IJAO

Int J Artif Organs 2016; 39(6): 294-299

DOI: 10.5301/ijao.5000512

ISSN 0391-3988 ORIGINAL RESEARCH ARTICLE

Effect of extremely low frequency electromagnetic field

on MAP2 and Nestin gene expression of hair follicle
dermal papilla cells
Marzie Moraveji1,2, Nooshin Haghighipour1, Hamid Keshvari2, Tannaz Nourizadeh Abbariki1, Mohammad Ali Shokrgozar1,
Amir Amanzadeh1
1
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran - Iran
2
Department of Biomedical Engineering, Amirkabir University of Technology, Tehran - Iran

Abstract
Introduction: In recent years, the extremely low frequency electromagnetic field (ELF-EMF) has attracted a great
deal of scientific interest. The ELF-EMF signal is able to control ion transport across ion channels and therefore
induce cell differentiation.
Aim: The purpose of this study was to investigate the effect of ELF-EMF (50 Hz, 1 mT) on MAP2 and Nestin gene
expression of dermal papilla mesenchymal cells (DPCs).
Methods: In order to examine the effect of chemical and electromagnetic factors on gene expression, 4 experi-
mental groups, namely chemical (cell exposure to chemical signals), EMF (exposing cells to ELF-EMF), chemical-
EMF (subjecting cells to chemical signals and ELF-EMF) and control (with no treatment) groups, were prepared,
treated for 5 days, and studied. To assess the effect of extended test time on the expression of neural differentia-
tion markers (Nestin and MAP2), an EMF group was prepared and treated for a period of 14 consecutive days. The
beneficial role of EMF in inducing neural differentiation was shown by real-time PCR analysis.
Results: The higher expression of MAP2 after 14 days compared to that after 5 days and decrease of cell prolifera-
tion on days 5 to 20 were indicative of the positive effect of extending treatment time on neural differentiation
by evaluation of gene expression in EMF group.
Keywords: Chemical signaling, Differentiation, Extremely low frequency electromagnetic field, Necroptosis

Introduction
Cell sources and signaling factors are 2 main parts of neural
tissue engineering. A variety of cell types have been examined
for application in neural tissue engineering and cell therapy,
including embryonic stem cells, neural stem/progenitor cells
(NSPCs) (1), induced pluripotent stem cells (iPSCs) (2), mes-
enchymal stem cells isolated from umbilical cord blood (3),
bone marrow (4, 5), adipose (6), placenta (7), bulge area (8)
as well as olfactory ensheathing cells (OECs) (9). Among these
cell types, NSPCs have exhibited the highest potential for dif-
ferentiating into functional neurons but they are not easily ac-
cessible in the body, which limits their application in this field.
On the other hand, hair follicle dermal papilla mesenchymal
Accepted: July 5, 2016
Published online: August 10, 2016
Corresponding author:
Nooshin Haghighipour
National Cell Bank of Iran
Pasteur Institute of Iran
69 Pasteur Ave
Tehran, Iran
haghighipour@pasteur.ac.ir
stem cells (DPCs) play major roles in hair formation, growth
and cycling. Ectoderm is one of the germ layers, which de-
velops into neural crest, skin, hair and epidermis. Because of
the common origin of hair and neural cells, they also express
some common genes (10). In this regard, DPCs, which are the
cell source for hair formation and are also easily accessible in
the body, can be considered as an appropriate cell source for
neural differentiation.
Although chemical factors are the most widely studied
signaling factors in the literature, in recent years electrical
and magnetic signals have been also reported to affect cell
behavior. EMF has been used to induce cells toward neural
(11-13), chondrogenic (14), osteogenic (15) and skeletal/
cardiac muscle cell (16, 17) differentiation. The membrane
voltage depends on the type of parenchymal cell and the
stage of stem cell differentiation (18). EMF has been sug-
gested to affect ion channels and the ion flow into or out of
the cell (19) and consequently changing the cell membrane
voltage. Briefly, EMF increases the intracellular calcium level
which leads cell polarization (19).
Among parenchymal cells, skeletal muscle cells, glia and
neuron cells are associated with the most negative membrane
voltages, respectively (18), and therefore EMF can be used as
a tool to induce neural differentiation. The expression of cal-
cium channel, voltage-dependent, L type, alpha 1C subunit,

