Extracted Text
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Electrical stimulation drives
chondrogenesis of mesenchymal
stem cells in the absence of
exogenous growth factors
Hyuck Joon Kwon
1
, Gyu Seok Lee
2
& Honggu Chun
3
Electrical stimulation (ES) is known to guide the development and regeneration of many tissues.
However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage
repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs)
have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during
chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels
of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen
levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition
of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by
driving Ca
2+
/ATP oscillations, which are known to be essential for prechondrogenic condensation. In
subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on
extracellular ATP signaling via P2X
4 receptors, and ES induced significant increases in TGF-β1 and BMP2
expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the
inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates
ES-driven condensation. These findings may contribute to the development of electrotherapeutic
strategies for cartilage repair using MSCs.
Articular cartilage is a unique load-bearing tissue that lacks vascular, neural, and lymphatic tissues. Articular
cartilage cannot spontaneously regenerate in vivo because chondral defects do not penetrate the subchondral
bone and therefore cannot be accessed by blood supply or mesenchymal stem cells (MSCs) from bone marrow
1,2
.
Hence, researchers and surgeons have developed various techniques to repair cartilage tissues
3
. However, most
current cartilage repair techniques eventually lead to the formation of fibrocartilage and cartilage degeneration
4
.
Accordingly, autologous chondrocyte implantation has been used for cartilage repair but is associated with sev-
eral disadvantages, including limited cell sources and frequent injury of healthy cartilage during surgery, further
encouraging formation of inferior fibrocartilage at defect sites
5
.
Owing to their capacity for self-renewal and differentiation into adipocytes, cartilage, bone, tendons, muscle,
and skin, MSCs are an attractive cell source for cartilage defect therapies
6–11
. Furthermore, because MSCs are
free of both ethical concerns and teratoma risks, MSCs have considerable therapeutic potential
12
. Therefore, it is
important to develop effective and safe methods for the induction of MSC chondrogenesis and for the production
of stable cartilaginous tissue by these cells. Multiple previous studies have demonstrated the effects of various
chemical factors, such as soluble growth factors, chemokines, and morphogens, on chondrogenesis. In particular,
transforming growth factors (TGF-β ) and bone morphogenetic proteins (BMPs) have been shown to play essen-
tial roles in the induction of chondrogenesis
13,14
. Although these growth factors have great therapeutic potential
for cartilage regeneration, growth factor-based therapies have several clinical complications, including high dose
requirements, low half-life, protein instability, higher costs, and adverse effects
15,16
. Recent studies demonstrate
1
Department of Physical Therapy and Rehabilitation, College of Health Science, Eulji University, Gyeonggi, Republic
of Korea.
2
Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam
National University, Daejeon, Republic of Korea.
3
Department of Bio-convergence Engineering, Korea University,
Seoul, Republic of Korea. Correspondence and requests for materials should be addressed to H.J.K. (email: kwonhj@
eulji.ac.kr) or H.C. (email: chunhonggu@korea.ac.kr)
received: 13 April 2016
accepted: 21 November 2016
Published: 22 December 2016
OPEN
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that physical factors regulate cell differentiation and tissue development
17–21
, suggesting therapeutic potential of
physical factors as alternatives to chemical agents for cartilage regeneration.
Endogenous electrical signals have been observed in articular cartilage during physiological processes,
prompting the application of various electrical stimulation (ES) and electromagnetic field (EMF) inducers to
in vitro chondrogenesis and in vivo cartilage repair
22–24
. In particular, studies using animal models show that ES
and EMF improve healing of cartilage defects by increasing cell proliferation, glycosaminoglycan synthesis, and
the expression of extracellular matrix genes, and by reducing the production of inflammatory mediators
25–29
.
However, the precise roles of ES and EMF in cartilage repair remains unclear, and the effects of ES and EMF have
only been observed in the surrounding cartilage, and not in articular defects. These observations indicate that ES
or EMF alone have limited therapeutic efficacy for the repair of large osteochondral defects. Autologous chondro-
cyte transplants have been successfully used to repair large osteochondral defects
30
. Thus, multiple studies have
investigated the effects of ES on proliferation and synthesis of cartilage extracellular matrix proteins in chondro-
cytes
31–34
. However, chondrocytes gradually decrease in number with age
33
, and it is difficult to obtain sufficient
chondrocyte numbers to repair large defects due to limited life span and de-differentiation with downregulation
of cartilage-marker genes during culture
34,35
. As an alternative, MSCs with self-renewing abilities can differentiate
into chondrocytes and offer a reliable resource for ES-based therapies for damaged cartilage defects. However,
although recent studies have reported the effects of ES on proliferation and differentiation of MSCs
36,37
, it remains
unclear whether ES induces chondrogenic differentiation of MSCs.
Our previous studies demonstrated that intracellular ATP levels oscillate during chondrogenic differentiation
and the ATP oscillations play critical roles in prechondrogenic condensation
38,39
, and that extracellular ATP sig-
naling mediates the ATP oscillations during chondrogenesis
40
. ES has been shown to activate extracellular ATP
signaling in a variety of cell types
41–43
. Therefore, we hypothesized that ES induces ATP oscillations in MSCs by
stimulating extracellular ATP signaling and consequently drives MSC chondrogenesis. In the present study, we
demonstrated that ES induces ATP oscillations and promotes MSC chondrogenesis in the absence of exogenous
growth factors. Moreover, ES induced MSC chondrogenesis more effectively than treatments with chondrogenic
medium (CM) supplemented with soluble growth factors. Accordingly, further experiments showed that extracel-
lular ATP signaling via P2X
4 receptors was responsible for ATP oscillations and mediated chondrogenesis follow-
ing ES. In addition, ES-driven chondrogenesis depended on both TGF-β and BMP signaling pathways. However,
TGF-β signaling, but not BMP signaling, was involved in ES-driven condensation. The present data suggest that
ES has high potential as an MSC-based therapy for cartilage regeneration.
Results
ES induces calcium/ATP oscillations and MSC condensation. Flow cytometry analysis showed that
the expanded MSCs were positive for typical MSC markers (Sca-1, CD44, CD73) but showed low expression of
markers of hematopoietic stem cells (CD34), macrophages (CD11b), and granulocytes (CD45), which confirmed
that the expanded MSCs exibited the characteristics of MSCs (Supplementary Fig. S1). To determine whether
ES induces ATP oscillations in MSCs, we monitored temporal changes in intracellular ATP levels using a biolu-
minescent ATP-dependent luciferase (Luc) reporter gene fused to a constitutive ACTIN promoter (P
ACTIN-Luc).
Following transfection of MSCs with P
ACTIN-Luc, bioluminescence intensity was measured in real-time during
ES of 0, 1, 5 or 25 V/cm at 5 Hz (Fig. 1a). In these experiments, ES of 5 V/cm induced ATP oscillations of ~ 5 min
periods, whereas ES of 0, 1, or 25 V/cm did not (Fig. 1b). Because ATP oscillations were driven by changes in Ca
2+
concentrations during chondrogenesis
38
, we examined Ca
2+
oscillations using the bioluminescent Ca
2+
reporter
Aequorin (AQ) gene fused to a CMV promoter (P
CMV-AQ)
44
. ES of 5 V/cm consistently induced Ca
2+
oscillations,
whereas ES of 0, 1, or 25 V/cm did not (Fig. 1c), suggesting that optimized ES can drive fluctuations of both Ca
2+
and ATP. We previously showed that growth factors such as TGF-βs and insulin induce prechondrogenic conden-
sation by generating Ca
2+
/ATP oscillations
39
. Consistently, ES of 5 V/cm which induced Ca
2+
/ATP oscillations
led to compact condensation of MSCs, whereas ES of 0, 1, or 25 V/cm did not (Fig. 1d). Moreover, time-course
observations showed that ES of 5 V/cm induced gradual aggregation of MSCs into compact structures within 3
days, corresponding with the effects of chondrogenic medium (CM) supplemented with growth factors such as
TGF-βs and insulin (Supplementary Fig. S2). These results indicate that ES induces prechondrogenic condensa-
tion by driving Ca
2+
/ATP oscillations, even in the absence of exogenous growth factors.
ES induces MSC chondrogenesis. In further experiments, the effects of optimized ES on chondrogenic
differentiation were examined. Since ES for 3 days had little effect on cell damage (< 5%) but induced significant
cell death (almost 50%) after 7 days (Supplementary Fig. S3), ES was performed for 3 days. Gene expression of
chondrogenic markers such as type II collagen (COL2A1), aggrecan (AGC), and SRY (Sex Determining Region
Y)-Box 9 (SOX9)
45–47
was analyzed at 1 and 3-day of ES treatment and 7-day post-ES treatment. Quantitative
real-time RT-PCR analyses showed that ES significantly enhanced gene expression of chondrogenic markers
within 3 days of ES treatment, revealing a 66-fold increase in COL2A1, a 43-fold increase in AGC, and a 35-fold
increase in SOX9 expression at 3-day of ES (Fig. 2a). Moreover, increases of chondrogenic marker expression in
MSCs treated with ES for 3 days were much higher than those in the CM exposed cells, and were greater than
or equal to expression levels in MCSs that were fully differentiated into chondrocytes following treatment with
CM for 14 days (Fig. 2a). These data suggest that ES induces MSC chondrogenesis more effectively than CM.
