co
i
Sh
iu
ong
t-se
Dec
e 2
hepatocytes, the precise mechanisms controlling this pro-
cess remain unclear yet.
ing cellular proliferation and differentiation in adult stem
cell niches, including the skin and hair follicle [12], the
mammary gland [13] and hematopoietic tissues [14]. All
these studies indicated that Wnt signaling might play
an important role in the fate determination and differen-
tiation of stem cells. The aim of our study is to explore
*
Corresponding author.
E-mail address: zhaoguoqiang_sysu@126.com (G. Zhao).
Available online at www.sciencedirect.com
Biochemical and Biophysical Research ComThere is growing evidence to suggest that bone marrow-
derived mesenchymal stem cells (BMSCs) may retain the
potential to transdifferentiate from one phenotype to
another, presenting exciting possibilities for cellular ther-
apy [1]. Krause et al. recently reported that BMSCs had
tremendous differentiation capacity to transform into epi-
thelial cells of liver [2]. It has also been demonstrated that
human umbilical cord blood-derived MSCs can differenti-
ate into functional hepatocyte-like cells in vitro [3,4]. Our
previous work had established a special experimental sys-
tem in which BMSCs could differentiate into hepatocytes
successfully [5]. Though BMSCs can differentiate into
Wnt signaling was known to be involved in numerous
events of animal embryonic development, including the
proliferation of stem cells and the specification of the
neural crest [6,7]. A binding of Wnts to Frizzled may ini-
tiate a signal cascade resulting in the accumulation of
non-phosphorylated b-catenin and its translocation into
the nucleus, where it binds to the N-terminus of LEF/
TCF (lymphoid enhancer factor/T cell factor) transcrip-
tion factors and forms a heterodimer with the TCF/
LEF transcription factor for activation of target genes,
such as c-myc and cyclin D1 [8–11]. Another evidences
also showed that Wnt signaling was involved in regulat-Abstract
Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be able to differentiate into hepatocytes, but the
precise mechanisms controlling this process are unclear. Our aim is try to explore the role of Wnt signaling on the differentiation of
BMSCs into hepatocytes. Our study demonstrated that BMSCs could successfully differentiate into hepatocytes under in vitro induction
of the tissue extract of damaged liver. The mRNA level of Wnt-1, Wnt-5a, Frizzled1, DSH (disheveled), GSK-3b (glycogen synthase
kinase 3 beta) and b-catenin on day 21 when the differentiation direction was determined, was lower than that on days 0, 7, and 11. Fur-
thermore, blocking Wnt-1 signaling by treating BMSCs with Dkk1 could induce BMSCs to express albumin earlier and up-regulation of
Wnt signaling by treating BMSCs with Wnt-1 could inhibit BMSCs to differentiate into hepatocytes. Above results indicated that inhi-
bition on Wnt signaling can promote BMSCs to differentiate into hepatocytes.
 2007 Elsevier Inc. All rights reserved.
Keywords: Adult stem cell; Mesenchymal stem cell; Directing-differentiation; Hepatocyte; Wnt; b-CateninDown-regulation of Wnt signaling
mesenchymal stem cells to d
Zunfu Ke, Feng Zhou, Liantang Wang,
Fang Tang, Dawei L
Department of Pathology, Medical School of Sun Yat-sen University, Zh
Department of Pathology, The First Affiliated Hospital of Sun Ya
Received 12
Available onlin0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2007.12.134uld promote bone marrow-derived
fferentiate into hepatocytes
aohong Chen, Fang Liu, Xinjuan Fan,
, Guoqiang Zhao
*
shan 2nd Road 74, Guangzhou 510080, Province Guangdong, PR China
n University, Guangzhou 510080, Province Guangdong, PR China
ember 2007
January 2008
www.elsevier.com/locate/ybbrc
munications 367 (2008) 342–348

the role of Wnt signaling on the differentiation of
BMSCs into hepatocytes.
