Life Sciences 102 (2014) 16–27
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Egr1 mediated the neuronal differentiation induced by extremely low-frequency electromagnetic fields Yeju Seong, Jihye Moon, Jongpil Kim ⁎ Department of Biomedical Engineering, Dongguk University, Seoul 100-715, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 November 2013 Accepted 17 February 2014 Available online 3 March 2014 Keywords: Human bone marrow–mesenchymal stem cell Mouse neural stem cell Neuronal differentiation Transcription factor Egr1 Extremely low-frequency electromagnetic fields
a b s t r a c t Aim: There is a specific frequency of extremely low-frequency electromagnetic field (ELF-EMF) that promotes neuronal differentiation. Although several mechanisms are known to regulate ELF-EMF-induced neuronal differentiation, a key factor that mediates neurogenic potentials by the ELF-EMF is largely unknown. Also, the potential use of ELF-EMF exposure in cell transplantation assays is yet to be determined, including their possible use in ELFEMF based therapy of neurological diseases. The aim of this study is to understand the underlying mechanisms that mediate ELF-EMF-induced neuronal differentiation and also to harness these mechanisms for cell transplantation assays. Main method: Human bone marrow–mesenchymal stem cells (hBM–MSCs) were exposed to ELF-EMF (50 Hz frequency, 1 mT intensity) for 8 days. The hBM–MSC derived neurons were then analyzed by general molecular biology techniques including immunofluorescence and quantitative RT-PCR. To assess changes in gene expression induced by ELF-EMF exposure, we analyzed the transcriptome of neuronal cells after an 8-day ELF-EMF exposure (50 Hz, 1 mT) and compared the transcriptional profiles to control cells. Key finding: We found that early growth response protein 1 (Egr1) is one of the key transcription factors in ELFEMF-induced neuronal differentiation. In addition, we show that transplantations of ELF-EMF-induced neurons significantly alleviate symptoms in mouse models of neurodegenerative disease. Significance: These findings indicate that a specific transcriptional factor, Egr1, mediates ELF-EMF-induced neuronal differentiations, and demonstrate the promise of ELF-EMF based cell replacement therapies for neurodegenerative diseases. © 2014 Elsevier Inc. All rights reserved.
Introduction Stem cell based cell replacement therapies are a highly promising method of repairing neurons which have been damaged through neurodegenerative diseases (Körbling and Estrov, 2003; Lindvall et al., 2004). In particular, human bone marrow (hBM)-derived mesenchymal stem cells (MSCs) have the ability to differentiate into several neural types, and can be readily prepared for autologous cells, showing great potential in cell replacement therapies for neurodegenerative diseases (Joyce et al., 2010). Several studies suggest that MSCs have the ability to induce improvements in models of neurological diseases (Karussis et al., 2008; Torrente and Polli, 2008). However, limitations exist in the ability of MSCs to be stably engrafted in cell replacement procedures, since their efficacy relies on the secretions of cytokines for direct differentiation (Salem and Thiemermann, 2010; Uccelli et al., 2008). Several chemicals and additional differentiation factors have been identified to create a better environment for neuronal differentiation for more still yield low efficiency therapy (Sanchez-Ramos et al., 2000;
⁎ Corresponding author. E-mail address: [email protected] (J. Kim).
http://dx.doi.org/10.1016/j.lfs.2014.02.022 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Woodbury et al., 2000) and show evidence of in vivo toxicity (Burdon et al., 2011; Lu et al., 2004). Extremely low-frequency electromagnetic field (ELF-EMF) exposure is known to have effects on several biological processes, such as cell proliferation, differentiation, gene expression pattern, and regulation of transcription factor (Blank, 1993; Goodman et al., 1995). Particularly, recent results indicate that a specific frequency of ELF-EMF treatment can have strong neurogenic effects in stem cells (Güven et al., 2005). For example, 50 Hz ELF-EMF exposure has been shown to promote neuronal differentiation in rat chromaffin cells (Morgado-Valle et al., 1998) and neural stem cells (Piacentini et al., 2008). Neural differentiation on PC12 cells has also been observed following ELF-EMF exposure (Morabito et al., 2010; Zhang et al., 2005). In our previous study, we also showed the increased neuronal differentiation in hBM–MSCs following ELF-EMF exposure (50 Hz, 1 mT) (Cho et al., 2012), providing a feasible non-chemical solution for neuronal differentiation of stem cells for cell replacement therapy. Despite various studies exploring the effect of ELF-EMF exposure on neuronal differentiation, the underlying mechanism for this pathway is still largely unknown. Several studies suggest that the intracellular Ca2+ signaling plays a critical role in ELF-EMF induced neuronal differentiation (Cuccurazzu et al., 2010). Other studies also have shown that
Y. Seong et al. / Life Sciences 102 (2014) 16–27
changes in the K+ current by K+ channel activation mediate ELF-EMF effects in neuronal differentiation (Blank and Soo, 1993; Funk and Monsees, 2006). Additionally, it has been reported that free radicals or ROSs play an important role in ELF-EMF based neural differentiation (Park et al., 2013). However, the epigenetic mechanisms governing neuronal cell fate changes by ELF-EMF exposure remain unclear. In this study we investigate the epigenetic mechanisms of ELF-EMF (50 Hz, 1 mT) induced neuronal differentiation. Initially, we show that ELF-EMF exposure leads to efficient neuronal differentiation in both human BM–MSC and mouse neural stem cells. In addition, early growth response protein 1 (Egr1), a strong early neurogenic transcription factor, is identified as a mediator in ELF-EMF-induced neuronal differentiations. The overexpression of Egr1 combined with ELF-EMF exposure significantly increases the efficiency of cell replacement therapy. Taken together, our results provide the molecular basis of ELF-EMF-induced neuronal differentiation for efficient cell replacement therapy.