Moreover, it was found that the chondrogenic markers were highly expressed for as long as 7 days after the last
ES treatment (Fig. 2a), which confirmed that chondrogenesis was induced in MSCs by ES. In addition, ES led to
significant decreases in the expression of type I collagen (COL1; Fig. 2b). This result indicates that ES induces
differentiation of MSCs into not fibrocartilaginous tissues but hyaline cartilaginous tissues
48–51
. In contrast, ES
did not significantly change the expression of the osteogenic marker alkaline phosphatase (ALP), or the adipo-
genic marker adipocyte protein 2 (aP2; Fig. 2b), indicating that ES does not induce osteogenesis or adipogenesis.
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Additionally, immunostaining and alcian blue staining analyses showed significantly higher expression of type
II collagen and GAGs in ES-treated MSCs compared with control cells (Fig. 3a–c). Taken together, these data
suggest that ES induces MSC chondrogenesis for hyaline cartilage regeneration even in the absence of exogenous
growth factors.
Extracellular ATP signaling via P2X
4 receptor mediates ATP oscillations, condensation, and
subsequent chondrogenesis following ES.
Extracellular ATP signaling via the P2X
4 receptor report-
edly plays key roles in prechondrogenic condensation by mediating ATP oscillations
36
. In the present study, ES
enhanced the expression of P2X
4 receptor mRNA in MSCs (Fig. 4a), suggesting that extracellular ATP signaling
Figure 1. Electrical stimulation induces ATP/Ca
2+
oscillations and MSC condensation. (a) Schematic of the
real-time bioluminescence monitoring system for ES treated MSCs transfected with bioluminescence reporters (b) Real-time monitoring of intracellular ATP levels in MSCs under ES (0, 1, 5, and 25 V/cm, 8 ms, 5 Hz) using
a bioluminescence ATP reporter (P
ACTIN-Luc) (c) Real-time monitoring of intracellular Ca
2+
levels under ES
(0, 1, 5, and 25 V/cm, 8 ms, 5 Hz) using a bioluminescence Ca
2+
reporter (P
CMV-AQ) (d) Condensation
behaviors of MSCs in micromass culture were examined using phase contrast images during culture for 3 days under ES (0, 1, 5, and 25 V/cm, 8 ms, 5 Hz); Scale bars, 500 μm.
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via the P2X
4 receptor is involved in ATP oscillations, MSC condensation, and chondrogenesis following ES. In
agreement, the P2X
4 purinergic receptor inhibitor 5-BDBD inhibited ES-driven ATP oscillations (Fig. 4b). In sub-
sequent experiments, it was examined whether extracellular ATP signaling via the P2X
4 receptor was associated
with ES-driven condensation and subsequent chondrogenesis. 5-BDBD almost completely inhibited ES-driven
condensation, and apyrase significantly suppressed this process (Fig. 4c). In addition, ES did not enhance the
expression of the chondrogenic markers COL2A1, AGC, and SOX9 in MSCs treated with either apyrase or
5-BDBD (Fig. 4d). Hence, extracellular ATP signaling via P2X
4 receptors mediates ES-driven MSC condensation
and chondrogenesis.
Intercellular communications mediates ES-driven chondrogenesis. It was known that ES influ-
ences intercellular communications such as paracrine signaling
52
and gap junction
53
. We found that BFA, which
blocks classical secretion of paracrine factors, suppressed ES-driven condensation and ES-driven increases of
COL2A1, AGC, and SOX9 expression (Fig. 5a,b). In addition, the gap-junction inhibitor carbenoxolone also sup-
pressed ES-driven condensation and ES-driven increases of expression of the chondrogenic markers (Fig. 5a,b).
This result indicates that ES induces chondrogenesis by activating the release of paracrine factors and the
gap-junction activity.
TGF-β signaling mediates MSC condensation and chondrogenesis following ES. TGF-β sign-
aling reportedly induces prechondrogenic condensation and chondrogenesis through ATP oscillations
39
. The
present study showed that ES led to much higher mRNA expression of TGF-β1 (74 fold) than expression in
control cells (Fig. 6a), suggesting that TGF-β signaling is involved in ES-driven MSC condensation and chondro-
genesis. In agreement, inhibition of TGF-β signaling by SB-431542 almost completely blocked ES-driven con-
densation and significantly suppressed ES-driven increases of COL2A1, AGC, and SOX9 expression (Fig. 6b,c).
Although these data indicate that ES induces MSC condensation and chondrogenesis by activating TGF-β signa-
ling, SB-431542 did not completely suppress ES-driven induction of chondrogenic markers (Fig. 6c), suggesting
that other growth factors and cytokines also mediate the actions of ES.
Figure 2. ES enhances mRNA expression of chondrogenic markers in MSCs. Relative levels of mRNA
were determined by quantitative real-time PCR in relation to beta-actin. (a) Gene expression of type II collagen (COL2A1), aggrecan (AGC), and Sox9 in MSCs was determined at various time points during ES treatment (5 V/cm, 8 ms, 5 Hz) and CM treatment. Data are presented as means ± standard deviations (S.D.).
Statistical analyses were performed using ANOVA (Dunnett’s test); **p < 0.01, *p < 0.05 versus Ctl (control;
MSCs cultured in maintenance medium for 3 days). (b) Gene expression of type 1 collagen (COL1), alkaline phosphatase (ALP), and adipocyte protein (aP2) was determined at various time points during ES treatment (5 V/cm, 8 ms, 5 Hz). Data show mean ± S.D. Statistical analyses were performed using ANOVA (Dunnett’s
test); **p < 0.01, *p < 0.05 versus control (MSCs cultured in maintenance medium for 3 days).
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BMP signaling mediates ES-induced chondrogenesis, but not ES-induced condensation. BMPs
have been shown to play important roles in cartilage development
54,55
. Moreover, the present experiments showed
that in comparison with non-treated controls, ES increased BMP2 expression by 42 fold (Fig. 7a). In addition, the
inhibitor of BMP signaling noggin suppressed ES-driven increases in COL2A1, AGC, and SOX9 mRNA expres-
sion (Fig. 7c). However, noggin did not suppress ES-driven condensation (Fig. 7b), indicating that BMP signaling
mediates ES-driven chondrogenesis, but not ES-driven condensation.
Discussion
ES is a versatile treatment that remains poorly understood in the context of stem cell-based therapy. Herein,
we demonstrate that ES significantly enhances the expression of chondrogenic markers (Figs 2a and 3a,b), but
significantly decreases COL1 expression in MSCs (Fig. 2b). These data indicate that ES induces MSC differen-
tiation into hyaline chondrogenic cells, and provide evidence of the potential of electrically stimulated MSCs to
efficiently regenerate hyaline cartilage in the absence of additional exogenous chemical factors.
Our previous results demonstrated that ATP oscillations driven by chondrogenic growth factors such as TGF
beta and insulin play essential roles for prechondrogenic condensation that is the initial step of chondrogenesis by
inducing oscillatory expression of proteins involved in actin dynamics, cell migration, and adhesion which leads
to collective migration and adhesion
38,56
. The present results demonstrate that ES generates Ca
2+
/ATP oscillations
in MSCs even in the absence of exogenous growth factors (Fig. 1b). Since ES directly regulates voltage-gated Ca
2+
channels
57
, ES can drive Ca
2+
oscillations by modulating voltage-gated Ca
2+
channels. In addition, since extracel-
lular ATP signaling modulates Ca
2+
flux by producing diacylglycerol and inositol 1,4,5-triphosphate, activating
protein kinase C, and by mobilizing intracellular Ca
2+
in multiple cell types
58
, ES can induces Ca
2+
oscillation
by extracellular ATP signaling via the P2X
4 receptor, which is supported by the present result that P2X
4 ATP
Figure 3. ES enhances expression of COL2A1 and GAGs in MSCs. (a) Immunofluorescent staining
(COL2A1; green) and alcian blue staining (glycosaminoglycan (GAG); blue) of micromass cultures after culture in maintenance medium (control) or maintenance medium with ES (5 V/cm, 8 ms, 5 Hz) for 3 days (ES). Nuclei
were stained blue using Hoechst 33342; Scale bars, 500 μm. (b) Immunofluorescent staining (COL2A1; green)
and alcian blue staining (GAG; blue) of paraffin-embedded sections of micromass cultures after culture in
maintenance medium (control) or maintenance medium with ES (5 V/cm, 8 ms, 5 Hz) for 3 days (ES). Nuclei
were stained blue using Hoechst 33342; Scale bars, 100 μm. (c) Quantitative analysis of immunostaining
intensity and alcian blue staining intensity. Data are presented as means ± S.D. and differences were identified
using Students t-test, **p < 0.01.
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signaling mediates the actions of ES (Fig. 4). Increased Ca
2+
levels activate ATP-consuming processes such as ion
pumping and exocytosis
59
, decrease glucose consumption by inhibiting glycolytic enzymes
60
, and decrease mito-
chondrial ATP production by abolishing mitochondrial membrane potential
61
, indicating the negative effects
of Ca
2+
on ATP levels. Accordingly, Ca
2+
oscillations can drive ATP oscillations. In addition, previous studies
demonstrated that pulsed electrical fields or pulsed electromagnetic fields modulate cAMP levels by activating
adenosine receptors such as A
2A, A
2b, and A
3 receptors, which leads to activation of anti-inflammatory pathways
and cellular proliferation in cartilage
62–65
. Our previous results showed that ATP oscillations are dependent on
cAMP dynamics
38,40
. These results suggest that ES drive ATP oscillations by modulating cAMP levels.