© 2016 Wichtig Publishing

Moraveji et al
(Cav1.2) subunit and Cav1.2 channel-mediated Ca2+influx have
been reported to be increased during neurogenesis in re-
sponse to the application of EMF (50 Hz) (20). Moreover, a
simultaneous activity of 2 subunits of voltage gated K+(Kv3)
channel (Kv3.4) and Cav1.2 channels during the formation of
pioneer axons and neuronal network has been reported else-
where (21). Therefore, EMF is able to induce cell polarization
and increase membrane voltage, which will subsequently lead
to the differentiation and maturation of neural cells. In one
of the pioneer studies, Piacentini et al (20) reported the ef-
fect of EMF (1 mT; 50 Hz) on inducing neural differentiation
with an increase in Cav1.2 channel activity, which leads to
the expression of MAP2 and β-III-tubulin genes (20). Several
studies have addressed the neuronal effect of 50 Hz ELF-EMF
(11-13, 20, 22), indicating that at such a frequency, lower field
strengths are more beneficial. For instance, as opposed to a
field strength of 20 mT, which shows no neural differentiation
effect (22), low field strengths of 1 or 2 mT are effective in
inducing such differentiation (12, 13).
The aim of this research is to assess the neural differentia-
tion inducing effect of chemical, EMF and chemical-EMF sig-
nals on DPCs by quantification and comparison of MAP2 and
Nestin gene expression levels in these experimental groups
using real-time PCR.
Materials and methods
Isolation of DP cells
DPCs were isolated from hair follicles of healthy men aged
30 to 35 years, according to a protocol previously described
by Wu et al (23). Briefly, the biopsy obtained from each sub-
ject was rinsed with D-Hank’s balanced salt solution and then
cut into strips. Dermal subcutaneous fat dissection strips
were incubated with 0.3% dispase at 4°C for 16 to 18 hours.
Epithelial cells were isolated from the dermal sheath. In the
final enzymatic step, dermal sheathes were incubated with
0.1% collagenase I in DMEM (Gibco) medium (supplemented
with 10% fetal bovine serum) at 37°C for 8 hours. After the
complete digestion of fibrous sheath, D-Hank’s was added
to stop the enzymatic process. Following centrifugation the
suspension and the complete separation of DPCs, these iso-
lated cells were cultured in 0.1% gelatin-coated flasks and in-
cubated with DMEM/F12 supplemented with 1% OPI (Gibco)
and 10% FBS.
Characterization of DPCs
In order to characterize the isolated DPCs, the produc-
tion of proteoglycans, glycosaminoglycans and alkaline
phosphatase was examined. Toluidine blue O (24), alcian
blue (24) and NBT/BCIP (25) were used to stain proteogly-
cans, glycosaminoglycans (GAG) and alkaline phosphatase,
respectively. Briefly, for each staining, cells were fixed with
0.1% glutaraldehyde in PBS, stained with the stain solution
and then washed with PBS. pH of alcian blue and toluidine
blue O solutions were 2.5 and 2.0 to 2.5, respectively. In the
case of toluidine blue O staining, after the mentioned steps,
dehydration through 95% alcohol and then washing with
PBS were also performed.
© 2016 Wichtig Publishing
295
ELF-EMF device
Extremely low frequency-pulsed electromagnetic field
was used in this study using a device that was previously de-
signed and fabricated at the National Cell Bank of Iran. It con-
sisted of a multi-turn solenoid, electrical current source, and
an operative to generate nonsinusoidal, pulsed electromag-
netic fields. This device was placed inside an incubator with
appropriate conditions for cell culture. Cells were exposed to
EMF, with the frequency and flux density of 50 Hz and 1 mT,
respectively (pulse period of 40 ms and Duty cycle (On/Off) of
25/15), for 7 hours per day in test periods of 5, 14 or 20 days
(see Supplementary Figure S1, available online as supplemen-
tary material at www.artificial-organs.com).
Experimental groups