Materials and methods
17
Viral infection An Egr1-knockdown lentiviral vector encoding a specific hairpin for the Egr1 gene and the Egr1 cDNA were cloned into the EcoRI cloning site of FUW lentiviral vectors. For lentiviral vector infections of hBM–MSCs, cells were seeded in six-well plates at a density of 3 × 104 cells per well and infected on 3 consecutive days. Medium changes were performed 12–24 h after infection. Fresh differentiation medium was added every other day for 8 days after infection. Immunofluorescence Cells were fixed in 4% paraformaldehyde in PBS and immunostained according to the standard protocols using the following primary antibodies: anti-TuJ1 (1:300 dilution), anti-TH (1:300 dilution) (Sigma-Aldrich, USA), anti-vMAT2 (1:300 dilution) (Millipore, USA), anti-NeuroD1 (1:300 dilution), anti-MAP2 (1:300 dilution) (Cell Signaling, USA) and appropriate fluorescent secondary antibodies (1:500 dilution) (Invitrogen, USA).
Cell culture & ELF-EMF exposure
Quantitative real time-PCR analysis
Human bone marrow–mesenchymal stem cells (hBM–MSCs) were purchased from CEFO, Co. Ltd. (Seoul, Korea) and were cultured in StemMACS MSC Expansion Medium (Miltenyi Biotec, Germany). Cells were grown at 37 °C for 3–4 days until confluent, and then were subcultured with Accutase (Innovative Cell Technology, USA). To induce neuronal differentiation by ELF-EMF exposure, hBM–MSCs were seeded at a density of 3 × 104 cells/cm2 at 35 mm cell culture plates and were changed into differentiation medium (DMEM/F12 (Gibco, USA), valproic acid 2 μM (Sigma-Aldrich, USA), forskolin 10 μM (LC laboratories, USA), hydrocortisone 1 μM (Sigma-Aldrich, USA), insulin 10 mg/ml (Welgene, Korea) and 1% penicillin/streptomycin (Gibco, USA)). A day after subculture, the cells were moved to the EMF generator and were continuously exposed to ELF-EMF (F = 50 Hz; Bm = 1 mT) for 8 days (Supplementary Fig. 4B). We utilized an ELF-EMF device which was previously described (Cho et al., 2012). Briefly, hBM–MSCs were exposed to continuous sinusoidal ELFEMF (50 Hz, 1 mT) in a system formed by two Helmholtz coils (15 cm inner diameter) oriented to produce a vertical magnetic field. This system was supplied by the COMSOL 3.4 (MA) and Tesla meter TM-701 (Japan) for magnetic flux density distribution or measured data, respectively. The culture was located in a cell culture incubator with 5% CO2 at 37 °C. Control cultures were grown in a separate incubator without Helmholtz coils.
Total RNA was isolated using a PureLink RNA Mini Kit (Invitrogen, USA). 400 ng of DNase treated RNA was reverse transcribed using a First Strand Synthesis Kit (Bioneer, Korea). Quantitative RT-PCR analysis was performed in triplicate using 1/40 of the reverse transcription reaction in a StepOnePlus Real-Time PCR System with SYBR Fast qPCR Kit (KAPA Biosystems, USA) (Table 1). The qPCR used a GAPDH control and a standard curve. Electrophysiology For electrophysiological recordings, ELF-EMF-induced hBM–MSCs were grown on 12 mm glass coverslips and transferred to recording medium containing the extracellular solution (in mM): 130 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.35, 325 mosM). Patch pipettes were filled with the following intracellular solution (in mM): 110 potassium gluconate, 20 KCl, 2 Mg-ATP, 10 sodium phosphocreatine, 1.0 EGTA, 0.3 GTP–Tris, and 20 HEPES (pH 7.25, 320 mosM). Electrodes were fabricated from borosilicate capillary glass tubing (Warner Instruments) with a capillary glass puller (Sutter Instruments). After establishing whole-cell mode, the cell membrane capacitance and series resistance were compensated to 75% electronically using a patch clamp amplifier (Axopatch 200B; Molecular Devices). Data generation and acquisition were performed using pClamp10 software on an IBM
Table 1 Primer sequence used for quantitative real-time PCR. Genes
Species
Sense
Antisense
Size