We demonstrated that pharmacological inhibition of P2X
4-mediated ATP oscillations suppressed ES-driven
condensation (Fig. 4b,c). Previous study showed that Ca
2+
/ATP oscillations induced synchronized secretion of
adhesion molecules and prechondrogenic condensation
38
. In agreement, extracellular ATP signaling reportedly
mediates chemotaxis and morphological changes from spread to spherical shapes, and Ca
2+
oscillations play
Figure 4. Extracellular ATP signaling via P2X
4 receptors mediates ES-driven ATP oscillations,
prechondrogenic condensation, and chondrogenesis. (a) Real-time gene expression analyses of P2X
4
receptors in micromass cultures of MSCs after culture for 3 days in maintenance medium (control) or maintenance medium under ES. Data are presented as means ± S.D. and differences were identified using
Students t-test, *p < 0.05 (b) Effects of 5-BDBD on ES-driven ATP oscillations; MSCs in micromass culture
were treated with 5-BDBD after induction of P
ACTIN-Luc oscillations by ES. Bioluminescence monitoring was
performed after application of ES to MSCs (time = 0 h). (c) Effects of apyrase and 5-BDBD on ES-driven MSC
condensation; MSCs in micromass culture were examined using phase contrast images after 3 days culture in maintenance medium (control), with ES (ES), with ES plus apyrase (ES + apyrase), or with ES plus 5-BDBD
(ES + 5-BDBD); Scale bars, 500 μm. (d) Suppressive effects of apyrase and 5-BDBD on ES-induced type II
collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days culture in maintenance medium (control), with ES (ES), with ES plus apyrase (ES + apyrase), or with ES plus 5-BDBD (ES + 5-BDBD), gene
expression was analyzed in MSCs using real-time PCR. Data are presented as means ± S.D. and differences were
identified using ANOVA; **p < 0.01, *p < 0.05.
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critical roles in cell-cell communications that lead to platelet aggregation
66–68
. Hence, ES may induce synchro-
nized secretion of adhesion molecules and paracrine signaling, cell migration, and spherical morphogenesis by
activating extracellular ATP signaling and Ca
2+
/ATP oscillations, leading to prechondrogenic condensation.
In the present study, ES induced chondrogenesis by stimulating both TGF-β and BMP signaling (Figs 6 and 7)
69–71
.
It was known that TGF-β signaling reportedly stimulated prechondrogenic condensation by inducing the pro-
duction of fibronectin and N-cadherin, and subsequently enhanced the expression of chondrogenic markers in
various in vitro models
69,70
, and BMPs also promote chondrogenesis and regulate formation of cartilage elements
in the limb
71
. Moreover, BMP signaling was shown to enhances TGF-β-induced chondrogenesis
72
. In addition, ES
activates voltage-sensitive sodium and calcium ion channels to induce Ca
2+
influx
57
. Hence, because Ca
2+
influx
activates exocytotic secretion
73
, increased Ca
2+
influx following ES may enhance secretion of TGF-β s and BMPs,
likely contributing significantly to the induction of MSC chondrogenesis. These facts can explain why ES led to
stronger and more rapid induction of chondrogenesis than CM supplemented with TGF-β1 (Fig. 2a).
Many studies have shown that TGF-β signaling precedes BMP signaling and effectively initiates MSC con-
densation, leading to increases in the size and numbers of MSC aggregates, while BMP signaling is more effective
in aggregated MSCs than in low density MSCs and increases sizes but not numbers of MSC aggregates
69–71,74
.
We also previously demonstrated that TGF-β signaling but not BMP signaling drives ATP oscillations, leading
to prechondrogenic condensation
39
. These data suggest differential effects of TGF-β and BMP signaling path-
ways on chondrogenesis. Consistent with these results, the present result showed that pharmacological inhibition
of TGF-β signaling suppressed ES-driven condensation (Fig. 6b), whereas inhibition of BMP signaling did not
(Fig. 7b), indicating that ES-driven condensation is mediated by TGF-β signaling, but is not mediated by BMP
signaling. TGF-β signaling has been shown to enhance extracellular ATP levels and thus activate extracellular
ATP signaling
75
. Accordingly, TGF-β signaling is stimulated by ES and then activates P2X
4 signaling to conse-
quently induce MSC condensation, which suggests that P2X
4 signaling mediates the differential effects between
TGF-β and BMP signaling on chondrogenesis.
Based upon the findings from previous studies and the present study, the actions of ES for MSC chondrogen-
esis could be proposed: ES drives ATP/Ca
2+
oscillations, leading to MSC condensation through TGF-β signaling
and P2X
4 signaling, and subsequently induces chondrogenesis through TGF-β signaling, BMP signaling and P2X
4
signaling (Fig. 8).
Figure 5. Paracrine signals and gap junction mediates MSC condensation and chondrogenesis following
ES. (a) Effects of BFA and CBX on ES-driven MSC condensation; MSCs in micromass culture were examined
using phase contrast images after 3 days culture in maintenance medium (control), with ES (ES), with ES plus BFA (ES + BFA), or with ES plus CBX (ES + CBX); Scale bars, 500 μm. (b) Suppressive effects of BFA and CBX
on ES-induced type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days culture in
maintenance medium (control), with ES (ES), with ES plus BFA (ES + BFA), or with ES plus CBX (ES + CBX),
gene expression was analyzed in MSCs using real-time PCR. Data are presented as means ± S.D. and differences
were identified using ANOVA; **p < 0.01, *p < 0.05.
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In summary, in this paper we demonstrate for the first time that ES drives Ca
2+
/ATP oscillations, leading to
MSC chondrogenesis in the absence of exogenous cytokine or growth factor supplements, and optimized ES
regimes for induction of MSC chondrogenesis. Subsequently, we showed that P2X
4 signaling mediates ES-driven
ATP oscillations and chondrogenesis, and TGF-β and BMP signaling both mediates ES-driven chondrogenesis
but have differential effects on ES-driven condensation. These data will facilitate the development of a novel
ES-based technology for cell therapy and ES-based rehabilitation for cartilage repair. However, further studies are
required to establish ES-based therapeutic strategies with the potential to overcome limitations of cartilage repair.
Methods
Cell culture and light microscopy observations. Mouse MSCs which were produced from bone marrow
that was isolated from C57BL/6 mice were purchased from Invitrogen (Carlsbad, CA, USA), and were expanded
in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich, St. Louis,
MO, USA) with GlutaMAX-I supplemented with 10% fetal bovine serum (FBS; Invitrogen). All experiments
were performed in micromass cultures. Briefly, the expanded MSCs (passage 3–7) were harvested and resus-
pended in maintenance medium at 2 × 10
7
cells/ml. Droplets (10 μL) were carefully placed in each dish and cells
were allowed to adhere at 37 °C for 1 h. Subsequently, 3 mL of maintenance medium were added to control and
ES groups, while 3 mL of chondrogenic medium (CM; DMEM/F12, 1% ITS (Sigma-Aldrich), 10-ng/ml TGF-β
1 (Peprotech, Rocky Hill, NJ, USA), 0.9-mM sodium pyruvate (Sigma-Aldrich), 50-μg/ml l-ascorbic acid-2-
phosphate (Sigma-Aldrich), 10
−7
-M dexamethasone (Sigma–Aldrich), and 40-μg/ml l-proline (Sigma-Aldrich))
were added to CM group. To investigate the effects of chemical compounds on MSCs, culture medium was
replaced with medium supplemented with 100-unit/ml apyrase (Sigma-Aldrich), which catalyzes the hydrolysis
of ATP to AMP and inorganic phosphate, 100-μM 5-(3-bromophenyl)-1,3-dihydro-2Hbenzofuro[3,2-e]-1,4-di-
azepin-2-one (5-BDBD; Tocris Bioscience, Bristol, United Kingdom), which is an inhibitor of P2X
4 puriner-
gic receptors, 100 ng/ml noggin (R&D Systems), which is a BMP-specific antagonist protein, 10-μM SB-431542
(Sigma–Aldrich), which is an inhibitor of TGF-beta type I receptor, 100ng/ml brefeldin A (BFA), which is a
inhibitor for protein secretion, and 100-μM carbenoxolone, which is a gap junction inhibitor. After 3, 7, and 14
days culture, microscope observations were performed using a phase contrast microscope (Nikon, Tokyo, Japan).