This study was designed to evaluate the expression of neural
genes after 5 consecutive days of treatment in 4 experimental
groups including the control, chemical, EMF, and chemical-EMF
groups. In order to investigate the effect of extended test time
on mRNA levels of neural differentiation genes, an EMF group
was prepared and treated for 14 consecutive days, and the re-
sults were compared with those of the control group.
In chemical tests, DPCs were cultured in DMEM supple-
mented with EGF (Gibco), bFGF (Gibco), and DMSO (ICN Bio-
medicals), while in the EMF group, cells were incubated with
DMEM and exposed to an EM field (1 mT, 50 Hz sinusoidal,
7 hours per day). In the chemical-EMF experimental group,
DPCs were incubated with chemical neural differentiation
medium and subjected to the EM field. In the EMF and chem-
ical-EMF groups, samples were placed at the center of a uni-
form field area. The percentage of CO2 and the temperature
of the incubator were set to 5% and 37°C, respectively. During
the test, the cell culture medium was replaced every 2 days.
Upon completion of tests, real-time PCR method was used to
study the gene expression levels of Nestin (neural progenitor
cell marker) and MAP2 (mature neuron marker).
Cell proliferation analysis
Cell proliferation in the EM group was studied using MTT
(3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bro-
mide) assay at days 5, 14 and 20. Furthermore, the optical
density of the colored solution was measured at day 0, which
corresponded to the control group. Upon completion of the
test for each group, after removal of the culture medium and
rinsing once with PBS 1X, 100 μL of MTT solution was added
to each well. After 4 hours, the MTT solution was replaced
with 100 μL of isopropanol for 20 minutes. Finally, light ab-
sorption was measured at 545 and 630 nm wavelengths.
RNA isolation and reverse transcription polymerase
chain reaction
The total RNA of each test group was extracted using RNeasy
Plus mini kit (Qiagen), and measured using a nanodrap instru-
ment (NanoDrap). 1 μg of total RNA was utilized to synthesize
cDNA by means of a Takara kit (Takara Bio USA). mRNA levels of
neural differentiation genes along with GAPDH as the reference
296
gene were evaluated in triplicate utilizing SYBR® Green Mas-
ter Mix and ABI Step One real time-PCR instrument, both pur-
chased from Applied Biosystems. Primer sequences, designed
using Beacon Designer (BD) software and checked by primer ex-
press and gene runner software, are as follows: MAP2, forward
5’-ACGGCTGGAGAAGGCTGAA-3’ and reverse 5’-CCACG CT T
GCTGTATTACTGTAGG-3’; Nestin, forward 5’-AAGAGAACC AGGA
GCCACTGAAG-3’ and reverse 5’- ACCTCCGTCGCTGTTGAGTCT-3’
and GAPDH, forward 5’- ACACCC AC T CCTCCACCTTTG-3’ and
reverse 5’ TCCACCACCCTGTTGCTGTAG-3’. mRNA levels of genes
were quantified via comparative Ct method (2–ΔΔCt) and Ct val-
ues of each target marker were normalized relative to GAPDH.
Thereafter, resulting values were normalized to the obtained
results for the control group.
Statistical analysis
Results of 3 independent tests, carried out in triplicate
were expressed in the form of mean ± SD. Student’s t-test
was utilized for comparing the mean values corresponding to
experimental groups, by making p<0.05 assumption.
Results
Histological analysis of DPCs
Results of cell staining with toluidine blue O, alcian blue
and NBT/BCIP demonstrated the production of proteoglycans,
glycosaminoglycans and alkaline phosphatase, respectively
(see Supplementary Figure S2, available online as supple-
mentary material at www.artificial-organs.com). These results
confirmed the dermal papilla cell phenotype of isolated cells,
and therefore, they could be used in subsequent experiments.
Effect of ELF-EMF on cell proliferation
Using MTT assay, the rate of cell proliferation as well as
the metabolic events leading to apoptosis and necrosis were
Effect of ELF-EMF on neural differentation