GAPDH Tubb3 MAP2 NeuroD1 CHAT Pitx3 ID2 ID3 Smad7 JunB Egr1 GAPDH CHAT GAD67 HB9 Pitx3 Egr1 DBP
Human Human Human Human Human Human Human Human Human Human Human Mouse Mouse Mouse Mouse Mouse Mouse Mouse
cgagatccctccaaaatcaa agctcacccagcagatgttc ctgcacactcacatccacct gttctcaggacgaggagcac tacaggctccaccgaagact agctagaggcgaccttcca cgtgaggtccgttaggaaaa actcagcttagccaggtgga gtggatggtgtgtgggtgta ccatcagctacctcccacac tgaacaacgagaaggtgctg aacgaccccttcattgacct cctgccagtcaactctagcc gcacagagaccgacttctcc ctcatgctcaccgagactca gaggaatcgctaccctgaca aaaggtggtttccaggttcc cattccaggccatgagactt
ttcacacccatgacgaacat gggatccactccacgaagta tctccgttgatcccattctc gtctcttgggcttttgatcg catccttcaggagcagaagc aagctgcctttgcatagctc gcaggatttccatcttgctc aagctccttttgtcgttgga tgctgcataaactcgtggtc actttgatgcgctcttggtc tgggttggtcatgctcacta ccctgttgctgtagccgtat ggaagccggtatgatgagaa aaaatcgagggtgacctgtg ccattgctgtacgggaagtt gggtacacctcctcgtaggg actgagtggcgaaggcttta tggctgcttcattgttcttg
170 175 166 168 178 172 179 167 185 178 173 280 222 294 287 201 166 161
18
Y. Seong et al. / Life Sciences 102 (2014) 16–27
computer equipped with an analog to digital converter (Digidata 1440A; Axon Instruments). Recordings were conducted on day 8 based on the neuron-like morphology including elevated cell bodies and the presence of branched and extended neurites. Once currentclamp mode was obtained, the cell was maintained at a potential of approximately −65 to −70 mV. Current injection protocol steps were applied ranging from − 100 pA to + 120 pA. Inward sodium current
and outward potassium current were measured in voltage-clamp mode with voltage step protocols ranging from −55 to +55 mV. Cell transplantation and behavior analysis All animal procedures were performed according to the guidelines and were approved by the Committee on Animal Care at Dongguk
ELF-EMF 50 Hz, 1 mT (8 days)
A
Neurons
Human BM-MSCs
C
Control
Tuj1
Tuj1 / DAPI
Tuj1
ELF-EMF
BF
Tuj1 / DAPI
80
Tuj1 + cells per field (%)
BF
**
60
40
20
0
NeuroD1 + cells per field (%)
B
80
60
**
40
20
0
D Tuj1
NeuroD1
Merge
F
+55 mV
-100 mV
n=12
n=12
n=12
500 pA
100 ms
RMF (mV)
20 mV
n=12
AP amplitude (mV)
E
DAPI
10 ms -50 mV
Fig. 1. ELF-EMF exposure on neuronal differentiation of hBM–MSCs. (A) Schematic procedure of ELF-EMF (50 Hz, 1 mT) exposure in neuronal differentiation of human BM–MSCs. After changing neuronal culture media, the hBM–MSCs were exposed to ELF-EMF for 8 days. (B) (upper) Morphology of control hBM–MSCs and ELF-EMF exposed hBM–MSCs (lower). Immunofluorescence images of ELF-EMF-induced neurons using antibody against Tuj1. TuJ1 expressing neurons are present in the hBM–MSC culture after an 8-day ELF-EMF (50 Hz, 1 mT) exposure. BF: bright field, scale bar = 50 μm (left, middle) and 100 μm (right). (C) Quantitative analysis of Tuj1 and NeuroD1 positive cells in the ELF-EMF-induced neuronal differentiations. Data shown are means ± SEM; Student's t-test, **p b 0.05. Three independent experiments of three sets each were performed with 10 visual fields per set. (D) Immunofluorescence images for neuronal marker, NeuroD1 in the ELF-EMF-induced neurons. Scale bar = 100 μm. (E) Electrophysiological properties of ELF-EMF-induced functional neurons using wholecell patch-clamp analysis. Representative images of action potentials (top panel) and voltage-dependent membrane currents (bottom panel) detected in ELF-EMF-induced neurons. (top panel) Bottom traces represent current injections (−20 pA to +120 pA), and top traces indicated voltage recordings. (bottom panel) Depolarizing voltage steps elicited fast inward sodium currents (bottom traces) and slow inactivating outward potassium currents (top traces). (F) Quantification of membrane properties in hBM–MSC derived neurons after an 8-day ELF-EMF exposure and primary embryonic neurons. Data are presented as mean ± SEM, n = 12. RMP: resting membrane potential, AP: action potential.
