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Introduction
Mesenchymal stem cells (MSC), separated from other
cells in bone marrow by virtue of their adherence to the
plastic walls of tissue culture containers, are capable of
differentiating into skeletal muscle cells as well as osteoblasts,
chondrocytes, and adipocytes under appropriate culture
conditions[1_3]. MSC are also
easily obtained from various sources and propagated manifold
ex vivo to clinically relevant
numbers[4]. Furthermore, MSC avoid ethical and
immunological hurdles associated with embryonic stem cells,
making them attractive candidates for tissue
repair and gene therapy[5].
Several studies have shown that transplanted MSC can
contribute to muscle cells and restore the sarcolemmal
expression of dystrophin in the mdx mouse, a model of
Duchenne muscular dystrophy (DMD)[6,7]. MSC as the
source of cell therapy to treat muscle diseases are promising
in principle, but the process is rather inefficient when
comparing the number of cells implanted with the amount of
formed muscle cells. A key to improving the current
protocols lies in exploiting the molecular mechanisms governing
the distinct steps of myogenic differentiation in
MSC[8].
The wingless-related MMTV integration site (Wnt)
family includes over 20 cysteine-rich secreted glycoproteins and
plays an important role in embryogenesis, including the
generation of cell polarity, specification of cell fate, and the
regulation of proliferation and
differentiation[9,10]. Wnt target gene expression by several different signaling
pathways[11]. The canonical pathway is the best characterized at the present
time. In the absence of Wnt signaling, β-catenin is
phosphorylated by glycogen synthase kinase-3β, in association
with axin and adenomatous polyposis coli. These proteins
target β-catenin for degradation by the ubiquitin proteasome
pathway[12]. When Wnt bind to Frizzled-Low-density
lipoprotein receptor-related protein 5/6 (LRP5/6) complex,
disheveled protein (a component of the Wnt signaling pathway) is
activated and inhibits the phosphorylation of β-catenin. It
results in β-catenin stabilization and accumulation in the
cytoplasm. The stabilized β-catenin enters the nucleus to
bind with members of the T-cell factor (TCF) and lymphoid
enhancer factor transcription factor family and induces the
expression of target genes[13].
Wnt signaling has been associated with myogenesis in
embryogenesis and postnatal muscle regeneration. During
embryonic development, Wnt1 and Wnt3a expressed in the
dorsal neural tube, and Wnt7a expressed in surface ectoderm,
have been shown to activate the expression of myogenic
regulatory factor genes in the paraxial
mesoderm[14,15]. On the other hand, myogenesis is severely reduced in presomitic
mesoderm and newly-formed somites by soluble
Frizzled-related proteins that are Wnt
antagonists[16]. Furthermore, defects in myogenesis are observed in Wnt1/Wnt3a
double-knockout mouse embryos[17]. In postnatal muscle
regenera-tion, it has been shown that Wnt proteins induce myogenic
differentiation in CD45+ stem
cells[18]. Although these data demonstrate the important roles of Wnt in the regulation of
myogenic differentiation in embryogenesis and muscle
regeneration, little is known about Wnt signaling inducing
myogenic differentiation in bone morrow-derived MSC.
Recent experiments have shown that Wnt signaling has
the capacity to promote proliferation and regulate the
invasion of human MSC[19]. Moreover, Wnt signaling has an
inhibitory effect on osteogenic and adipogenic
differentiation in human MSC[20,21]. These studies suggest that Wnt
signaling plays a potentially important role in the control of
the stem cell properties of MSC.
In this study, we examined the possible roles of Wnt3a in
myogenic differentiation, proliferation, and migration of rat
MSC (rMSC). The results demonstrated that Wnt3a was
sufficient in inducing myogenic differentiation in rMSC by
activating the muscular regulatory genes. Thus, to our
knowledge, our data suggest that canonical Wnt signaling
is sufficient in inducing myogenic lineage commitment in
rMSC. In addition, the present study showed that Wnt3a
inhibited adipogenic differentiation, but promoted the
proliferation and migration of rMSC.
Materials and methods
Isolation and culture of rMSC All of the animal
experiments were approved by the Animal Care and
Experimentation Committee of Sun Yat-sen University (Guangzhou,
China). Adult male Sprague-Dawley rats (60_80 g) were
obtained from Sun Yat-sen University Laboratorial Animal
Center. The primary MSC were isolated from Sprague-Dawley
rats according to the method described by Wakitani
et al with some
modifications[22]. In brief, femora and tibiae of
male Sprague-Dawley rats were collected; the adherent soft
tissues were carefully removed to ensure that the marrow
preparations were not contaminated by myogenic precursors.
