proneural genes and the specification of neural cell types. Nat Rev Neurosci 3 (7) 517–530. doi: 10.1038/nrn874
[41]
Ross SE, Greenberg ME, Stiles CD (2003) Basic Helix-Loop-Helix Factors in Cortical Development. Neuron 39: 13–25. doi: 10.1016/s0896-6273(03)00365-9
[42]
Harry GJ, Bartenbach M, Haines W, A B (2000) Developmental profiles of growth-associated protein (Gap43), Ngfb, Bndf and Ntf4 mRNA levels in the rat forebrain after exposure to 60 Hz magnetic fields. Radiat Res 153: 642–647. doi: 10.1667/0033-7587(2000)153[0642:dpogap]2.0.co;2
[43]
Del Vecchio G, Giuliani A, Fernandez M, Mesirca P, Bersani F, et al. (2009) Continuous exposure to 900 MHz GSM-modulated EMF alters morphological maturation of neural cells. Neurosci Lett 455: 173–177. doi: 10.1016/j.neulet.2009.03.061