Fig. 1 - Effect of ELF-EMF on cell prolif-
eration. Cell proliferation was decreased
over time, due to DPC differentiation
(p<0.05).
measured. Results of this experiment depended on the dif-
ference in proliferation rate, as no cell death was observed.
Based on the proliferation rate of cells exposed to EMF after
days 5, 14 and 20, a decrease in cell proliferation over time
compared to the control group was demonstrated (Fig. 1).
This observation indicates more differentiation of cells with
increasing test time.
Morphology of DPCs
Figure 2 shows cell morphology in the experimental
groups. As shown in Figures 2B and D, after 5 and 14 days in
the EMF group the unipolar and multipolar morphologies of
neurons could be observed, respectively. In the chemical-EMF
group, a large number of dead cells were observed, which can
be related to the death of neurons.
Expression of neural genes
The expression of MAP2 and Nestin genes was stud-
ied using real-time PCR technique. According to the results
(Fig. 3A), the highest expression of MAP2 was observed in
the chemical group at the day 5, in comparison to the control
group, while no expression of this gene was observed in the
chemical-EMF group.
The mRNA level of MAP2 in the EMF group was increased
approximately 2.5- fold after 14 days in comparison to that
after 5 days. The highest expression of Nestin was observed
in the chemical-EMF group, compared to the control group
(Fig. 3B).
Discussion
In the present study, we demonstrated the effect of
ELF-EMF on neural differentiation by evaluation of MAP2
and Nestin gene expression. The simultaneous effects of
chemical and electromagnetic signals were also studied
for the first time. Briefly, dermal papilla cells were exposed
© 2016 Wichtig Publishing
Moraveji et al
Fig. 2 - The morphology of the cells into four groups: (A) Chemical (5 days), (B) EMF (5 days), (C) Chemical-EM (5 days), (D) EM (14 days) and
(E) Control. In the EMF group, the morphologies of cells are unipolar and multipolar after 5 and 14 days, respectively. (Magnification = 200 x).
to an electromagnetic field (50 Hz; 1 mT, 7 hours per day)
for 5 and 14 days. Meanwhile, the effects of chemical and
chemical-electromagnetic factors were also studied. The ex-
pression levels of MAP2 and Nestin genes were quantified
to compare the results of applying such signaling factors.
The effect of ELF-EMF on cell differentiation (26, 27),
proliferation (28), migration (29), apoptosis (30) and other
intracellular activities has been previously studied, but the
mechanisms underlying such observed cellular responses as
well as the corresponding signaling pathways are still unclear.
Among such studies, neural differentiation using EMF signal
via reactive oxygen species (ROS)-mediated activation of epi-
dermal growth factor receptor (EGFR) has been reported (11).
Recently, the effect of ELF-EMF on cell reprogramming com-
bined with the octamer-binding transcription factor 4 (OCT4)
has been proven (31). Thus, depending on the conditions, ELF-
EMF can play different roles. According to the results of pres-
ent study, proliferation of DPCs is decreased over time, which
may be due to the differentiation of these cells into neural
cells such as neurons.
Neuronal differentiation was evaluated by quantification
of Nestin and MAP2 expression levels. In EMF group, an in-
crease in MAP2 expression and no decrease in Nestin mRNA
level were observed at day 14 compared to those at day 5.
Increase in the expression of MAP2 can be assigned to an im-
proved neural differentiation.
Inducing cells toward neural differentiation in the chem-
ical-EMF group resulted in an increase in Nestin expression
while no expression of MAP2 was observed. Considering the
intracellular signaling pathway in this group, EMF (11) and EGF
(32, 33) simultaneously produce ROS, which activate a P13k/