Materials and methods
Isolation and culture of BMSCs. BMSCs were harvested from bone
marrow of the tibias of 2- to 3-month-old male Sprague–Dawley rats by
inserting a 21-gauge needle into the shaft of the bone and flushing it with
30 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with fetal bovine serum. After centrifugation and re-suspension, isolated
cells were grown in a culture flask and cultured under a routine condition
(37 C, 5% CO
2
). Non-adherent cells were discarded after 48 h. After
about 8 days, isolated colonies of BMSCs were apparent. BMSCs from
passage 4 were used for differentiation protocol.
control. PCR products were resolved by 2% agarose gel electrophoresis
and visualized after being stained with Goldview under UV illumination.
The gel images were captured and analyzed by the Syngene CCDBIO
acquisition system (Hitachi Genetics, Alameda, CA).
Reverse dot blot hybridization. Six key genes (Wnt-1, Wnt-5a, Friz-
zled1, DSH, GSK-3b, and b-catenin) in Wnt pathway were selected as
target genes. Oligonucleotides were designed according to their gene
sequences (Table 2) and synthesized by Sangon Biological Technology
Company (Shanghai, China). Total RNA of BMSCs harvested at days 0,
7, 11, and 21 were used as template for cDNA reverse transcription. The
total cDNAs were simultaneously labeled with non-radioactive DIG-
dUTP and used as probes.
The target genes were denatured at 96 C for 10 min, chilled on ice for
5 min and then added on the nylon membrane. The nylon membrane was
prehybridized with freshly prepared hybridization solution at 50 C for at
least 1 h. Hybridization was performed at 48.5 C for at least 16 h, after
0
-3
0
C-3
’
Z. Ke et al. / Biochemical and Biophysical Research Communications 367 (2008) 342–348 343Analysis of BMSCs by FCM. Flow cytometry (FCM) was used to
detect the cell surface markers specific for BMSCs. BMSCs of the fourth
passage were harvested with 0.25% trypsin and resuspended in PBS at
2 · 10
6
cells per reaction tube. The BMSCs were fixed in 4% cold para-
formaldehyde for 30 min and washed with PBS containing 2% FBS. Cells
were incubated with mouse anti-rat CD29 (1:1000, Santa Cruz, CA, USA),
anti-CD44 (1:1000, Santa Cruz, CA, USA), anti-CD45 (1:1000, Santa
Cruz, CA, USA) antibodies and FITC-labeled goat anti-mouse secondary
antibodies (1:100, Santa Cruz, CA, USA). The samples were characterized
by FCM.
Stimulating culture of BMSCs. BMSCs were cultured sequentially in a
specialized medium (DMEM-Fl2) containing 150 mg/L tissue extract of
damaged liver according to a protocol in our laboratory. In the control
group, the medium contained 150 mg/L tissue extract of normal liver
instead of tissue extract of damaged liver. BMSCs were harvested on days
0, 7, 11, and 21.
RNA isolation and reverse transcription-polymerase chain reaction.