Y. Seong et al. / Life Sciences 102 (2014) 16–27
University. Mice were anesthetized with Avertin, and placed in a stereotaxic frame and 6-hydroxydopamine (6-OHDA) (Sigma-Aldrich, USA) was injected into the midbrain region (SN) (AP: −3.1 mm; ML: ±1.1 mm; DV: −4.4 mm). 4 weeks after 6-OHDA was lesioned, the mice were injected with apomorphine (Santa Cruz Biotechnology, USA, 4 mg/kg i.p. injection) and apomorphine-induced turning behavior was assessed before the cell grafting. Mice exhibiting net clockwise turns were lesioned in the left striatum, while mice exhibiting net counter-clockwise turns were lesioned in the right striatum (ipsilateral). The hBM–MSCs cells were resuspended in medium at a density of about 10,000 cells/μl and the cells were grafted into the lesioned striatum (AP: +0.4 mm; ML: ±1.5 mm; DV: −2.8 mm) with 2 or 3 μl of cell suspension. Apomorphine induced rotational behavior was measured again at 4 and 8 weeks after grafting. The mouse was kept in the field of ELFEMF (50 Hz frequency, 2 mT intensity) every two days for 2 or 4 weeks.
19
The number of clockwise and counter-clockwise turns was counted and expressed as the number of net rotations per 30 min to the hemisphere. Results ELF-EMF exposure on neuronal differentiation of human BM–MSCs Human BM–MSCs (hBM–MSCs) are capable of differentiating into several tissue types, such as adipocytes, osteocytes, chondrocytes, neurons and myocytes (Pittenger et al., 1999). In particular, exposure to specific neurotropic factors can trigger human BM–MSC differentiation into neurons, albeit with limitations in efficiency and toxicity (Long et al., 2005). In order to examine whether ELF-EMF exposure can alternatively influence neural differentiation of hBM–MSCs without neurotropic factors, we first induced neural differentiation of hBM–MSCs via
ELF-EMF 50 Hz, 1 mT (6 days)
A
ESC derived NSCs
Control
C
ELF-EMF Tuj1
Tuj1
TH
120
Tuj1 + cells per field (%)
B
Neurons
TH
120
**
100
TH + cells per field (%)
Mouse ESCs
80 60 40 20
100
50
0
*
150
GAD67 100
50
0
*
150
HB9 100
50
0
80 60 40 20 0
**
Relative gene expression (%)
ChAT
Relative gene expression (%)
Relative gene expression (%)
150
Relative gene expression (%)
0
D
*
100
150
Pitx3
* 100
50
0
Fig. 2. ELF-EMF exposure on neuronal differentiation of mouse neural stem cells. (A) Schematic procedure of ELF-EMF-induced neuronal differentiations of mouse neural stem cells. Mouse ES-derived neuronal precursors were differentiated into functional neurons under the exposure to ELF-EMF (50 Hz, 1 mT) for 6 days. (B) Representative immunofluorescence images for neuronal markers Tuj1 and TH in control neurons and ELF-EMF exposed neurons. Scale bar = 50 μm. (C) Quantitative analysis of Tuj1, and TH positive cells in the control neurons and ELFEMF-induced neurons. Proportion of Tuj1 and TH positive expressions. Data shown are means ± SEM; Student's t-test, *p b 0.5, **p b 0.05. Three independent experiments of three sets each were performed with 10 visual fields per set. (D) Quantitative real-time PCR of neuronal marker genes, ChAT, GAD67, HB9 and Pitx3 in ELF-EMF-induced neurons. Expression levels were normalized to Gapdh. Data are presented as mean ± SEM; Student's t-test, *p b 0.5, **p b 0.05, n = 3.
20
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exhibited neuronal morphology and an increased number of neuronspecific class III beta-tubulin (Tuj1) + cells (Fig. 1B and Supplementary Fig. 1A). Additionally, we observed that ELF-EMF exposed hBM–MSCs showed significant increase in the expression of endogenous neurogenic differentiation 1 (NeuroD1), a key marker for neuronal differentiation
ELF-EMF (50 Hz, 1 mT) exposure. After changing the neuronal culture media without neurotropic factors, hBM–MSCs were exposed to different frequencies (0 Hz, 50 Hz, 100 Hz, 200 Hz, at 1 mT intensity) of ELFEMF for 8 days (Fig. 1A and Supplementary Fig. 1A)). We observed that the culture treated with 50 Hz frequency of ELF-EMF exposure only
A
B 7
Signaling molecule
11
Transcription factor
5
Extracellular matrix
3
Protease
8
Nucleic acid binding
2
Cell adhesion molecule
3
Receptor
2
Transporter
2
Kinase Transferase
1
Select regulatory molecule
1
Phosphatase
1
Oxidoreductase
1
Miscellaneous function
1
Hydrolase
1
Defense/immunity protein
1 1
Cytoskeletal protein
0
ELF-EMF
5
10
15
Control
C Human BM-MSC derived neurons (Control vs ELF-EMF) 16
no significant IFCI>=2
Egr1
14
**
10 16
8
150
ID1 100
50
0
**
10
12
14
150
HES1 100
50
0
**
16
Relative gene expression (%)
Egr1
14
Relative gene expression (%)
12
150
0
Smad7 JunB
Relative gene expression (%)
Relative gene expression (%)
10
Egr1 ID2
8
8
RELB
50
BHLHB2
ID1 JunB
100
FOS
12
TCEA3
8
ID3
HES1
10
12
DBP
D
no significant
IFCI>=1.5 IFCI>=1.5 & adj.p
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Egr1 mediated the neuronal differentiation induced by extremely low-frequency electromagnetic fields Yeju Seong, Jihye Moon, Jongpil Kim ⁎ Department of Biomedical Engineering, Dongguk University, Seoul 100-715, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 November 2013 Accepted 17 February 2014 Available online 3 March 2014 Keywords: Human bone marrow–mesenchymal stem cell Mouse neural stem cell Neuronal differentiation Transcription factor Egr1 Extremely low-frequency electromagnetic fields