AKT/mTOR pathway (34). Being activated by 2 stimulators, this
© 2016 Wichtig Publishing
297
Fig. 3 - mRNA expression levels in
treatment groups. Real-time PCR data
of MAP2 (A) and Nestin (B) genes ex-
pression in experimental groups.
(*shows p<0.05)
pathway affects the programmed necrosis or necroptosis of
differentiated neurons (35) (Fig. 4). Neurons are more sensi-
tive to the levels of ROS but neural stem/progenitor cells have
a high capacity of ROS, regulating their self-renewal and neu-
rogenesis through the PI3K/Akt pathway (36). Therefore, cells
in the chemical-EMF may try to survive by transforming into
neural stem/progenitor cells.
In a previous study, bone marrow-derived mesenchy-
mal stem cells (BMSCs) were exposed to EMF (50 Hz;1 mT)
(12), in which cell type and the time schedule of treatment
(application of EMF for 12 consecutive days), differed from
our study and the expression of MAP2 was increased by
almost 1.5 times. The higher expression of MAP2 observed
in our research compared to that in the mentioned work
may have originated from the duration of cell exposure to
EMF, however the exact mechanisms underlying observa-
tion of such different results are unclear. The difference
may also arise from the different cell sources used in the
2 studies, considering the fact that DPCs express more
common active genes with neural cells, and therefore can
exhibit a higher expression of MAP2 gene compared to
BMSCs.
Cell communication, in particular between neurons,
is of great importance in neural differentiation and tissue
engineering. Indeed, there has been no successful work in
neuronal differentiation and neuron cell therapy without
achieving proper neuron-neuron signaling. Neurons can
communicate with each other through chemical synapses
and the release of neurotransmitter molecules by a neuron
in the synaptic cleft space that binds to their receptors on
the other cell. Such a release significantly depends on the
calcium concentration. Briefly, neurotransmitter molecules
298
Fig. 4 - A schematic of the proposed mechanism for neuron necrop-
tosis. Effect of EMF and EGF on ROS production and necroptosis of
neuron and consequently no expression of MAP2 gene in chemical-
EMF group.
are located inside the vesicles. Calcium ions bind to proteins
on the surface of vesicles and change their conformation,
leading to the fusion of vesicles with cell membrane and the
release of neurotransmitter molecules (37). Therefore cal-
cium play major roles in cell communication and ELF-EMF
can be used as a tool to open calcium channels and facilitate
the entrance of calcium ions. This mechanism implies the
beneficial role of using an electromagnetic wave as a dif-
ferential factor in neural tissue engineering. ELF-EMF was
able to result in cell differentiation and cell communication,
simultaneously.
Conclusions
The results of the present paper indicate the significant
role of ELF-EMF in MAP2 and Nestin gene expression. Appli-
cation of ELF-EMF is a low-cost method compared to the utili-
zation of chemical signaling factors. Contrary to expectations,
the results showed that the synergistic effect of the 2 signals
leads to no expression of the MAP2 gene. The exact intracel-
lular mechanisms underlying the results are still unclear and
require further investigation in the future. The findings of this
work may be applied in neural tissue engineering to provide
functional neural cells differentiated from suitable cell sourc-
es, especially dermal papilla mesenchymal cells (DPCs), which
express some common genes with neural cells and are readily
accessible in the body.
Effect of ELF-EMF on neural differentation
Disclosures
Financial support: This work was supported by the National Cell
Bank in the Pasteur Institute of Iran.
Conflict of interest: None of the authors has financial interest
related to this study to disclose.
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