Both ends of the bones were cut with bone scissors. The
bone marrow plugs were hydrostatically expelled from the
bones by syringes filled with growth medium. The bone
marrow cells were centrifuged and resuspended twice in
growth medium. After the cells were resuspended, the cells
were introduced into 25 cm2 tissue culture flasks in 6 mL
growth medium. Three days later, the medium was changed
and the non-adherent cells were discarded. The medium
was completely replaced every 3 d. Approximately 7_10 d
after seeding, the culture flasks became nearly confluent and
the adherent cells were released from the dishes with 0.25%
trypsin (Gibco Laboratories, Grand Island, New York, USA),
split 1:2, and seeded into fresh culture flasks. All of the
experiments described below were performed using cells from
the third to the fifth passage. The cells were cultured in
Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories, Grand Island, New York, USA) and 10% fetal
calf serum (FCS; Hyclone, Logan, Utah, USA). Every 3 d the
medium was changed once. The cells were grown at 37 °C in
a humidified atmosphere with 5% CO2.
Preparation of Wnt3a-conditioned medium and analysis
of the Wnt3a protein The mouse L cells were obtained from
the American Type Culture Collection (CRL-2648, Manassas,
VA, USA). The cells were cultured in DMEM supplemented
with 10% FCS at 37 °C. pGKWnt3a and pGKneo (control)
plasmids were generously provided by Prof Shinji TAKADA
(Okazaki Institute for Integrative Biosciences, National
Institutes of Natural Sciences, Okazaki, Aichi, Japan).
pGKWnt3a was constructed by inserting the mouse Wnt3a
cDNA, whose expression was driven by a promoter of rat
phosphoglycerokinase gene (PGK promoter) and terminated
at a transcriptional terminator sequence of the bovine growth
hormone gene, into pGKneo, containing the neomycin
phosphotransferase gene (neo) driven by the PGK promoter.
The L cells were transfected with pGKWnt3a or pGKneo
plasmids by Lipofectamine 2000 according to manufacturer's
instruction (Invitrogen, Carlsbad, California, USA). Briefly,
the L cells were seed on 35 mm culture plates at a density of
5×105 cells per cm2. Lipofectamine diluted in Opti-MEM
(Invitrogen, Carlsbad, California, USA) was applied to
the plasmid mixture and the formulation was continued
for 25 min. In total, 1 µg pGKWnt3a or pGKneo plasmids with 8 µL
Lipofectamine were applied in a final volume of 1.0 mL/well.
The transfection media were replaced with growth medium
after 4 h of incubation at 37 °C. The cells were selected by
G418 (0.4 g/L, Sigma, Saint Louis, Missouri, USA) for 3 weeks
and stably transfected clones were then selected. The L
cells transfected with pGKWnt3a or pGKneo plasmids were
termed L-Wnt3a or control L-cells. The Wnt3a-conditioned
medium (Wnt3a-CM) and the control L-conditioned medium
(L-CM) were prepared as described
previously[23]. Briefly,
1×106 L-Wnt3a or L cells were seeded in 100 mm dishes
containing DMEM with 10% FCS. After 3 d of culture, the cells
were cultured with fresh medium and incubated for 1 more
day. Then the medium was collected, centrifuged at
1000×g for 10 min, and filtered through a nitrocellulose membrane.
The conditioned media were stored at -80 °C until use. The
abundance of the Wnt3a protein in Wnt3a-CM and L-CM,
as well as in the extracts of L-Wnt3a and control L-cells, was
detected by Western blot analysis. The Wnt3a protein in
Wnt3a-CM and L-CM was diluted 1:1 in Western blot
sampling buffer, containing 20% glycerin, 10%
2-mercapto-ethanol, 4% SDS, and bromophenol blue, was boiled for 5
min at 95 °C and frozen at -80 °C until required. The cell
lysates were prepared by lysing the cells on ice in Western
blot sampling buffer, then boiled for 5 min at 95 °C and frozen
at -80 °C until required. The Western blot analysis was
performed with a primary rabbit anti-Wnt3a antibody (1:500,
Santa Cruz Biotechnology, Santa Cruz, California, USA) and
a secondary antirabbit peroxidase antibody (1:3000, Santa
Cruz Biotechnology, Santa Cruz, California, USA).
Wnt3a-CM was depleted of the Wnt3a protein by incubation with 4
µg/mL rabbit anti-Wnt3a antibody (Santa Cruz
Biotechno-logy, Santa Cruz, California, USA) at 4 °C overnight. The
conditioned medium was called Wnt3a-depleted-conditioned
medium (Wnt3a depl-CM) and was used to perform
antibody blocking experiments.
Flow cytometry (FACS) analysis An analysis of cell
surface molecules was performed on passage 3 cultures of rMSC
using flow cytometry and the following procedure. The
medium was removed from flasks; the cell layers were washed
twice with phosphate-buffered saline (PBS) and detached
from the flasks by incubation with a solution of 0.25% trypsin
for 3_5 min at room temperature. The cells were collected by
centrifugation and washed in flow cytometry buffer
consisting of 2% bovine serum albumin (BSA) and 0.1% sodium
azide in PBS. Then the rMSC were incubated with
fluorescein-5-isothiocyanate (FITC)-conjugated monoclonal
anti-bodies, including anti-CD11b, anti-CD29, anti-CD44, and
anti-CD45 (Chemicon, Temecula, CA, USA). All incubations with
antibodies were performed for 30 min, after which the cells
were washed with flow cytometry buffer. The washed cells
were pelleted and resuspended in flow cytometry buffer
containing 1% paraformaldehyde for 15 min. Non-specific
fluorescence was determined using equal aliquots of the cell
preparation that were incubated with antimouse monoclonal
antibodies. Data were acquired and analyzed on FACSCalibur
with CellQuest software (Becton Dickinson, San Jose, CA,
USA).