a b s t r a c t Aim: There is a specific frequency of extremely low-frequency electromagnetic field (ELF-EMF) that promotes neuronal differentiation. Although several mechanisms are known to regulate ELF-EMF-induced neuronal differentiation, a key factor that mediates neurogenic potentials by the ELF-EMF is largely unknown. Also, the potential use of ELF-EMF exposure in cell transplantation assays is yet to be determined, including their possible use in ELFEMF based therapy of neurological diseases. The aim of this study is to understand the underlying mechanisms that mediate ELF-EMF-induced neuronal differentiation and also to harness these mechanisms for cell transplantation assays. Main method: Human bone marrow–mesenchymal stem cells (hBM–MSCs) were exposed to ELF-EMF (50 Hz frequency, 1 mT intensity) for 8 days. The hBM–MSC derived neurons were then analyzed by general molecular biology techniques including immunofluorescence and quantitative RT-PCR. To assess changes in gene expression induced by ELF-EMF exposure, we analyzed the transcriptome of neuronal cells after an 8-day ELF-EMF exposure (50 Hz, 1 mT) and compared the transcriptional profiles to control cells. Key finding: We found that early growth response protein 1 (Egr1) is one of the key transcription factors in ELFEMF-induced neuronal differentiation. In addition, we show that transplantations of ELF-EMF-induced neurons significantly alleviate symptoms in mouse models of neurodegenerative disease. Significance: These findings indicate that a specific transcriptional factor, Egr1, mediates ELF-EMF-induced neuronal differentiations, and demonstrate the promise of ELF-EMF based cell replacement therapies for neurodegenerative diseases. © 2014 Elsevier Inc. All rights reserved.
Introduction Stem cell based cell replacement therapies are a highly promising method of repairing neurons which have been damaged through neurodegenerative diseases (Körbling and Estrov, 2003; Lindvall et al., 2004). In particular, human bone marrow (hBM)-derived mesenchymal stem cells (MSCs) have the ability to differentiate into several neural types, and can be readily prepared for autologous cells, showing great potential in cell replacement therapies for neurodegenerative diseases (Joyce et al., 2010). Several studies suggest that MSCs have the ability to induce improvements in models of neurological diseases (Karussis et al., 2008; Torrente and Polli, 2008). However, limitations exist in the ability of MSCs to be stably engrafted in cell replacement procedures, since their efficacy relies on the secretions of cytokines for direct differentiation (Salem and Thiemermann, 2010; Uccelli et al., 2008). Several chemicals and additional differentiation factors have been identified to create a better environment for neuronal differentiation for more still yield low efficiency therapy (Sanchez-Ramos et al., 2000;
⁎ Corresponding author. E-mail address: [email protected] (J. Kim).
http://dx.doi.org/10.1016/j.lfs.2014.02.022 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Woodbury et al., 2000) and show evidence of in vivo toxicity (Burdon et al., 2011; Lu et al., 2004). Extremely low-frequency electromagnetic field (ELF-EMF) exposure is known to have effects on several biological processes, such as cell proliferation, differentiation, gene expression pattern, and regulation of transcription factor (Blank, 1993; Goodman et al., 1995). Particularly, recent results indicate that a specific frequency of ELF-EMF treatment can have strong neurogenic effects in stem cells (Güven et al., 2005). For example, 50 Hz ELF-EMF exposure has been shown to promote neuronal differentiation in rat chromaffin cells (Morgado-Valle et al., 1998) and neural stem cells (Piacentini et al., 2008). Neural differentiation on PC12 cells has also been observed following ELF-EMF exposure (Morabito et al., 2010; Zhang et al., 2005). In our previous study, we also showed the increased neuronal differentiation in hBM–MSCs following ELF-EMF exposure (50 Hz, 1 mT) (Cho et al., 2012), providing a feasible non-chemical solution for neuronal differentiation of stem cells for cell replacement therapy. Despite various studies exploring the effect of ELF-EMF exposure on neuronal differentiation, the underlying mechanism for this pathway is still largely unknown. Several studies suggest that the intracellular Ca2+ signaling plays a critical role in ELF-EMF induced neuronal differentiation (Cuccurazzu et al., 2010). Other studies also have shown that
Y. Seong et al. / Life Sciences 102 (2014) 16–27
changes in the K+ current by K+ channel activation mediate ELF-EMF effects in neuronal differentiation (Blank and Soo, 1993; Funk and Monsees, 2006). Additionally, it has been reported that free radicals or ROSs play an important role in ELF-EMF based neural differentiation (Park et al., 2013). However, the epigenetic mechanisms governing neuronal cell fate changes by ELF-EMF exposure remain unclear. In this study we investigate the epigenetic mechanisms of ELF-EMF (50 Hz, 1 mT) induced neuronal differentiation. Initially, we show that ELF-EMF exposure leads to efficient neuronal differentiation in both human BM–MSC and mouse neural stem cells. In addition, early growth response protein 1 (Egr1), a strong early neurogenic transcription factor, is identified as a mediator in ELF-EMF-induced neuronal differentiations. The overexpression of Egr1 combined with ELF-EMF exposure significantly increases the efficiency of cell replacement therapy. Taken together, our results provide the molecular basis of ELF-EMF-induced neuronal differentiation for efficient cell replacement therapy.