Figure 6. TGF-β signaling mediates MSC condensation and chondrogenesis following ES. (a) Real-time
gene expression analysis of TGF-β 1 in micromass cultures of MSCs after culture for 3 days in maintenance
medium (control) or with ES (ES); Data are presented as means ± S.D. and differences were identified using
Students t-test, **p < 0.01 (b) Effects of SB-431542 (SB) on ES-driven MSC condensation; MSCs in micromass
culture were examined using phase contrast images after 3 days culture in maintenance medium (control), with
ES (ES), or with ES plus SB-431542 (ES + SB); Scale bars, 500 μm. (c) Effects of SB on ES-induced enhancement
of type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression; After 3 days culture in micromass cultures
without treatment (control), with ES (ES), or with ES plus SB-431542 (ES + SB), gene expression was analyzed
in MSCs using real-time PCR. Data are presented as means ± S.D. Differences were identified using ANOVA;
**p < 0.01, *p < 0.05.
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Flow cytometry. The cell surface markers of MSCs were analyzed using a FACS Calibur flow cytometer (BD
Biosciences, San Jose, CA, USA). Briefly, cells that reached 90% confluence were harvested using 0.25% EDTA
and washed twice in Dulbecco’s phosphate buffered saline supplemented with 10% FBS. The cells for detecting
CD11b, CD34, CD45, Sca-1, CD44 and CD73 were labeled directly with BB515 or PE-conjugated CD markers
Figure 7. BMP signaling mediates ES-driven chondrogenesis, but not ES-driven condensation. (a) Real-
time gene expression analysis of BMP2 in micromass cultures of MSCs after 3 days in maintenance medium (control) or in maintenance medium with ES (ES). Data are presented as means ± S.D. and differences were
identified using Students t-test, **p < 0.01 (b) Effect of noggin (Nog) on ES-induced MSC condensation; MSCs
in micromass culture were examined using phase contrast images after 3 days culture in maintenance medium
(control), with ES, or with Nog and ES; Scale bars, 500 μm. (c) Effect of Nog on ES-induced enhancement of
type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days micromass culture of MSCs
with no treatment (control), with ES (ES), or with ES and Nog, gene expression was analyzed using real-time
PCR. Data are presented as means ± S.D. Differences were identified using ANOVA; **p < 0.01, *p < 0.05.
Figure 8. Proposed model of the functions of electrotransduction for MSC chondrogenesis. ES drives ATP/
Ca
2+
oscillations, leading to MSC condensation through TGF-β signaling and P2X
4 signaling, and subsequently
induces chondrogenesis through TGF-β signaling, BMP signaling and P2X
4 signaling.
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(rat anti-mouse CD11b [1: 100, BD Pharmingen; BD Biosciences, Franklin Lake, NJ, USA], rat anti-mouse CD34
[1: 100, BD Pharmingen], rat anti-mouse CD45 [1: 100, BD Pharmingen], rat anti-mouse Sca-1 [1: 100, BD
Pharmingen], rat anti-mouse CD44 [1: 100, BD Pharmingen], rat anti-mouse CD73 [1: 100, BD Pharmingen]).
Electric stimulation. ES was applied to MSCs using a C-Pace EP culture pacer (IonOptix, MA, USA), which
is a multi-channel stimulator designed for chronic stimulation of bulk quantities of cells in culture. This instru-
ment emits bipolar pulses to culture media immersed carbon electrodes of a C-dish. ES was applied to MSCs cul-
tured under conditions of high-density micromass (2 × 10
7
cells/ml) under electrical fields of 0, 1, 5, or 25 V/cm,
with a duration of 8 ms and a frequency of 5.0 Hz. At indicated time points, MSCs were harvested in Trizol
(Invitrogen) for real-time PCR analyses or were fixed using paraformaldehyde in phosphate-buffered saline (pH 7.4)
for immunocytochemical analyses and alcian blue staining.
Transfection of cells with reporter genes and bioluminescence monitoring. For real-time moni-
toring of intracellular ATP levels in MSCs, MSCs were transfected with a bioluminescent luciferase reporter gene
(Luc) fused to an ACTIN promoter (P
ACTIN-Luc) using Lipofectamine LTX (Invitrogen) and then the medium was
replaced with recording medium (DMEM/F12 containing 10% FBS, 0.1-mM luciferin (Wako, Osaka, Japan), and
50-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH (pH = 7.0)). For real-time moni-
toring of intracellular Ca
2+
levels in MSCs, MSCs were transfected with a aequorin gene (AQ) fused to an CMV
promoter (P
CMV-AQ) using Lipofectamine LTX (Invitrogen) and then the medium was replaced with recording
medium (DMEM/F12 containing 10% FBS, 5-μM coelenterazine (Invitrogen), and 50-mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES)-NaOH (pH = 7.0)). Bioluminescence intensity was continuously meas-
ured using a dish-type luminescence detector (Kronos; ATTO, Osaka, Japan) at 1 min intervals under ES.
Lactate Dehydrogenase (LDH) Release Assays. LDH release assays were performed to assess the cyto-
toxicity of ES using LDH-cytotoxicity assay kits (DoGen, Korea) according to the manufacturer’s instructions.
After ES for 3 or 7 days, supernatants from each dish were transferred to fresh, flat bottom 96-well culture plates
containing 100-μL reaction mixtures, and were incubated for 30 min at room temperature. Formazan absorbance
was then measured at 480 nm using a microplate reader (TECAN, Switzerland).
Real-time PCR analysis. Total RNA was isolated from various MSCs cultures using the Direct-zol™
RNA
MiniPrep (Zymo Research Corporation, Irvine, CA, U.S.A.) according to the manufacturer’s protocol. RNA
concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington,
DE, USA), and reverse transcription reactions were performed using 0.2 μg of total RNA with a TOPscriptTM
cDNA synthesis kit (enzynomics, Daejeon, Korea). The real-time PCRs for beta-actin, collagen II, and aggrecan
were performed using the TOPrealTM qPCR 2X Pre MIX (enzynomics). Primer sequences are listed in Table 1.
Real-time PCRs were performed using a StepOnePlus
™
instrument (Applied Biosystems, Grand Island, NY,
USA) at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 10 s, extension at 60 °C for 15 s, and
annealing at 72 °C for 15 s. Gene expression levels were normalized to that of beta-actin and relative gene expres-
sion was calculated using the ddCT method.
Immunofluorescence staining and alcian blue staining. MSCs were fixed in 4% paraformaldehyde
for 20 min at room temperature and were washed three times in phosphate buffered saline (PBS). Some samples
were dehydrated through a graded ethanol series, infiltrated with xylene, embedded in paraffin, and sectioned at
a thickness of 7-μ m. After blocking in PBS containing 5% goat serum and 0.3% Triton X-100 for 60 min at room
temperature, cells were incubated with rabbit anti-type II collagen antibody (1:500; EnoGene Biotech, New York,
NY, USA) at 4 °C overnight, were washed three times in PBS containing 0.1% Triton X-100, and were then incu-
bated with Alexa488-conjugated secondary antibody (1:200; Invitrogen) for 60 min at room temperature in the
dark. Subsequently, cells were washed three times in PBS containing 0.1% Triton X-100 and nuclei were stained
with Hoechst 33258 (Dojindo, Tokyo, Japan). To visualize accumulation of sulfated glycosaminoglycans (GAGs),
cells were rinsed with PBS, fixed in paraformaldehyde for 20 min, stained with Alcian Blue Solution (pH 2.5;
Nacalai tesque, INC, Japan) overnight at room temperature, and were then rinsed with distilled water three times.
Accumulations of glycosaminoglycans were captured using a digital camera (Olympus, Tokyo, Japan). Expression
Gene Forward primers Reverse primers Accession No.
COL2A1 AGGGCAACAGCAGGTTCACATAC TGTCCACACCAAATTCCTGTTCA NM031163
Aggrecan AGTGGATCGGTCTGAATGACAGG AGAAGTTGTCAGGCTGGTTTGGA NM007424
SOX9 GAGGCCACGGAACAGACTCA CTTCAGATCAACTTTGCCAGCTT NM011448
P2X
4 AGACGGACCAGTGATGCCTAAC TGGAGTGGAGACCGAGTGAGA NM011026
TGF-β1 GCTTCAGACAGAAACTCACT GAACACTACTACATGCCATTAT BC013738
BMP2 ACTTTTCTCGTTTGTGGAGC GAACCCAGGTGTCTCCAAGA NM007553
ALP CCAACTCTTTTGTGCCAGAGA GGCTACATTGGTGTTGAGCTTT NM007431
COL1 GCTCCTCTTAGGGGCCACT CCACGTCTCACCATTGGG NM007742
aP2 GTGTGATGCCTTTGTGGGAAC CCTGTCGTCTGCGGTGATT NM024406
β-actin AGGTCATCACTATTGGCAACGA ATGGATGCCACAGGATTCCA NM007393
Table 1. The primer sequences for Real-time PCR analysis.
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levels of type II collagen and GAGs were quantified using immunofluorescence and alcian blue intensity profiles
with the NIH IMAGE J program, and data were transferred into Microsoft Excel for further analyses.
Statistical analysis. The results are presented as means ± SD for all samples. The statistical differences
between groups were analyzed by Students t-test, and multiple comparisons were performed by Fisher’s protected
least significant difference (PLSD) or Dunnett’s test. A value of p < 0.05 was considered to indicate statistical
significance.
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Acknowledgements
This work was supported by Basic Science Research Program (2014R1A1A1002054) and Global Frontier Project
(NRF-2013M3A6A4046061) through the National Research Foundation of Korea (NRF) funded by the Ministry
of Science, ICT and Future Planning.
Author Contributions
H.J.K. and H.G.C. designed research; H.J.K. and G.S.L. performed research; H.J.K. and H.G.C. analyzed data;
H.J.K. and H.G.C. wrote the paper. All authors discussed the results and commented on the paper.
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Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Kwon, H. J. et al. Electrical stimulation drives chondrogenesis of mesenchymal stem
cells in the absence of exogenous growth factors. Sci. Rep. 6, 39302; doi: 10.1038/srep39302 (2016).