Measurement of rMSC proliferation To study the
effect of Wnt3a on rMSC proliferation, rMSC at passage 3
were inoculated on 24-well plates at a density of
1×104 cells/cm2 in the growth medium supplemented with varying
concentrations of control L-CM, Wnt3a-CM, or Wnt3a
depl-CM. After 96 h, the cells were harvested using trypsin and
counted in a hemocytometer. For the determination of
5-bromodeoxyuridine (BrdU) incorporation, the cells were
grown in 6-well plates and treated as described earlier. After
72 h, BrdU (Sigma, Saint Louis, Missouri, USA) was added
at 5 µg/mL. The cells were then incubated for 4 h and fixed.
The detection of BrdU was performed with a mouse
anti-BrdU (Sigma, Saint Louis, Missouri, USA), and a secondary
anti mouse-cyanine (CY) 3 (Santa Cruz Biotechnology, Santa
Cruz, California, USA) was used for visualization. Six
areas/well were randomly selected and automatically counted with
a computer-connected light microscope. All proliferation
assays were performed in triplicate.
Skeletal myogenic differentiation To induce myogenic
differentiation, the cells were plated at a density of
5×104 cells/mL in 6-well tissue culture plates and allowed to adhere
for 24 h at 37 °C, at which time the cells were switched to
myogenic medium consisting of DMEM, 2% horse serum,
and varying concentrations of control L-CM, Wnt3a-CM, or
Wnt3a depl-CM. The medium was changed every 3_4 d and
the cells were analyzed for the expression of the
muscle-specific markers desmin and myosin heavy chain (MHC)
after up to 10 d in culture.
Osteogenic differentiation To induce osteogenic
differentiation, the cells were plated at a density of
5×104 cells/mL in 6-well tissue culture plates and allowed to adhere
for 24 h. Then rMSC were incubated in growth medium
supplemented with 0.01 µmol/L dexamethasone, 10 mmol/L
glycerophosphate, and 50 mmol/L L-ascorbic
acid-2-phosphate (Sigma, Saint Louis, Missouri, USA). Osteogenic
differentiation was observed after 3 weeks by the deposit of a
mineralized hydroxyapatite extracellular matrix, as detected
by microscopy after staining with 40 mmol/L Alizarin Red S
(Sigma, Saint Louis, Missouri, USA).
Adipogenic differentiation To induce adipogenic
differentiation, the cells were plated at a density of
5×104 cells/mL in 6-well tissue culture plates and allowed to adhere
for 24 h. Then the cells were switched to adipogenic medium
(DMEM, 10% FBS, 1 µmol/L dexamethasone, 0.5 mmol/L
methylisobutylxanthine, and 10 µg/mL insulin; Sigma, Saint
Louis, Missouri, USA) for 21 d and analyzed by Oil Red O
(Sigma, Saint Louis, Missouri, USA) staining. To study the
effect of Wnt3a on rMSC adipogenesis, the cells were seeded
at a density of 5×104 cells/mL in 6-well tissue culture plates
and cultured for 3 weeks in adipogenic medium,
supplemented with different concentrations of control L-CM,
Wnt3a-CM, or Wnt3a depl-CM. The medium was refreshed
every 3_4 d and lipid formation was detected after 21 d by Oil
Red O staining. The adipocytes were washed with PBS,
fixed for 30 min in formol (3.7% formalin plus
CaCl23.2H2O), and stained for 10 min in freshly-filtered Oil Red O solution
(stock: 500 mg Oil Red O, 99 mL isopropanol, 1 mL water;
stain: 42 mL stock plus 28 mL water). Oil Red O staining was
quantified by extraction with 4% Igepal (Sigma, Saint Louis,
Missouri, USA) in isopropanol for 15 min and measurement
of absorbance at 520 nm. Values represent the mean±SD.
Transwell migration assay Transwell migration assays
were performed with 6.5 mm diameter, Falcon
cell culture inserts (8 µm pore size; Becton Dickinson, San Jose, California,
USA) and 24-well cell culture plates. rMSC were trypsinized,
resuspended in serum-free DMEM, and transferred to the
upper chamber (5×104 cells resuspended in 1.0 mL
pre-equilibrated DMEM). An equal volume maintenance medium
supplemented with varying concentrations of control L-CM,
Wnt3a-CM, or Wnt3a depl-CM was added to
the lower chamber. The cells were allowed to migrate for 24 h in
a humidified CO2 incubator at 37 °C. Following
incubation, the media were aspirated, and the cells remaining on the
upper surface of the filter were removed with a cotton swab;
the cells that had migrated to the lower surface were stained
with hematoxylin for 30 min. The average numbers of
migrated cells were determined by counting the cells in 6
random high-power fields (×200). The transwell assays for
each condition were performed in triplicate and
representative results are shown.