Materials and methods
17
Viral infection An Egr1-knockdown lentiviral vector encoding a specific hairpin for the Egr1 gene and the Egr1 cDNA were cloned into the EcoRI cloning site of FUW lentiviral vectors. For lentiviral vector infections of hBM–MSCs, cells were seeded in six-well plates at a density of 3 × 104 cells per well and infected on 3 consecutive days. Medium changes were performed 12–24 h after infection. Fresh differentiation medium was added every other day for 8 days after infection. Immunofluorescence Cells were fixed in 4% paraformaldehyde in PBS and immunostained according to the standard protocols using the following primary antibodies: anti-TuJ1 (1:300 dilution), anti-TH (1:300 dilution) (Sigma-Aldrich, USA), anti-vMAT2 (1:300 dilution) (Millipore, USA), anti-NeuroD1 (1:300 dilution), anti-MAP2 (1:300 dilution) (Cell Signaling, USA) and appropriate fluorescent secondary antibodies (1:500 dilution) (Invitrogen, USA).
Cell culture & ELF-EMF exposure
Quantitative real time-PCR analysis
Human bone marrow–mesenchymal stem cells (hBM–MSCs) were purchased from CEFO, Co. Ltd. (Seoul, Korea) and were cultured in StemMACS MSC Expansion Medium (Miltenyi Biotec, Germany). Cells were grown at 37 °C for 3–4 days until confluent, and then were subcultured with Accutase (Innovative Cell Technology, USA). To induce neuronal differentiation by ELF-EMF exposure, hBM–MSCs were seeded at a density of 3 × 104 cells/cm2 at 35 mm cell culture plates and were changed into differentiation medium (DMEM/F12 (Gibco, USA), valproic acid 2 μM (Sigma-Aldrich, USA), forskolin 10 μM (LC laboratories, USA), hydrocortisone 1 μM (Sigma-Aldrich, USA), insulin 10 mg/ml (Welgene, Korea) and 1% penicillin/streptomycin (Gibco, USA)). A day after subculture, the cells were moved to the EMF generator and were continuously exposed to ELF-EMF (F = 50 Hz; Bm = 1 mT) for 8 days (Supplementary Fig. 4B). We utilized an ELF-EMF device which was previously described (Cho et al., 2012). Briefly, hBM–MSCs were exposed to continuous sinusoidal ELFEMF (50 Hz, 1 mT) in a system formed by two Helmholtz coils (15 cm inner diameter) oriented to produce a vertical magnetic field. This system was supplied by the COMSOL 3.4 (MA) and Tesla meter TM-701 (Japan) for magnetic flux density distribution or measured data, respectively. The culture was located in a cell culture incubator with 5% CO2 at 37 °C. Control cultures were grown in a separate incubator without Helmholtz coils.
Total RNA was isolated using a PureLink RNA Mini Kit (Invitrogen, USA). 400 ng of DNase treated RNA was reverse transcribed using a First Strand Synthesis Kit (Bioneer, Korea). Quantitative RT-PCR analysis was performed in triplicate using 1/40 of the reverse transcription reaction in a StepOnePlus Real-Time PCR System with SYBR Fast qPCR Kit (KAPA Biosystems, USA) (Table 1). The qPCR used a GAPDH control and a standard curve. Electrophysiology For electrophysiological recordings, ELF-EMF-induced hBM–MSCs were grown on 12 mm glass coverslips and transferred to recording medium containing the extracellular solution (in mM): 130 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.35, 325 mosM). Patch pipettes were filled with the following intracellular solution (in mM): 110 potassium gluconate, 20 KCl, 2 Mg-ATP, 10 sodium phosphocreatine, 1.0 EGTA, 0.3 GTP–Tris, and 20 HEPES (pH 7.25, 320 mosM). Electrodes were fabricated from borosilicate capillary glass tubing (Warner Instruments) with a capillary glass puller (Sutter Instruments). After establishing whole-cell mode, the cell membrane capacitance and series resistance were compensated to 75% electronically using a patch clamp amplifier (Axopatch 200B; Molecular Devices). Data generation and acquisition were performed using pClamp10 software on an IBM
Table 1 Primer sequence used for quantitative real-time PCR. Genes
Species
Sense
Antisense
Size