RNA isolation and reverse transcription-polymerase chain reaction (RT-
PCR) was used to determine mRNA transcripts in BMSCs after differ-
entiation for 7, 11 and 21 days. In brief, total RNA was isolated using the
RNeasy RNA isolation kit (Qiagen, Hilden, Germany) and treated with
DNase I (Ambion, Austin, TX). cDNA was synthesized with 2 lg DNase-
treated total RNA by oligo(dT)
12–18
primer using Superscript II RNase-
reverse transcriptase (Invitrogen, CA). Gene-specific primers for albumin,
M
2
-PK (M
2
-type isozyme of rat pyruvate kinase), GST-p (glutathione S-
transferase p), Bst-1 (bone marrow stromal cell antigen-1) and GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) were designed using the
Primer Premier software (Premier Biosoft International, Palo Alto, CA) as
listed in Table 1. PCRs were performed in 1· PCR buffer (50 ll) con-
taining AmpliTaq Gold DNA polymerase (Applied Biosystems, CA),
200 lM dNTPs, 1.5 mM MgCl
2
, and 0.8 lM of each respective primer
pair, using the ABI 9700 Thermocycler (Applied Biosystems). The PCR
was carried out at 95 C for 4 min, followed by 35–40 cycles at 95 C for
30 s, 55–60 C for 45 s, and 72 C for 60 s and a final elongation step at
72 C for 10 min. GAPDH was included in all experiments as an internal
Table 1
Oligonucleotide primer sequences used in this study
Gene Oligonucleotide sequences
M
2
-PK Sense 5
0
-CCATCTACCACTTGCAGTTATTCGA-3
Anti-sense 5
0
-TCATGGTACAGGCACTACACGC
GST-p Sense 5
0
-GATGAGGGTAAATATTTGCATCG-3
0
Anti-sense 5
0
-TGAGTCCACACCTCTGTCTACCG
Albumin Sense 5
0
-GAGCCCGAAAGAAACGAGTGTT-3
0
Anti-sense 5
0
-GGGGAATCTCTGGCTCATACG-3
Bst-1 Sense 5
0
-TGTGCTCCCCTCAGACTATGA-3
0
Anti-sense 5
0
-GGATCTTAACACTGCCTTCGC-3
0
0 0GAPDH Sense 5 -CCATGGAGAAGGCTGGG-3
Anti-sense 5
0
-CAAAGTTGTCATGGATGACC-3
0which, the membrane was washed in 2· SSC for 5 min at room temper-
ature twice. The membrane was washed briefly in buffer 1 (0.1 M maleic
acid, 0.15 M NaCl, pH 7.5), and then incubated in buffer 2 (1% blocking
reagent in buffer 1) at room temperature for 30 min. The membrane was
incubated in 20 ml diluted antibody-conjugate (Roche, diluted to 75 mU/
ml in buffer 2) at room temperature for 30 min. After incubation, the
membrane was washed in buffer 1 at room temperature for 15 min twice
and then incubated in 1 ml CDP-Star solution (Roche Applied Science,
Mannheim, Germany) at room temperature for 15 min. The fluorescent
signal was detected by sealing the membrane and exposing on X-ray film.
Western blot. Cell lysates were prepared in RIPA buffer containing 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and pro-
teinase inhibitor mix (Roche Diagnostics, Mannheim, Germany) on ice for
1 h. After microcentrifugation, the protein content was determined by the
Bradford assay (Bio-Rad Laboratories, Hercules, CA). The samples were
fractionated on 10% SDS–PAGE gels and transferred to polyvinylidene
difluoride membrane. After washing, the membrane was blocked with 10%
skim milk at room temperature for 3 h and incubated with rabbit
monoclonal antibody against Wnt-1 (1:2000 dilution; Sigma), rabbit
polyclonal antibody against b-catenin (1:2000 dilution, Sigma), and goat
anti-b-actin polyclonal antibody (1:1000 dilution; Sigma) at 4 C over-
night. After washing, the membrane was incubated with horseradish
peroxidase-conjugated anti-rabbit/goat antibodies (1:5000 dilution;
Sigma) at room temperature for 2 h. After washing, the immunoreactive
bands were detected by ECL chemiluminescence reagents. The density of
the bands was quantified with a laser densitometer (ATTO densitograph
4.0, Tokyo, Japan).
BMSCs coculture with Dkk1 and Wnt-1. We cultured BMSCs together
with Dkk1 (R&D, USA) at the concentration of 20 ng/ml and Wnt-1
(R&D, USA) at the concentration of 40 ng/ml in DMEM with tissue
extract of damaged liver BMSCs were cultured in four different ways,
respectively, (1) Dkk1 was added to medium; (2) Wnt-1 was added to
medium; (3) both Dkk1 and Wnt-1 were added to medium; and (4) in the
control group, BMSCs were cultured in DMEM with tissue extract of
Length (bp) T
m
(C) No. of cycles
431 55  35
0
354 57 C35
389 60 C35
485 56 C35180 58 C30