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Acta Pharmacol Sin 2007 Nov; 28 (11): 1761–1774
©2007 CPS and SIMM
Full-length article
Wnt3a signaling promotes proliferation, myogenic differentiation, and
migration of rat bone marrow mesenchymal stem cells
1
Yan-chang SHANG
2,3
, Shu-hui WANG
4
, Fu XIONG
5
, Cui-ping ZHAO
2
, Fu-ning PENG
2
, Shan-wei FENG
2
, Mei-shan LI
2
,

Yon g
LI
5
, Cheng ZHANG
2,5,6
2
Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080,China;
3
Department of Geriatric Neurology,
Chinese People’s Liberation Army General Hospital, Beijing 100853, China;
4
Department of Neurology, Beijing Friendship Hospital, Capital
Medical University, Beijing 100050, China;
5
Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou
510080, China
Abstract
Aim: To investigate the effects of the wingless-related MMTV integration site 3A
(Wnt3a) signaling on the proliferation, migration, and the myogenic and adipogenic
differentiation of rat bone marrow mesenchymal stem cells (rMSC). Methods:
Primary MSC were isolated and cultured from Sprague-Dawley rats and character-
ized by flow cytometry. Mouse L cells were transfected with Wnt3a cDNA, and
conditioned media containing active Wnt3a proteins were prepared. Cell prolif-
eration was evaluated by cell count and 5-bromodeoxyuridine incorporation assay.
The migration of rMSC was performed by using a transwell migration and wound
healing assay. The myogenic and adipogenic differentiation in rMSC were exam-
ined by light microscopy, immunofluorescence, and RT–PCR at different time
points after myogenic or adipogenic introduction. Results: Wnt3a signaling in-
duced Ε-catenin nuclear translocation and activated the Wnt pathway in rMSC.
In the presence of Wnt3a, rMSC proliferated more rapidly than the control cells,
keeping their differentiation potential. Moreover, Wnt3a signaling induced 2.62%
and 3.76% of rMSC-expressed desmin and myosin heavy chain after being cul-
tured in myogenic medium. The myogenic differentiation genes, including Pax7,
MyoD, Myf5, Myf4, and myogenin, were activated after Wnt3a treatment. On the
other hand, Wnt3a inhibited the adipogenic differentiation in rMSC through the
downregulated expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha)
and peroxisome proliferator-activated receptor gamma (PPARgamma). Furthermore,
Wnt3a promoted the migration capacity of rMSC. Conclusion: The results indi-
cate that Wnt3a signaling can induce myogenic differentiation in rMSC. Wnt3a
signaling is also involved in the regulation of the proliferation and migration of
rMSC. These results could provide a rational foundation for cell-based tissue
repair in humans.
Key words
Wingless-related MMTV integration site
(Wnt); mesenchymal stem cells; myogenic
differentiation; adipogenic differentiation;
proliferation; migration
1
Project supported by the National Natural
Science Foundation of China (No 30370510
and 30170337) and the Key Project of the
State Ministry of Public Health (No 2001321).
6
Correspondence to Prof Cheng ZHANG.
Phn 86-20-8733-4329.
Fax 86-20-8733-3122.
E-mail zhangch6@mail.sysu.edu.cn
Received 2007-03-08
Accepted 2007-06-14
doi: 10.1111/j.1745-7254.2007.00671.x
Introduction
Mesenchymal stem cells (MSC), separated from other
cells in bone marrow by virtue of their adherence to the plas-
tic walls of tissue culture containers, are capable of differen-
tiating 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 rel-
evant numbers
[4]
. Furthermore, MSC avoid ethical and im-
munological 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

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Acta Pharmacologica Sinica ISSN 1671-4083Shang YC et al
in principle, but the process is rather inefficient when com-
paring the number of cells implanted with the amount of
formed muscle cells. A key to improving the current proto-
cols lies in exploiting the molecular mechanisms governing
the distinct steps of myogenic differentiation in MSC
[8]
.
The wingless-related MMTV integration site (Wnt) fam-
ily includes over 20 cysteine-rich secreted glycoproteins and
plays an important role in embryogenesis, including the gen-
eration of cell polarity, specification of cell fate, and the regu-
lation 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, Ε-catenin is phos-
phorylated by glycogen synthase kinase-3 Ε, in association
with axin and adenomatous polyposis coli. These proteins
target Ε-catenin for degradation by the ubiquitin proteasome
pathway
[12]
. When Wnt bind to Frizzled-Low-density lipo-
protein receptor-related protein 5/6 (LRP5/6) complex, dishev-
eled protein (a component of the Wnt signaling pathway) is
activated and inhibits the phosphorylation of Ε-catenin. It
results in Ε-catenin stabilization and accumulation in the
cytoplasm. The stabilized Ε-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 inva-
sion of human MSC
[19]
. Moreover, Wnt signaling has an
inhibitory effect on osteogenic and adipogenic differentia-
tion 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 pro-
liferation and migration of rMSC.
Materials and methods
Isolation and culture of rMSC All of the animal experi-
ments were approved by the Animal Care and Experimenta-
tion 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 cm
2
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% CO
2
.
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