Wound healing assay For the wounding healing
assay, rMSC were plated in 6-well tissue culture plates and allowed
to grow to a confluency of 70%_80%. The experimental
wounds were made by dragging pipette tips across the
cell culture. The cultures were then rinsed with PBS and
replaced with fresh maintenance medium supplemented with
varying concentrations of control L-CM, Wnt3a-CM, or
Wnt3a depl-CM. The wound healing at approximately the
same fields was recorded under a bright field
at selected time-points. The wound gap was measured, and the percentage
of wound repair was determined for each time-point. All
treatments were assessed in triplicate.
Immunofluorescence analysis For the
immunofluorescence analysis, the cells grown on glass coverslips were
fixed in methanol/acetone (1:1, v/v) for 10 min at room
temperature. Blocking was carried out in 3% BSA in PBS for
30 min and the primary antibody was diluted at 1:50
(β-catenin, Cell Signaling Technology, Danvers, Massachusetts, USA)
and 1:200 (desmin and MHC; Santa Cruz Biotechnology,
Santa Cruz, California, USA) in a solution containing 3%
BSA. Incubation was carried out at room temperature for 1 h
or at 4 °C overnight. After 3 washes in PBS, the secondary
antibody (CY3-conjugated goat antirabbit, FITC-conjugated
goat antirabbit, Santa Cruz Biotechnology, Santa Cruz,
California, USA) was used at a 1:200 dilution in 3% BSA, and
the incubation lasted for 1 h at room temperature. The nuclear
localization of immunostaining was confirmed by
counterstaining with 4',6-diamidino-2-phenylindole (DAPI, Sigma,
Saint Louis, Missouri, USA) at a 1:5000 dilution in PBS. The
immunofluorescence images were recorded using an Olympus
immunofluorescence microscope (Olympus Optical
Com-pany, Ltd, Tokyo, Japan). The percentages of
β-catenin-, desmin-, and MHC-positive cells were calculated from the
ratio of positive cells to the total cells counted. Six random
fields at 20-fold magnification were examined under the
microscope at the indicated time-points. Values represent the
mean±SD.
RT-PCR Total RNA was extracted from the cells at the
indicated time-points using Trizol reagent (Invitrogen,
Carlsbad, California, USA) and treated with RNase-free
DNase (Fermentas, Hanover, Maryland, USA) to remove any
contaminating genomic DNA according to the manufacturer's
protocol. One microgram of total cellular RNA was reversely
transcribed using the Revert Aid TM H Minus First Strand
Synthesis Kit (Fermentas, Hanover, Maryland, USA) for 60
min at 42 °C. cDNA was heated at 70 °C for 10 min. The
first-strand synthesized cDNA was used directly for PCR
amplification. Each PCR reaction was carried out in 25 µL
mixture. The primer sequences used for the PCR
amplification were designed based on published cDNA sequences
and are as follows: 5'-GGC TTT CAA CCA TCT CAT TC-3'
and 5'-GTT GGT CAG AAG TCC CAT TAC-3' for Pax3,
generating a 343 bp fragment; 5'-TTC GGG AAG AAA GAG GAC
G-3' and 5'-ATG GTT GAT GGC GGA AGG-3' for Pax7,
generating a 512 bp fragment; 5'-CTA CAG CGG CGA CTC AGA
CG-3' and 5'-TTG GGG CCG GAT GTA GGA-3' for MyoD, generating a 563 bp
fragment[24]; 5'-TTA GAA GTG GCA GAG GGC TC-3' and 5'-AGG TGC GCA GGA AAT CCG CA-3' for
Myf4, generating a 475 bp fragment[25]; 5'-GAG CCA AGA
GTA GCA GCC TTC G-3' and 5'-GTT CTT TCG GGA CCA GAC AGG G-3' for Myf5, generating a 440 bp
fragment[26]; 5'-ACT ACC CAC CGT CCA TTC AC-3' and 5'-TCG GGG CAC
TCA CTG TCT CT-3' for myogenin, generating a 233 bp
fragment[24]; 5'-CTC AGG CTT CAA GAT TTG GTG G-3' and
5'-TTG TGC CTC TCT TCG GTC ATT C-3' for MHC, generating
a 265 bp fragment; 5'-GCC TTG CTG TGG GGA TGT CT-3'
and 5'-CGA AAC TGG CAC CCT TGA AAA AT-3' for
peroxisome proliferator-activated receptor gamma
(PPARγ), generating a 355 bp fragment; and 5'-GGC GGG AAC GCA ACA
ACA-3' and 5'-GAG ATC CAG CGA CCC TAA ACC A-3' for
CCAAT/enhancer-binding protein alpha (C/EBPα),
generating a 292 bp fragment. To control the amount of RNA in the
different samples, the expression of GADPH was amplified
with the following primers: 5'-ACC ACA GTC CAT GCC ATC
AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3' and
generating a 451 bp fragment[27]. All the primers were synthesized
by Saibaisheng (Beijing, China). The amplification products
were electrophoresed on 1.5% agarose gels and visualized
by ethidium bromide staining followed by UV light
illumina-tion.