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© The Author(s) 2016
www.nature.com/scientificreports
Electrical stimulation drives
chondrogenesis of mesenchymal
stem cells in the absence of
exogenous growth factors
Hyuck Joon Kwon
1
, Gyu Seok Lee
2
& Honggu Chun
3
Electrical stimulation (ES) is known to guide the development and regeneration of many tissues.
However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage
repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs)
have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during
chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels
of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen
levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition
of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by
driving Ca
2+
/ATP oscillations, which are known to be essential for prechondrogenic condensation. In
subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on
extracellular ATP signaling via P2X
4 receptors, and ES induced significant increases in TGF-β1 and BMP2
expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the
inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates
ES-driven condensation. These findings may contribute to the development of electrotherapeutic
strategies for cartilage repair using MSCs.
Articular cartilage is a unique load-bearing tissue that lacks vascular, neural, and lymphatic tissues. Articular
cartilage cannot spontaneously regenerate in vivo because chondral defects do not penetrate the subchondral
bone and therefore cannot be accessed by blood supply or mesenchymal stem cells (MSCs) from bone marrow
1,2
.
Hence, researchers and surgeons have developed various techniques to repair cartilage tissues
3
. However, most
current cartilage repair techniques eventually lead to the formation of fibrocartilage and cartilage degeneration
4
.
Accordingly, autologous chondrocyte implantation has been used for cartilage repair but is associated with sev-
eral disadvantages, including limited cell sources and frequent injury of healthy cartilage during surgery, further
encouraging formation of inferior fibrocartilage at defect sites
5
.
Owing to their capacity for self-renewal and differentiation into adipocytes, cartilage, bone, tendons, muscle,
and skin, MSCs are an attractive cell source for cartilage defect therapies
6–11
. Furthermore, because MSCs are
free of both ethical concerns and teratoma risks, MSCs have considerable therapeutic potential
12
. Therefore, it is
important to develop effective and safe methods for the induction of MSC chondrogenesis and for the production
of stable cartilaginous tissue by these cells. Multiple previous studies have demonstrated the effects of various
chemical factors, such as soluble growth factors, chemokines, and morphogens, on chondrogenesis. In particular,
transforming growth factors (TGF-β ) and bone morphogenetic proteins (BMPs) have been shown to play essen-
tial roles in the induction of chondrogenesis
13,14
. Although these growth factors have great therapeutic potential
for cartilage regeneration, growth factor-based therapies have several clinical complications, including high dose
requirements, low half-life, protein instability, higher costs, and adverse effects
15,16
. Recent studies demonstrate
1
Department of Physical Therapy and Rehabilitation, College of Health Science, Eulji University, Gyeonggi, Republic
of Korea.
2
Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam
National University, Daejeon, Republic of Korea.
3
Department of Bio-convergence Engineering, Korea University,
Seoul, Republic of Korea. Correspondence and requests for materials should be addressed to H.J.K. (email: kwonhj@
eulji.ac.kr) or H.C. (email: chunhonggu@korea.ac.kr)
received: 13 April 2016
accepted: 21 November 2016
Published: 22 December 2016
OPEN
www.nature.com/scientificreports/ 2SCIENTIFIC REPOrTS |�393v2 I� 1v.1v3~/srep393v2
that physical factors regulate cell differentiation and tissue development
17–21
, suggesting therapeutic potential of
physical factors as alternatives to chemical agents for cartilage regeneration.
Endogenous electrical signals have been observed in articular cartilage during physiological processes,
prompting the application of various electrical stimulation (ES) and electromagnetic field (EMF) inducers to
in vitro chondrogenesis and in vivo cartilage repair
22–24
. In particular, studies using animal models show that ES
and EMF improve healing of cartilage defects by increasing cell proliferation, glycosaminoglycan synthesis, and
the expression of extracellular matrix genes, and by reducing the production of inflammatory mediators
25–29
.
However, the precise roles of ES and EMF in cartilage repair remains unclear, and the effects of ES and EMF have
only been observed in the surrounding cartilage, and not in articular defects. These observations indicate that ES
or EMF alone have limited therapeutic efficacy for the repair of large osteochondral defects. Autologous chondro-
cyte transplants have been successfully used to repair large osteochondral defects
30
. Thus, multiple studies have
investigated the effects of ES on proliferation and synthesis of cartilage extracellular matrix proteins in chondro-
cytes
31–34
. However, chondrocytes gradually decrease in number with age
33
, and it is difficult to obtain sufficient
chondrocyte numbers to repair large defects due to limited life span and de-differentiation with downregulation
of cartilage-marker genes during culture
34,35
. As an alternative, MSCs with self-renewing abilities can differentiate
into chondrocytes and offer a reliable resource for ES-based therapies for damaged cartilage defects. However,
although recent studies have reported the effects of ES on proliferation and differentiation of MSCs
36,37
, it remains
unclear whether ES induces chondrogenic differentiation of MSCs.
Our previous studies demonstrated that intracellular ATP levels oscillate during chondrogenic differentiation
and the ATP oscillations play critical roles in prechondrogenic condensation
38,39
, and that extracellular ATP sig-
naling mediates the ATP oscillations during chondrogenesis
40
. ES has been shown to activate extracellular ATP
signaling in a variety of cell types
41–43
. Therefore, we hypothesized that ES induces ATP oscillations in MSCs by
stimulating extracellular ATP signaling and consequently drives MSC chondrogenesis. In the present study, we
demonstrated that ES induces ATP oscillations and promotes MSC chondrogenesis in the absence of exogenous
growth factors. Moreover, ES induced MSC chondrogenesis more effectively than treatments with chondrogenic
medium (CM) supplemented with soluble growth factors. Accordingly, further experiments showed that extracel-
lular ATP signaling via P2X
4 receptors was responsible for ATP oscillations and mediated chondrogenesis follow-
ing ES. In addition, ES-driven chondrogenesis depended on both TGF-β and BMP signaling pathways. However,
TGF-β signaling, but not BMP signaling, was involved in ES-driven condensation. The present data suggest that
ES has high potential as an MSC-based therapy for cartilage regeneration.
Results
ES induces calcium/ATP oscillations and MSC condensation. Flow cytometry analysis showed that
the expanded MSCs were positive for typical MSC markers (Sca-1, CD44, CD73) but showed low expression of
markers of hematopoietic stem cells (CD34), macrophages (CD11b), and granulocytes (CD45), which confirmed
that the expanded MSCs exibited the characteristics of MSCs (Supplementary Fig. S1). To determine whether
ES induces ATP oscillations in MSCs, we monitored temporal changes in intracellular ATP levels using a biolu-
minescent ATP-dependent luciferase (Luc) reporter gene fused to a constitutive ACTIN promoter (P
ACTIN-Luc).
Following transfection of MSCs with P
ACTIN-Luc, bioluminescence intensity was measured in real-time during
ES of 0, 1, 5 or 25 V/cm at 5 Hz (Fig. 1a). In these experiments, ES of 5 V/cm induced ATP oscillations of ~ 5 min
periods, whereas ES of 0, 1, or 25 V/cm did not (Fig. 1b). Because ATP oscillations were driven by changes in Ca
2+
concentrations during chondrogenesis
38
, we examined Ca
2+
oscillations using the bioluminescent Ca
2+
reporter
Aequorin (AQ) gene fused to a CMV promoter (P
CMV-AQ)
44
. ES of 5 V/cm consistently induced Ca
2+
oscillations,
whereas ES of 0, 1, or 25 V/cm did not (Fig. 1c), suggesting that optimized ES can drive fluctuations of both Ca
2+
and ATP. We previously showed that growth factors such as TGF-βs and insulin induce prechondrogenic conden-
sation by generating Ca
2+
/ATP oscillations
39
. Consistently, ES of 5 V/cm which induced Ca
2+
/ATP oscillations
led to compact condensation of MSCs, whereas ES of 0, 1, or 25 V/cm did not (Fig. 1d). Moreover, time-course
observations showed that ES of 5 V/cm induced gradual aggregation of MSCs into compact structures within 3
days, corresponding with the effects of chondrogenic medium (CM) supplemented with growth factors such as
TGF-βs and insulin (Supplementary Fig. S2). These results indicate that ES induces prechondrogenic condensa-
tion by driving Ca
2+
/ATP oscillations, even in the absence of exogenous growth factors.
ES induces MSC chondrogenesis. In further experiments, the effects of optimized ES on chondrogenic
differentiation were examined. Since ES for 3 days had little effect on cell damage (< 5%) but induced significant
cell death (almost 50%) after 7 days (Supplementary Fig. S3), ES was performed for 3 days. Gene expression of
chondrogenic markers such as type II collagen (COL2A1), aggrecan (AGC), and SRY (Sex Determining Region
Y)-Box 9 (SOX9)
45–47
was analyzed at 1 and 3-day of ES treatment and 7-day post-ES treatment. Quantitative
real-time RT-PCR analyses showed that ES significantly enhanced gene expression of chondrogenic markers
within 3 days of ES treatment, revealing a 66-fold increase in COL2A1, a 43-fold increase in AGC, and a 35-fold
increase in SOX9 expression at 3-day of ES (Fig. 2a). Moreover, increases of chondrogenic marker expression in
MSCs treated with ES for 3 days were much higher than those in the CM exposed cells, and were greater than
or equal to expression levels in MCSs that were fully differentiated into chondrocytes following treatment with
CM for 14 days (Fig. 2a). These data suggest that ES induces MSC chondrogenesis more effectively than CM.