GAPDH Tubb3 MAP2 NeuroD1 CHAT Pitx3 ID2 ID3 Smad7 JunB Egr1 GAPDH CHAT GAD67 HB9 Pitx3 Egr1 DBP
Human Human Human Human Human Human Human Human Human Human Human Mouse Mouse Mouse Mouse Mouse Mouse Mouse
cgagatccctccaaaatcaa agctcacccagcagatgttc ctgcacactcacatccacct gttctcaggacgaggagcac tacaggctccaccgaagact agctagaggcgaccttcca cgtgaggtccgttaggaaaa actcagcttagccaggtgga gtggatggtgtgtgggtgta ccatcagctacctcccacac tgaacaacgagaaggtgctg aacgaccccttcattgacct cctgccagtcaactctagcc gcacagagaccgacttctcc ctcatgctcaccgagactca gaggaatcgctaccctgaca aaaggtggtttccaggttcc cattccaggccatgagactt
ttcacacccatgacgaacat gggatccactccacgaagta tctccgttgatcccattctc gtctcttgggcttttgatcg catccttcaggagcagaagc aagctgcctttgcatagctc gcaggatttccatcttgctc aagctccttttgtcgttgga tgctgcataaactcgtggtc actttgatgcgctcttggtc tgggttggtcatgctcacta ccctgttgctgtagccgtat ggaagccggtatgatgagaa aaaatcgagggtgacctgtg ccattgctgtacgggaagtt gggtacacctcctcgtaggg actgagtggcgaaggcttta tggctgcttcattgttcttg
170 175 166 168 178 172 179 167 185 178 173 280 222 294 287 201 166 161
18
Y. Seong et al. / Life Sciences 102 (2014) 16–27
computer equipped with an analog to digital converter (Digidata 1440A; Axon Instruments). Recordings were conducted on day 8 based on the neuron-like morphology including elevated cell bodies and the presence of branched and extended neurites. Once currentclamp mode was obtained, the cell was maintained at a potential of approximately −65 to −70 mV. Current injection protocol steps were applied ranging from − 100 pA to + 120 pA. Inward sodium current
and outward potassium current were measured in voltage-clamp mode with voltage step protocols ranging from −55 to +55 mV. Cell transplantation and behavior analysis All animal procedures were performed according to the guidelines and were approved by the Committee on Animal Care at Dongguk
ELF-EMF 50 Hz, 1 mT (8 days)
A
Neurons
Human BM-MSCs
C
Control
Tuj1
Tuj1 / DAPI
Tuj1
ELF-EMF
BF
Tuj1 / DAPI
80
Tuj1 + cells per field (%)
BF
**
60
40
20
0
NeuroD1 + cells per field (%)
B
80
60
**
40
20
0
D Tuj1
NeuroD1
Merge
F
+55 mV
-100 mV
n=12
n=12
n=12
500 pA
100 ms
RMF (mV)
20 mV
n=12
AP amplitude (mV)
E
DAPI
10 ms -50 mV
Fig. 1. ELF-EMF exposure on neuronal differentiation of hBM–MSCs. (A) Schematic procedure of ELF-EMF (50 Hz, 1 mT) exposure in neuronal differentiation of human BM–MSCs. After changing neuronal culture media, the hBM–MSCs were exposed to ELF-EMF for 8 days. (B) (upper) Morphology of control hBM–MSCs and ELF-EMF exposed hBM–MSCs (lower). Immunofluorescence images of ELF-EMF-induced neurons using antibody against Tuj1. TuJ1 expressing neurons are present in the hBM–MSC culture after an 8-day ELF-EMF (50 Hz, 1 mT) exposure. BF: bright field, scale bar = 50 μm (left, middle) and 100 μm (right). (C) Quantitative analysis of Tuj1 and NeuroD1 positive cells in the ELF-EMF-induced neuronal differentiations. Data shown are means ± SEM; Student's t-test, **p b 0.05. Three independent experiments of three sets each were performed with 10 visual fields per set. (D) Immunofluorescence images for neuronal marker, NeuroD1 in the ELF-EMF-induced neurons. Scale bar = 100 μm. (E) Electrophysiological properties of ELF-EMF-induced functional neurons using wholecell patch-clamp analysis. Representative images of action potentials (top panel) and voltage-dependent membrane currents (bottom panel) detected in ELF-EMF-induced neurons. (top panel) Bottom traces represent current injections (−20 pA to +120 pA), and top traces indicated voltage recordings. (bottom panel) Depolarizing voltage steps elicited fast inward sodium currents (bottom traces) and slow inactivating outward potassium currents (top traces). (F) Quantification of membrane properties in hBM–MSC derived neurons after an 8-day ELF-EMF exposure and primary embryonic neurons. Data are presented as mean ± SEM, n = 12. RMP: resting membrane potential, AP: action potential.