Statistical analysis All data were presented as mean±SD.
The statistical differences among 3 or more groups were
determined by ANOVA, followed by a Dunnett's
post-hoc test of all groups versus the respective control group. The
statistical differences between 2 groups were performed by
Student's t-test. A value of P<0.05 was considered
statistically significant.
Results
Characterization of rMSC in culture To characterize
rMSC, we first performed FACS to detect the immunophenotype of isolated rMSC at passage 3. The results
showed that rMSC expressed CD29 (β1-integrin) and CD44.
The positive ratio of CD29 and CD44 were 97.8% and 95.1%,
respectively. In contrast, rMSC did not express CD11b
(Mac-1) and CD45, which are markers of hematopoietic stem cells
(Figure 1A). Furthermore, we induced cell differentiation of
isolated rMSC according to published
protocols[2]. In the presence of an osteogenic stimulus, rMSC developed into
osteoblastic cells, as judged by their ability to mineralize the
extracellular matrix (Figure 1B). After exposure to an
adipo-genic stimulus, the cells displayed an adipocyte phenotype,
as shown by the accumulation of neutral lipid droplets in the
cytoplasm (Figure 1C). These results confirm that the
isolated cells exhibit the previously reported properties of
MSC[2].
Activating Wnt signaling induces β-catenin nuclear
translocation in rMSC To explore the role of Wnt3a
signaling in the proliferation, differentiation, and migration of rMSC,
Wnt3a-CM were prepared and the presence of the Wnt3a
protein in Wnt3a-CM were confirmed by Western blot analysis
(Figure 2C). To determine whether Wnt3a-CM activate the
canonical pathway of Wnt signaling in rMSC, rMSC were
exposed to Wnt3a-CM and changes in β-catenin localization
were detected by fluorescent immunocytochemistry. The
results showed that β-catenin was detected in the cytoplasm
and at peripheral sites of cell-cell contact after exposure to
control conditioned medium. In comparison, β-catenin was
predominantly identified in the nucleus of cells, determined
by colocalization with nuclear staining by DAPI after rMSC
were cultured in Wnt3a-CM for 24 h (Figure 2A). These
observations were quantified, and the result showed that
Wnt3a-induced nuclear translocation was dose-dependent
and increased from 0% of cells with nuclear β-catenin in
L-CM-treated cells to 84.5%±4.37% in the 50%
Wnt3a-CM-treated cells (P<0.001; Figure 2B). To determine whether the
effect of Wnt3a-CM on β-catenin nuclear translocation in
rMSC was indeed due to the Wnt3a protein, we detected
β-catenin localization in rMSC after the cells were exposed to
Wnt3a-CM that had first been blocked from Wnt3a using a
Wnt3a antibody. As shown in Figure 2, the depletion of
Wnt3a completely abolished the ability of Wnt3a-CM to
induce β-catenin nuclear translocation in rMSC. These results demonstrate that Wnt3a possesses the ability to
induce β-catenin nuclear translocation and activate the Wnt
pathway in rMSC.
Effects of Wnt3a on rMSC proliferation To analyze the
effect of Wnt3a-CM on the proliferation of rMSC, we
cultured these cells in growth medium containing
differentiation concentrations of Wnt3a-CM or L-CM. The cells were
harvested using trypsin and counted in a hemocytometer at
the indicated time-points. Compared with the control L-CM,
Wnt3a led to a significant increase in the number of rMSC,
both at 25%
(3.04×104±0.43×104
vs
3.89×104±0.31×104,
P<0.05) and 50% conditioned medium
(3.16×104±0.39×104
vs
4.14×104±0.37×104
, P<0.05) after being cultured for 5 d (Figure
3A). To test whether the effect of Wnt3a-CM on
the proliferation of rMSC was specific to Wnt3a, rMSC were cultured
in growth medium containing different concentrations of
Wnt3a depl-CM. The result showed that the depletion of
Wnt3a significantly reduced the ability of Wnt3a-CM to
stimulate the proliferation of rMSC after 5 d of treatment (25%,
2.95×104±0.39×104
vs
3.89×104±0.31×104, P<0.05; 50%,
3.05×104±0.42×104
vs 4.14×104±0.37×104
, P<0.05; Figure 3A). These results suggest that Wnt3a is mitogenic to rMSC.
To further evaluate the Wnt effect on rMSC proliferation,
we measured the effect of Wnt3a-CM on BrdU
incorporation into rMSC. The incorporated BrdU
was detected by using a monoclonal antibody against BrdU. The proportion
of BrdU-positive cells increased about 1.42- and 1.75-fold in
the Wnt3a-treated cells compared with the control cells (25%,
12.5%±2.18% vs 17.8%±2.36%,
P<0.05; 50%, 11.8%±2.49% vs 20.6%±4.45%,
P<0.05; Figure 3B, 3C). After culturing rMSC
in Wnt3a depl-CM, the proportion of BrdU-positive cells
reduced to 10.8%±2.37% and 13.1%±3.04%
(P<0.05; Figure 3B,3C). These results further demonstrate that Wnt
signaling is capable of promoting the proliferation of rMSC.