Moreover, it was found that the chondrogenic markers were highly expressed for as long as 7 days after the last
ES treatment (Fig. 2a), which confirmed that chondrogenesis was induced in MSCs by ES. In addition, ES led to
significant decreases in the expression of type I collagen (COL1; Fig. 2b). This result indicates that ES induces
differentiation of MSCs into not fibrocartilaginous tissues but hyaline cartilaginous tissues
48–51
. In contrast, ES
did not significantly change the expression of the osteogenic marker alkaline phosphatase (ALP), or the adipo-
genic marker adipocyte protein 2 (aP2; Fig. 2b), indicating that ES does not induce osteogenesis or adipogenesis.
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Additionally, immunostaining and alcian blue staining analyses showed significantly higher expression of type
II collagen and GAGs in ES-treated MSCs compared with control cells (Fig. 3a–c). Taken together, these data
suggest that ES induces MSC chondrogenesis for hyaline cartilage regeneration even in the absence of exogenous
growth factors.
Extracellular ATP signaling via P2X
4 receptor mediates ATP oscillations, condensation, and
subsequent chondrogenesis following ES.
Extracellular ATP signaling via the P2X
4 receptor report-
edly plays key roles in prechondrogenic condensation by mediating ATP oscillations
36
. In the present study, ES
enhanced the expression of P2X
4 receptor mRNA in MSCs (Fig. 4a), suggesting that extracellular ATP signaling
Figure 1. Electrical stimulation induces ATP/Ca
2+
oscillations and MSC condensation. (a) Schematic of the
real-time bioluminescence monitoring system for ES treated MSCs transfected with bioluminescence reporters (b) Real-time monitoring of intracellular ATP levels in MSCs under ES (0, 1, 5, and 25 V/cm, 8 ms, 5 Hz) using
a bioluminescence ATP reporter (P
ACTIN-Luc) (c) Real-time monitoring of intracellular Ca
2+
levels under ES
(0, 1, 5, and 25 V/cm, 8 ms, 5 Hz) using a bioluminescence Ca
2+
reporter (P
CMV-AQ) (d) Condensation
behaviors of MSCs in micromass culture were examined using phase contrast images during culture for 3 days under ES (0, 1, 5, and 25 V/cm, 8 ms, 5 Hz); Scale bars, 500 μm.
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via the P2X
4 receptor is involved in ATP oscillations, MSC condensation, and chondrogenesis following ES. In
agreement, the P2X
4 purinergic receptor inhibitor 5-BDBD inhibited ES-driven ATP oscillations (Fig. 4b). In sub-
sequent experiments, it was examined whether extracellular ATP signaling via the P2X
4 receptor was associated
with ES-driven condensation and subsequent chondrogenesis. 5-BDBD almost completely inhibited ES-driven
condensation, and apyrase significantly suppressed this process (Fig. 4c). In addition, ES did not enhance the
expression of the chondrogenic markers COL2A1, AGC, and SOX9 in MSCs treated with either apyrase or
5-BDBD (Fig. 4d). Hence, extracellular ATP signaling via P2X
4 receptors mediates ES-driven MSC condensation
and chondrogenesis.
Intercellular communications mediates ES-driven chondrogenesis. It was known that ES influ-
ences intercellular communications such as paracrine signaling
52
and gap junction
53
. We found that BFA, which
blocks classical secretion of paracrine factors, suppressed ES-driven condensation and ES-driven increases of
COL2A1, AGC, and SOX9 expression (Fig. 5a,b). In addition, the gap-junction inhibitor carbenoxolone also sup-
pressed ES-driven condensation and ES-driven increases of expression of the chondrogenic markers (Fig. 5a,b).
This result indicates that ES induces chondrogenesis by activating the release of paracrine factors and the
gap-junction activity.
TGF-β signaling mediates MSC condensation and chondrogenesis following ES. TGF-β sign-
aling reportedly induces prechondrogenic condensation and chondrogenesis through ATP oscillations
39
. The
present study showed that ES led to much higher mRNA expression of TGF-β1 (74 fold) than expression in
control cells (Fig. 6a), suggesting that TGF-β signaling is involved in ES-driven MSC condensation and chondro-
genesis. In agreement, inhibition of TGF-β signaling by SB-431542 almost completely blocked ES-driven con-
densation and significantly suppressed ES-driven increases of COL2A1, AGC, and SOX9 expression (Fig. 6b,c).
Although these data indicate that ES induces MSC condensation and chondrogenesis by activating TGF-β signa-
ling, SB-431542 did not completely suppress ES-driven induction of chondrogenic markers (Fig. 6c), suggesting
that other growth factors and cytokines also mediate the actions of ES.
Figure 2. ES enhances mRNA expression of chondrogenic markers in MSCs. Relative levels of mRNA
were determined by quantitative real-time PCR in relation to beta-actin. (a) Gene expression of type II collagen (COL2A1), aggrecan (AGC), and Sox9 in MSCs was determined at various time points during ES treatment (5 V/cm, 8 ms, 5 Hz) and CM treatment. Data are presented as means ± standard deviations (S.D.).
Statistical analyses were performed using ANOVA (Dunnett’s test); **p < 0.01, *p < 0.05 versus Ctl (control;
MSCs cultured in maintenance medium for 3 days). (b) Gene expression of type 1 collagen (COL1), alkaline phosphatase (ALP), and adipocyte protein (aP2) was determined at various time points during ES treatment (5 V/cm, 8 ms, 5 Hz). Data show mean ± S.D. Statistical analyses were performed using ANOVA (Dunnett’s
test); **p < 0.01, *p < 0.05 versus control (MSCs cultured in maintenance medium for 3 days).
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BMP signaling mediates ES-induced chondrogenesis, but not ES-induced condensation. BMPs
have been shown to play important roles in cartilage development
54,55
. Moreover, the present experiments showed
that in comparison with non-treated controls, ES increased BMP2 expression by 42 fold (Fig. 7a). In addition, the
inhibitor of BMP signaling noggin suppressed ES-driven increases in COL2A1, AGC, and SOX9 mRNA expres-
sion (Fig. 7c). However, noggin did not suppress ES-driven condensation (Fig. 7b), indicating that BMP signaling
mediates ES-driven chondrogenesis, but not ES-driven condensation.
Discussion
ES is a versatile treatment that remains poorly understood in the context of stem cell-based therapy. Herein,
we demonstrate that ES significantly enhances the expression of chondrogenic markers (Figs 2a and 3a,b), but
significantly decreases COL1 expression in MSCs (Fig. 2b). These data indicate that ES induces MSC differen-
tiation into hyaline chondrogenic cells, and provide evidence of the potential of electrically stimulated MSCs to
efficiently regenerate hyaline cartilage in the absence of additional exogenous chemical factors.
Our previous results demonstrated that ATP oscillations driven by chondrogenic growth factors such as TGF
beta and insulin play essential roles for prechondrogenic condensation that is the initial step of chondrogenesis by
inducing oscillatory expression of proteins involved in actin dynamics, cell migration, and adhesion which leads
to collective migration and adhesion
38,56
. The present results demonstrate that ES generates Ca
2+
/ATP oscillations
in MSCs even in the absence of exogenous growth factors (Fig. 1b). Since ES directly regulates voltage-gated Ca
2+
channels
57
, ES can drive Ca
2+
oscillations by modulating voltage-gated Ca
2+
channels. In addition, since extracel-
lular ATP signaling modulates Ca
2+
flux by producing diacylglycerol and inositol 1,4,5-triphosphate, activating
protein kinase C, and by mobilizing intracellular Ca
2+
in multiple cell types
58
, ES can induces Ca
2+
oscillation
by extracellular ATP signaling via the P2X
4 receptor, which is supported by the present result that P2X
4 ATP
Figure 3. ES enhances expression of COL2A1 and GAGs in MSCs. (a) Immunofluorescent staining
(COL2A1; green) and alcian blue staining (glycosaminoglycan (GAG); blue) of micromass cultures after culture in maintenance medium (control) or maintenance medium with ES (5 V/cm, 8 ms, 5 Hz) for 3 days (ES). Nuclei
were stained blue using Hoechst 33342; Scale bars, 500 μm. (b) Immunofluorescent staining (COL2A1; green)
and alcian blue staining (GAG; blue) of paraffin-embedded sections of micromass cultures after culture in
maintenance medium (control) or maintenance medium with ES (5 V/cm, 8 ms, 5 Hz) for 3 days (ES). Nuclei
were stained blue using Hoechst 33342; Scale bars, 100 μm. (c) Quantitative analysis of immunostaining
intensity and alcian blue staining intensity. Data are presented as means ± S.D. and differences were identified
using Students t-test, **p < 0.01.
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signaling mediates the actions of ES (Fig. 4). Increased Ca
2+
levels activate ATP-consuming processes such as ion
pumping and exocytosis
59
, decrease glucose consumption by inhibiting glycolytic enzymes
60
, and decrease mito-
chondrial ATP production by abolishing mitochondrial membrane potential
61
, indicating the negative effects
of Ca
2+
on ATP levels. Accordingly, Ca
2+
oscillations can drive ATP oscillations. In addition, previous studies
demonstrated that pulsed electrical fields or pulsed electromagnetic fields modulate cAMP levels by activating
adenosine receptors such as A
2A, A
2b, and A
3 receptors, which leads to activation of anti-inflammatory pathways
and cellular proliferation in cartilage
62–65
. Our previous results showed that ATP oscillations are dependent on
cAMP dynamics
38,40
. These results suggest that ES drive ATP oscillations by modulating cAMP levels.