Y. Seong et al. / Life Sciences 102 (2014) 16–27
University. Mice were anesthetized with Avertin, and placed in a stereotaxic frame and 6-hydroxydopamine (6-OHDA) (Sigma-Aldrich, USA) was injected into the midbrain region (SN) (AP: −3.1 mm; ML: ±1.1 mm; DV: −4.4 mm). 4 weeks after 6-OHDA was lesioned, the mice were injected with apomorphine (Santa Cruz Biotechnology, USA, 4 mg/kg i.p. injection) and apomorphine-induced turning behavior was assessed before the cell grafting. Mice exhibiting net clockwise turns were lesioned in the left striatum, while mice exhibiting net counter-clockwise turns were lesioned in the right striatum (ipsilateral). The hBM–MSCs cells were resuspended in medium at a density of about 10,000 cells/μl and the cells were grafted into the lesioned striatum (AP: +0.4 mm; ML: ±1.5 mm; DV: −2.8 mm) with 2 or 3 μl of cell suspension. Apomorphine induced rotational behavior was measured again at 4 and 8 weeks after grafting. The mouse was kept in the field of ELFEMF (50 Hz frequency, 2 mT intensity) every two days for 2 or 4 weeks.
19
The number of clockwise and counter-clockwise turns was counted and expressed as the number of net rotations per 30 min to the hemisphere. Results ELF-EMF exposure on neuronal differentiation of human BM–MSCs Human BM–MSCs (hBM–MSCs) are capable of differentiating into several tissue types, such as adipocytes, osteocytes, chondrocytes, neurons and myocytes (Pittenger et al., 1999). In particular, exposure to specific neurotropic factors can trigger human BM–MSC differentiation into neurons, albeit with limitations in efficiency and toxicity (Long et al., 2005). In order to examine whether ELF-EMF exposure can alternatively influence neural differentiation of hBM–MSCs without neurotropic factors, we first induced neural differentiation of hBM–MSCs via
ELF-EMF 50 Hz, 1 mT (6 days)
A
ESC derived NSCs
Control
C
ELF-EMF Tuj1
Tuj1
TH
120
Tuj1 + cells per field (%)
B
Neurons
TH
120
**
100
TH + cells per field (%)
Mouse ESCs
80 60 40 20
100
50
0
*
150
GAD67 100
50
0
*
150
HB9 100
50
0
80 60 40 20 0
**
Relative gene expression (%)
ChAT
Relative gene expression (%)
Relative gene expression (%)
150
Relative gene expression (%)
0
D
*
100
150
Pitx3
* 100
50
0
Fig. 2. ELF-EMF exposure on neuronal differentiation of mouse neural stem cells. (A) Schematic procedure of ELF-EMF-induced neuronal differentiations of mouse neural stem cells. Mouse ES-derived neuronal precursors were differentiated into functional neurons under the exposure to ELF-EMF (50 Hz, 1 mT) for 6 days. (B) Representative immunofluorescence images for neuronal markers Tuj1 and TH in control neurons and ELF-EMF exposed neurons. Scale bar = 50 μm. (C) Quantitative analysis of Tuj1, and TH positive cells in the control neurons and ELFEMF-induced neurons. Proportion of Tuj1 and TH positive expressions. Data shown are means ± SEM; Student's t-test, *p b 0.5, **p b 0.05. Three independent experiments of three sets each were performed with 10 visual fields per set. (D) Quantitative real-time PCR of neuronal marker genes, ChAT, GAD67, HB9 and Pitx3 in ELF-EMF-induced neurons. Expression levels were normalized to Gapdh. Data are presented as mean ± SEM; Student's t-test, *p b 0.5, **p b 0.05, n = 3.
20
Y. Seong et al. / Life Sciences 102 (2014) 16–27
exhibited neuronal morphology and an increased number of neuronspecific class III beta-tubulin (Tuj1) + cells (Fig. 1B and Supplementary Fig. 1A). Additionally, we observed that ELF-EMF exposed hBM–MSCs showed significant increase in the expression of endogenous neurogenic differentiation 1 (NeuroD1), a key marker for neuronal differentiation
ELF-EMF (50 Hz, 1 mT) exposure. After changing the neuronal culture media without neurotropic factors, hBM–MSCs were exposed to different frequencies (0 Hz, 50 Hz, 100 Hz, 200 Hz, at 1 mT intensity) of ELFEMF for 8 days (Fig. 1A and Supplementary Fig. 1A)). We observed that the culture treated with 50 Hz frequency of ELF-EMF exposure only
A
B 7
Signaling molecule
11
Transcription factor
5
Extracellular matrix
3
Protease
8
Nucleic acid binding
2
Cell adhesion molecule
3
Receptor
2
Transporter
2
Kinase Transferase
1
Select regulatory molecule
1
Phosphatase
1
Oxidoreductase
1
Miscellaneous function
1
Hydrolase
1
Defense/immunity protein
1 1
Cytoskeletal protein
0
ELF-EMF
5
10
15
Control
C Human BM-MSC derived neurons (Control vs ELF-EMF) 16
no significant IFCI>=2
Egr1
14
**
10 16
8
150
ID1 100
50
0
**
10
12
14
150
HES1 100
50
0
**
16
Relative gene expression (%)
Egr1
14
Relative gene expression (%)
12
150
0
Smad7 JunB
Relative gene expression (%)
Relative gene expression (%)
10
Egr1 ID2
8
8
RELB
50
BHLHB2
ID1 JunB
100
FOS
12
TCEA3
8
ID3
HES1
10
12
DBP
D
no significant
IFCI>=1.5 IFCI>=1.5 & adj.p