Wnt3a-expanded rMSC retain their pluripotency
To demonstrate that rMSC after Wnt3a treatment maintain their
pluripotency for differentiation, we expanded rMSC in
proliferation medium with or without Wnt3a-CM and compared
their osteogenic and adipogenic differentiation
in vitro. After cultivation in the Wnt3a-CM for 6 d, rMSC were replated
and induced for osteogenic or adipogenic differentiation in
the absence of Wnt3a. When Wnt3a-expanded rMSC were
cultured in the osteogenic medium, the osteogenic
differentiation of these cells was similar to that of the control cells
(Figure 4). Similarly, when Wnt3a-expanded rMSC were
induced for adipogenic differentiation, an accumulation of fat
vacuoles in the cytoplasm were observed in these cells as
well as the control cells (Figure 4). These results suggest
that Wnt3a-expanded rMSC retain their pluripotency.
Wnt3a induces the myogenic differentiation of rMSC
Wnt signaling has been shown to play an important role in
the induction of myogenesis during embryonic development.
In order to investigate a possible role for Wnt3a in inducing
the myogenic differentiation of rMSC, we cultured rMSC in
myogenic medium supplemented with Wnt3a-CM. The treated cells were observed by phase-contrast microscopy
every day and further analyzed by immunofluorescence with
antibodies against desmin and MHC. After 7_10 d, we
observed that the morphology of a few Wnt3a-treated cells
changed from a fibroblast-like phenotype to elongated
mononucleated, myotubes-like shape. To further confirm
the myogenic nature of the mononucleated cells observed
by phase-contrast microscopy, immunofluorescence was examined in Wnt3a-treated rMSC with antibodies that
detected desmin and MHC (Figure 5A). As shown in Figure
5B, approximately 2%_3% of all rMSC stained positive for
desmin after 25% and 50% Wnt3a-CM treatment. The
positive ratio for MHC was 2.57%±0.66% and 3.76%±0.48% in
the presence of 25% and 50% Wnt3a-CM (Figure 5C). On
the other hand, no obvious morphological changes, desmin-,
or MHC-positive cells were observed in the L-CM treated
cells (Figure 5A).
To determine whether the role of Wnt3a-CM in inducing
the myogenic differentiation of rMSC was specific to Wnt3a,
we performed a myogenic differentiation experiment in rMSC
after the cells were exposed to Wnt3a-CM that had been
depleted of the Wnt3a protein by adding the Wnt3a antibody. As shown in Figure 5A, there were no desmin- or
MHC-positive cells observed in Wnt3a depl-CM treated cells.
Therefore, the depletion of Wnt3a completely abolished the
ability of Wnt3a-CM to induce myogenic differentiation in
rMSC. These findings suggest that the role of Wnt3a-CM
on myogenic differentiation in rMSC is specific to Wnt3a.
To examine whether the myogenic differentiation of
Wnt3a-treated rMSC was acquired through a cascade of
molecular events reminiscent of embryonic myogenesis, a
systematic study of the temporal expression pattern of
myogenic differentiation genes during the treatment of rMSC
with Wnt3a was examined by RT-PCR. As shown in Figure 6,
changes in the expression profiles of myogenic
differentiation genes were observed after Wnt3a treatment. RMSC
only expressed Pax3, but not Pax7 and other myogenic
regulatory factors, including MyoD, Myf4, Myf5, and myogenin
before Wnt3a treatment. Pax7 was activated at first after 2 d
Wnt3a treatment. Following the expression of Pax7, the
expression of MyoD and Myf5 were observed at 4 d treatment.
The expression of Myf4, myogenin was found after 6 d
treatment. MHC was also weakly expressed at d 6. None of
these transcription factors, except Pax3, was detected in rMSC
treated with L-CM (data not shown). These results
demonstrate that Wnt3a induces myogenic differentiation of rMSC
through triggering the expression of myogenic regulatory
factors.
Effect of Wnt3a on adipogenic differentiation of rMSC
Recently, the Wnt signaling pathway has been shown to
inhibit adipogenesis in 3T3-L1 pre-adipogenic cells and
human MSC[28,29]. Therefore, we examined whether Wnt3a
signaling had an inhibitory effect on rMSC adipogenesis. For
this analysis, rMSC were incubated in adipogenic
differentiation medium supplemented with Wnt3a-CM for 3 weeks.