We demonstrated that pharmacological inhibition of P2X
4-mediated ATP oscillations suppressed ES-driven
condensation (Fig. 4b,c). Previous study showed that Ca
2+
/ATP oscillations induced synchronized secretion of
adhesion molecules and prechondrogenic condensation
38
. In agreement, extracellular ATP signaling reportedly
mediates chemotaxis and morphological changes from spread to spherical shapes, and Ca
2+
oscillations play
Figure 4. Extracellular ATP signaling via P2X
4 receptors mediates ES-driven ATP oscillations,
prechondrogenic condensation, and chondrogenesis. (a) Real-time gene expression analyses of P2X
4
receptors in micromass cultures of MSCs after culture for 3 days in maintenance medium (control) or maintenance medium under ES. Data are presented as means ± S.D. and differences were identified using
Students t-test, *p < 0.05 (b) Effects of 5-BDBD on ES-driven ATP oscillations; MSCs in micromass culture
were treated with 5-BDBD after induction of P
ACTIN-Luc oscillations by ES. Bioluminescence monitoring was
performed after application of ES to MSCs (time = 0 h). (c) Effects of apyrase and 5-BDBD on ES-driven MSC
condensation; MSCs in micromass culture were examined using phase contrast images after 3 days culture in maintenance medium (control), with ES (ES), with ES plus apyrase (ES + apyrase), or with ES plus 5-BDBD
(ES + 5-BDBD); Scale bars, 500 μm. (d) Suppressive effects of apyrase and 5-BDBD on ES-induced type II
collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days culture in maintenance medium (control), with ES (ES), with ES plus apyrase (ES + apyrase), or with ES plus 5-BDBD (ES + 5-BDBD), gene
expression was analyzed in MSCs using real-time PCR. Data are presented as means ± S.D. and differences were
identified using ANOVA; **p < 0.01, *p < 0.05.
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critical roles in cell-cell communications that lead to platelet aggregation
66–68
. Hence, ES may induce synchro-
nized secretion of adhesion molecules and paracrine signaling, cell migration, and spherical morphogenesis by
activating extracellular ATP signaling and Ca
2+
/ATP oscillations, leading to prechondrogenic condensation.
In the present study, ES induced chondrogenesis by stimulating both TGF-β and BMP signaling (Figs 6 and 7)
69–71
.
It was known that TGF-β signaling reportedly stimulated prechondrogenic condensation by inducing the pro-
duction of fibronectin and N-cadherin, and subsequently enhanced the expression of chondrogenic markers in
various in vitro models
69,70
, and BMPs also promote chondrogenesis and regulate formation of cartilage elements
in the limb
71
. Moreover, BMP signaling was shown to enhances TGF-β-induced chondrogenesis
72
. In addition, ES
activates voltage-sensitive sodium and calcium ion channels to induce Ca
2+
influx
57
. Hence, because Ca
2+
influx
activates exocytotic secretion
73
, increased Ca
2+
influx following ES may enhance secretion of TGF-β s and BMPs,
likely contributing significantly to the induction of MSC chondrogenesis. These facts can explain why ES led to
stronger and more rapid induction of chondrogenesis than CM supplemented with TGF-β1 (Fig. 2a).
Many studies have shown that TGF-β signaling precedes BMP signaling and effectively initiates MSC con-
densation, leading to increases in the size and numbers of MSC aggregates, while BMP signaling is more effective
in aggregated MSCs than in low density MSCs and increases sizes but not numbers of MSC aggregates
69–71,74
.
We also previously demonstrated that TGF-β signaling but not BMP signaling drives ATP oscillations, leading
to prechondrogenic condensation
39
. These data suggest differential effects of TGF-β and BMP signaling path-
ways on chondrogenesis. Consistent with these results, the present result showed that pharmacological inhibition
of TGF-β signaling suppressed ES-driven condensation (Fig. 6b), whereas inhibition of BMP signaling did not
(Fig. 7b), indicating that ES-driven condensation is mediated by TGF-β signaling, but is not mediated by BMP
signaling. TGF-β signaling has been shown to enhance extracellular ATP levels and thus activate extracellular
ATP signaling
75
. Accordingly, TGF-β signaling is stimulated by ES and then activates P2X
4 signaling to conse-
quently induce MSC condensation, which suggests that P2X
4 signaling mediates the differential effects between
TGF-β and BMP signaling on chondrogenesis.
Based upon the findings from previous studies and the present study, the actions of ES for MSC chondrogen-
esis could be proposed: ES drives ATP/Ca
2+
oscillations, leading to MSC condensation through TGF-β signaling
and P2X
4 signaling, and subsequently induces chondrogenesis through TGF-β signaling, BMP signaling and P2X
4
signaling (Fig. 8).
Figure 5. Paracrine signals and gap junction mediates MSC condensation and chondrogenesis following
ES. (a) Effects of BFA and CBX on ES-driven MSC condensation; MSCs in micromass culture were examined
using phase contrast images after 3 days culture in maintenance medium (control), with ES (ES), with ES plus BFA (ES + BFA), or with ES plus CBX (ES + CBX); Scale bars, 500 μm. (b) Suppressive effects of BFA and CBX
on ES-induced type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days culture in
maintenance medium (control), with ES (ES), with ES plus BFA (ES + BFA), or with ES plus CBX (ES + CBX),
gene expression was analyzed in MSCs using real-time PCR. Data are presented as means ± S.D. and differences
were identified using ANOVA; **p < 0.01, *p < 0.05.
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In summary, in this paper we demonstrate for the first time that ES drives Ca
2+
/ATP oscillations, leading to
MSC chondrogenesis in the absence of exogenous cytokine or growth factor supplements, and optimized ES
regimes for induction of MSC chondrogenesis. Subsequently, we showed that P2X
4 signaling mediates ES-driven
ATP oscillations and chondrogenesis, and TGF-β and BMP signaling both mediates ES-driven chondrogenesis
but have differential effects on ES-driven condensation. These data will facilitate the development of a novel
ES-based technology for cell therapy and ES-based rehabilitation for cartilage repair. However, further studies are
required to establish ES-based therapeutic strategies with the potential to overcome limitations of cartilage repair.
Methods
Cell culture and light microscopy observations. Mouse MSCs which were produced from bone marrow
that was isolated from C57BL/6 mice were purchased from Invitrogen (Carlsbad, CA, USA), and were expanded
in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich, St. Louis,
MO, USA) with GlutaMAX-I supplemented with 10% fetal bovine serum (FBS; Invitrogen). All experiments
were performed in micromass cultures. Briefly, the expanded MSCs (passage 3–7) were harvested and resus-
pended in maintenance medium at 2 × 10
7
cells/ml. Droplets (10 μL) were carefully placed in each dish and cells
were allowed to adhere at 37 °C for 1 h. Subsequently, 3 mL of maintenance medium were added to control and
ES groups, while 3 mL of chondrogenic medium (CM; DMEM/F12, 1% ITS (Sigma-Aldrich), 10-ng/ml TGF-β
1 (Peprotech, Rocky Hill, NJ, USA), 0.9-mM sodium pyruvate (Sigma-Aldrich), 50-μg/ml l-ascorbic acid-2-
phosphate (Sigma-Aldrich), 10
−7
-M dexamethasone (Sigma–Aldrich), and 40-μg/ml l-proline (Sigma-Aldrich))
were added to CM group. To investigate the effects of chemical compounds on MSCs, culture medium was
replaced with medium supplemented with 100-unit/ml apyrase (Sigma-Aldrich), which catalyzes the hydrolysis
of ATP to AMP and inorganic phosphate, 100-μM 5-(3-bromophenyl)-1,3-dihydro-2Hbenzofuro[3,2-e]-1,4-di-
azepin-2-one (5-BDBD; Tocris Bioscience, Bristol, United Kingdom), which is an inhibitor of P2X
4 puriner-
gic receptors, 100 ng/ml noggin (R&D Systems), which is a BMP-specific antagonist protein, 10-μM SB-431542
(Sigma–Aldrich), which is an inhibitor of TGF-beta type I receptor, 100ng/ml brefeldin A (BFA), which is a
inhibitor for protein secretion, and 100-μM carbenoxolone, which is a gap junction inhibitor. After 3, 7, and 14
days culture, microscope observations were performed using a phase contrast microscope (Nikon, Tokyo, Japan).