The adipogenic differentiation was evidenced by the
positive Oil Red O staining of lipid droplets presented in the
cytoplasm of the cells. As shown in Figure 7A, the control
cells accumulated a lot of lipid droplets after the induction of
adipogenesis. In contrast, the accumulation of lipid
droplets was dramatically blocked in Wnt3a-treated rMSC. The
inhibitory effect of Wnt3a was dose-dependent. Lipid
formation in the control cells and Wnt3a-treated rMSC was
quantified and the result showed that the accumulation of
lipid decreased by 34.9% and 46.8% in the presence of 25%
and 50% Wnt3a-CM compared with the control cells (25%,
0.96±0.11 vs 0.62±0.04, P<0.01; 50%, 0.95±0.08
vs 0.51±0.04, P<0.01; Figure 7B). To determine whether the effect of
Wnt3a-CM on adipogenic differentiation in rMSC was due to Wnt3a,
rMSC were incubated in adipogenic differentiation medium
supplemented with 25% and 50% Wnt3a depl-CM. The
results showed that the depletion of Wnt3a eliminated the
inhibitory role of Wnt3a-CM on adipogenic differentiation
in rMSC (25%, 0.87±0.09 vs 0.62±0.04,
P<0.05; 50%, 0.93±0.07 vs 0.51±0.04, P<0.01; Figure 7A, 7B). These findings
suggest that the role of Wnt3a-CM on the adipogenic
differentiation in rMSC is specific to Wnt3a.
To further investigate the mechanism involved in the
inhibition of adipogenesis, we examined the effect of Wnt3a
on expression of C/EBPα and PPARγ by RT-PCR.
C/EBPα and PPARγ were the essential transcriptional activators and
markers of the terminal differentiation of adipogenesis.
Compared with the control cells, the expression of both
C/EBPα and PPARγ was downregulated in rMSC after Wnt3a
treatment (Figure 7C). These data demonstrate that Wnt
signaling has an inhibitory effect on the adipogenic differentiation
of rMSC.
Effect of Wnt3a on the migration of rMSC
Some therapeutic approaches have demonstrated that MSC are able to
regenerate injured tissues when applied from different sites
of application. The molecular mechanisms involved in the
control of the migration of MSC are widely unknown. Since
the Wnt signaling pathway was involved in the metastasis
of many kinds of cancer cells, we first conducted transwell
migration assays to investigate whether Wnt3a signaling
could function as a chemoattractant to mesenchymal stem
cells. As shown in Figure 8A, the presence of Wnt3a
significantly increased cell migration. In fact, the average migrated
cell number of rMSC increased 1.86- and 2.41-fold in the
presence of 25% and 50% Wnt3a-CM compared with the
controls (25%, 37.6±4.17 vs 70.2±13.03,
P<0.05; 50%, 35.7±7.24 vs 86.3±11.60,
P<0.01; Figure 8B). Compared with the
Wnt3a-CM group, the average migrated cell number
of rMSC
reduced to 32.3±5.38 and 38.5±6.14 in 25% and 50%Wnt3a
depl-CM treated groups (25%:P<0.05; 50%:
P<0.01; Figure 8). These results demonstrate that Wnt3a signaling is
strongly chemotactic for rMSC.
We next conducted a wound healing assay to determine
whether Wnt3a could promote rMSC migration. The wound
healing experiment is one of the most commonly used
methods to assess cell adhesion and migration under
in vitro conditions. As shown in Figure 9A, the ability of the cells to
migrate was significantly enhanced when they were treated
with Wnt3a-CM as compared with those treated with control
media. The stimulatory effect of Wnt3a on the migration of
rMSC was dose-dependent (Figure 9B). The specificity of
the treatment was shown by the reduction in the migration
of cells treated with Wnt3a-CM that had been depleted of
Wnt3a using the Wnt3a antibody (Figure 9A,9B). The
transwell migration and wound healing assays for each
condition were performed in triplicate. Taken together, these
findings suggest that Wnt3a is a significant factor in
regulating the migration of rMSCs in vitro.
Discussion
Wnt signals are involved in multiple developmental
processes, including differentiation, proliferation, and
migration of progenitor cells in the developing embryo. In this
study, we evaluated the effects of Wnt3a signaling on the
differentiation, proliferation, and migration of rMSC
in vitro. The results showed that the activation of the canonical Wnt
pathway induced myogenic differentiation of rMSC and
inhibited their adipogenic differentiation. Furthermore,
Wnt3a signaling had the ability to promote rMSC to expand
and regulate the migration of rMSC in vitro. The specificity
of the effects was demonstrated by the ability of a Wnt3a
antibody to block the effects of Wnt3a. These results
suggest that Wnt signaling play important roles in the
regulation of the proliferation, differentiation, and migration of
rMSC.
Because MSC have the ability to differentiate into
numerous mesenchymal tissue lineages, there has been much
interest in these cells and their potential application
in cytotherapy and gene therapy. How to rapidly expand MSC
using simple methods is an attractive characteristic of the
cells and it has been the subject of much investigation.
Recent experiments have suggested that Wnt signaling has
the capacity to promote self-renewal in various tissue stem
cells, including intestinal stem cells, skin stem cells, neural stem
cells, and hematopoietic stem cells in
vitro and in vivo[30_33]. In
the present study, we demonstrated that Wnt3a could
promote the proliferation of rMSC and that Wnt3a-expanded
cells preserve their multipotency. These findings indicate
that Wnt signaling has a mitogenic effect on rMSC. Thus,
our study provides evidence that the activation of canonical
Wnt signaling by Wnt3a is useful for expanding
MSC in vitro.