Figure 6. TGF-β signaling mediates MSC condensation and chondrogenesis following ES. (a) Real-time
gene expression analysis of TGF-β 1 in micromass cultures of MSCs after culture for 3 days in maintenance
medium (control) or with ES (ES); Data are presented as means ± S.D. and differences were identified using
Students t-test, **p < 0.01 (b) Effects of SB-431542 (SB) on ES-driven MSC condensation; MSCs in micromass
culture were examined using phase contrast images after 3 days culture in maintenance medium (control), with
ES (ES), or with ES plus SB-431542 (ES + SB); Scale bars, 500 μm. (c) Effects of SB on ES-induced enhancement
of type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression; After 3 days culture in micromass cultures
without treatment (control), with ES (ES), or with ES plus SB-431542 (ES + SB), gene expression was analyzed
in MSCs using real-time PCR. Data are presented as means ± S.D. Differences were identified using ANOVA;
**p < 0.01, *p < 0.05.
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Flow cytometry. The cell surface markers of MSCs were analyzed using a FACS Calibur flow cytometer (BD
Biosciences, San Jose, CA, USA). Briefly, cells that reached 90% confluence were harvested using 0.25% EDTA
and washed twice in Dulbecco’s phosphate buffered saline supplemented with 10% FBS. The cells for detecting
CD11b, CD34, CD45, Sca-1, CD44 and CD73 were labeled directly with BB515 or PE-conjugated CD markers
Figure 7. BMP signaling mediates ES-driven chondrogenesis, but not ES-driven condensation. (a) Real-
time gene expression analysis of BMP2 in micromass cultures of MSCs after 3 days in maintenance medium (control) or in maintenance medium with ES (ES). Data are presented as means ± S.D. and differences were
identified using Students t-test, **p < 0.01 (b) Effect of noggin (Nog) on ES-induced MSC condensation; MSCs
in micromass culture were examined using phase contrast images after 3 days culture in maintenance medium
(control), with ES, or with Nog and ES; Scale bars, 500 μm. (c) Effect of Nog on ES-induced enhancement of
type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days micromass culture of MSCs
with no treatment (control), with ES (ES), or with ES and Nog, gene expression was analyzed using real-time
PCR. Data are presented as means ± S.D. Differences were identified using ANOVA; **p < 0.01, *p < 0.05.
Figure 8. Proposed model of the functions of electrotransduction for MSC chondrogenesis. ES drives ATP/
Ca
2+
oscillations, leading to MSC condensation through TGF-β signaling and P2X
4 signaling, and subsequently
induces chondrogenesis through TGF-β signaling, BMP signaling and P2X
4 signaling.
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(rat anti-mouse CD11b [1: 100, BD Pharmingen; BD Biosciences, Franklin Lake, NJ, USA], rat anti-mouse CD34
[1: 100, BD Pharmingen], rat anti-mouse CD45 [1: 100, BD Pharmingen], rat anti-mouse Sca-1 [1: 100, BD
Pharmingen], rat anti-mouse CD44 [1: 100, BD Pharmingen], rat anti-mouse CD73 [1: 100, BD Pharmingen]).
Electric stimulation. ES was applied to MSCs using a C-Pace EP culture pacer (IonOptix, MA, USA), which
is a multi-channel stimulator designed for chronic stimulation of bulk quantities of cells in culture. This instru-
ment emits bipolar pulses to culture media immersed carbon electrodes of a C-dish. ES was applied to MSCs cul-
tured under conditions of high-density micromass (2 × 10
7
cells/ml) under electrical fields of 0, 1, 5, or 25 V/cm,
with a duration of 8 ms and a frequency of 5.0 Hz. At indicated time points, MSCs were harvested in Trizol
(Invitrogen) for real-time PCR analyses or were fixed using paraformaldehyde in phosphate-buffered saline (pH 7.4)
for immunocytochemical analyses and alcian blue staining.
Transfection of cells with reporter genes and bioluminescence monitoring. For real-time moni-
toring of intracellular ATP levels in MSCs, MSCs were transfected with a bioluminescent luciferase reporter gene
(Luc) fused to an ACTIN promoter (P
ACTIN-Luc) using Lipofectamine LTX (Invitrogen) and then the medium was
replaced with recording medium (DMEM/F12 containing 10% FBS, 0.1-mM luciferin (Wako, Osaka, Japan), and
50-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH (pH = 7.0)). For real-time moni-
toring of intracellular Ca
2+
levels in MSCs, MSCs were transfected with a aequorin gene (AQ) fused to an CMV
promoter (P
CMV-AQ) using Lipofectamine LTX (Invitrogen) and then the medium was replaced with recording
medium (DMEM/F12 containing 10% FBS, 5-μM coelenterazine (Invitrogen), and 50-mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES)-NaOH (pH = 7.0)). Bioluminescence intensity was continuously meas-
ured using a dish-type luminescence detector (Kronos; ATTO, Osaka, Japan) at 1 min intervals under ES.
Lactate Dehydrogenase (LDH) Release Assays. LDH release assays were performed to assess the cyto-
toxicity of ES using LDH-cytotoxicity assay kits (DoGen, Korea) according to the manufacturer’s instructions.
After ES for 3 or 7 days, supernatants from each dish were transferred to fresh, flat bottom 96-well culture plates
containing 100-μL reaction mixtures, and were incubated for 30 min at room temperature. Formazan absorbance
was then measured at 480 nm using a microplate reader (TECAN, Switzerland).
Real-time PCR analysis. Total RNA was isolated from various MSCs cultures using the Direct-zol™
RNA
MiniPrep (Zymo Research Corporation, Irvine, CA, U.S.A.) according to the manufacturer’s protocol. RNA
concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington,
DE, USA), and reverse transcription reactions were performed using 0.2 μg of total RNA with a TOPscriptTM
cDNA synthesis kit (enzynomics, Daejeon, Korea). The real-time PCRs for beta-actin, collagen II, and aggrecan
were performed using the TOPrealTM qPCR 2X Pre MIX (enzynomics). Primer sequences are listed in Table 1.
Real-time PCRs were performed using a StepOnePlus
™
instrument (Applied Biosystems, Grand Island, NY,
USA) at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 10 s, extension at 60 °C for 15 s, and
annealing at 72 °C for 15 s. Gene expression levels were normalized to that of beta-actin and relative gene expres-
sion was calculated using the ddCT method.
Immunofluorescence staining and alcian blue staining. MSCs were fixed in 4% paraformaldehyde
for 20 min at room temperature and were washed three times in phosphate buffered saline (PBS). Some samples
were dehydrated through a graded ethanol series, infiltrated with xylene, embedded in paraffin, and sectioned at
a thickness of 7-μ m. After blocking in PBS containing 5% goat serum and 0.3% Triton X-100 for 60 min at room
temperature, cells were incubated with rabbit anti-type II collagen antibody (1:500; EnoGene Biotech, New York,
NY, USA) at 4 °C overnight, were washed three times in PBS containing 0.1% Triton X-100, and were then incu-
bated with Alexa488-conjugated secondary antibody (1:200; Invitrogen) for 60 min at room temperature in the
dark. Subsequently, cells were washed three times in PBS containing 0.1% Triton X-100 and nuclei were stained
with Hoechst 33258 (Dojindo, Tokyo, Japan). To visualize accumulation of sulfated glycosaminoglycans (GAGs),
cells were rinsed with PBS, fixed in paraformaldehyde for 20 min, stained with Alcian Blue Solution (pH 2.5;
Nacalai tesque, INC, Japan) overnight at room temperature, and were then rinsed with distilled water three times.
Accumulations of glycosaminoglycans were captured using a digital camera (Olympus, Tokyo, Japan). Expression
Gene Forward primers Reverse primers Accession No.
COL2A1 AGGGCAACAGCAGGTTCACATAC TGTCCACACCAAATTCCTGTTCA NM031163
Aggrecan AGTGGATCGGTCTGAATGACAGG AGAAGTTGTCAGGCTGGTTTGGA NM007424
SOX9 GAGGCCACGGAACAGACTCA CTTCAGATCAACTTTGCCAGCTT NM011448
P2X
4 AGACGGACCAGTGATGCCTAAC TGGAGTGGAGACCGAGTGAGA NM011026
TGF-β1 GCTTCAGACAGAAACTCACT GAACACTACTACATGCCATTAT BC013738
BMP2 ACTTTTCTCGTTTGTGGAGC GAACCCAGGTGTCTCCAAGA NM007553
ALP CCAACTCTTTTGTGCCAGAGA GGCTACATTGGTGTTGAGCTTT NM007431
COL1 GCTCCTCTTAGGGGCCACT CCACGTCTCACCATTGGG NM007742
aP2 GTGTGATGCCTTTGTGGGAAC CCTGTCGTCTGCGGTGATT NM024406
β-actin AGGTCATCACTATTGGCAACGA ATGGATGCCACAGGATTCCA NM007393
Table 1. The primer sequences for Real-time PCR analysis.
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levels of type II collagen and GAGs were quantified using immunofluorescence and alcian blue intensity profiles
with the NIH IMAGE J program, and data were transferred into Microsoft Excel for further analyses.
Statistical analysis. The results are presented as means ± SD for all samples. The statistical differences
between groups were analyzed by Students t-test, and multiple comparisons were performed by Fisher’s protected
least significant difference (PLSD) or Dunnett’s test. A value of p < 0.05 was considered to indicate statistical
significance.
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Acknowledgements
This work was supported by Basic Science Research Program (2014R1A1A1002054) and Global Frontier Project
(NRF-2013M3A6A4046061) through the National Research Foundation of Korea (NRF) funded by the Ministry
of Science, ICT and Future Planning.
Author Contributions
H.J.K. and H.G.C. designed research; H.J.K. and G.S.L. performed research; H.J.K. and H.G.C. analyzed data;
H.J.K. and H.G.C. wrote the paper. All authors discussed the results and commented on the paper.
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Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Kwon, H. J. et al. Electrical stimulation drives chondrogenesis of mesenchymal stem
cells in the absence of exogenous growth factors. Sci. Rep. 6, 39302; doi: 10.1038/srep39302 (2016).
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