A crucial role of Wnt signalling for skeletal myogenesis
has been demonstrated in embryo
development[14,15,34]. It has been shown that either Wnt1 or Wnt3a in combination
with Sonic hedgehog is sufficient for activating skeletal
muscle-specific genes, such as MyoD and Myf5, in the
paraxial mesoderm in coculture
experiments[14]. Wnt derived from axial structures (neural tube and notochord)
preferentially activate myogenesis through a Myf5-dependent
pathway, while Wnt derived from dorsal ectoderm
preferentially activate myogenesis through a MyoD-dependent
pathway[15]. Other studies have shown that the ectopical
implantation of Wnt3a-expressing fibroblasts leads to increased
Pax-3 expression in adjacent
somites[34]. Wnt3a has also been shown to activate Pax3 and MyoD expressions and
results in the induction of myogenesis in P19 embryonal
carcinoma cells[35].
At present time, there is no study available on the role of
Wnt3a in the myogenic differentiation of adult stem cells.
To our knowledge, this study provides the experimental
evidence that Wnt3a can induce the myogenesis of rMSC.
Nevertheless, the efficiency of this induction was low. This
suggests that the induction conditions need to be further
optimized. In addition, it is possible that other molecules,
which cooperated with Wnt3a, may contribute to induce
myogenesis in MSC. This will require further studies. In line
with our results, one recent study reported that Wnt
molecules activated the myogenic recruitment of
CD45+ cells isolated from uninjured
muscles[18]; another study suggested that the activation of Wnt signaling induced the
transcription of different skeletal muscle marker genes in 2 stem cell
populations isolated from adult murine bone
marrow[36]. All these studies demonstrate that Wnt signaling is capable of
inducing myogenesis in adult stem cells.
Recent studies have demonstrated that Wnt signaling
controls the balance between myogenic and adipogenic
potential in many types of adult stem cells, including myoblasts,
pre-adipocytes, and C3H10T1/2 mesenchymal stem cells
in vitro and in
vivo[28,37,38]. Myoblasts isolated from
Wnt10b_/_ mice show increased adipogenic potential, likely
contributing to excessive lipid accumulation in actively
regenerating myofibers in vivo. The inhibition of the Wnt signaling
pathway by the overexpression of axin, dominant-negative
TCF4, or Dkk1 in 3T3-L1 pre-adipocytes and myoblasts
promotes these cells to differentiate into
adipocytes[28,38]. On the other hand, the activation of the Wnt signaling pathway
inhibits adipogenesis[28]. Moreover, the alteration in Wnt
signaling in myoblasts with age impairs muscle regenerative
capacity and increases muscle
adiposity[37]. These studies imply that Wnt signaling plays an important role in
mesodermal cell fate determination[28]. One recent study demonstrated
that the activation of the Wnt signaling pathway inhibited
adipogenic differentiation in human MSC
[21]. In this report, we found that Wnt3a inhibited the adipogenic
differentiation in rMSC, similar to the result in human MSC. In
embryogenesis, both adipocytes and myocytes are derived
from mesodermal precursor cells, which have the potential
to differentiate into the mesodermal cell types of adipocytes,
chondrocytes, osteoblasts, and myocytes. As our results
demonstrated that the activation of the Wnt signaling
pathway by Wnt3a induced myogenesis and inhibited
adipogenesis in rMSC, we hypothesized that the activation of
Wnt signaling might direct mesenchymal stem cell fate
towards myogenesis while preventing commitment to the
adipogenesis.
Different types of tissue engineering have been
examined using MSC in recent years. MSC have been shown to
regenerate injured tissues when applied from different sites
of application. However, the factors involved in the control
of the migration of MSC are widely unknown at present time.
It is well known that the Wnt signal is involved in cell
mobility during the development and metastasis of many kinds of
cancer cells[39_41]. In the present study, we analyzed the
effect of Wnt signaling on the migration of MSC. The
results from the transwell migration and wound healing
assays showed that Wnt3a could promote rMSC migration.
The ability of Wnt3a in stimulating the migration of rMSC is
reminiscent of the recent findings that Wnt3a induced the
migration of myeloma plasma cells through vascular
endothelia[40]. Furthermore, activating mutations in the Wnt
pathway result in extensive migration and are responsible for
increased metastasis in other tumor
cells[42]. Combining the results of published studies and the present study, it
becomes apparent that Wnt3a is capable of promoting the
migration of mesenchymal stem cells.
The ability of Wnt signaling to induce myogenic
differentiation and promote proliferation and migration in rMSC
may allow for its therapeutic application. Our ongoing
studies aim to further examine the effect of Wnt signaling on
transplanted rMSC maintenance, lineage commitment, and
differentiation in the mdx mouse model of DMD. These
studies could provide a rational foundation for cell-based tissue
repair in humans.
Acknowledgement
We thank Prof Shinji TAKADA for providing the pGKWnt3a and pGKneo plasmids.
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