Research in Veterinary Science 93 (2012) 404411

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Research in Veterinary Science

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Isolation, expansion, and differentiation of goat adipose-derived stem cells

Yu Ren a, Haiqing Wu a, Xueyuan Zhou a, Jianxun Wen a, Muzi Jin a, Ming Cang a, Xudong Guo b,
Qinglian Wang b, Dongjun Liu a,, Yuzhen Ma b,
a
b

Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, Hohhot 010021, China
Inner Mongolia Hospital, Inner Mongolia, Hohhot 010017, China

a r t i c l e

i n f o

Article history:
Received 22 February 2011
Accepted 8 August 2011

Keywords:
Adipose-derived stem cell
Isolation and culture in vitro
Cell identication
Directed differentiation culture

a b s t r a c t
A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells
had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same
conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained
negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after
osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining.
After osteogenic induction, ossication nodules of goat ADSCs were larger than in rats, with dense ALP
staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor
(PPARc2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More
cartilage lacunae with better morphology were seen following differentiation of goat ADSCs using the
hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced
cultures were better than for three-dimensional cultures.
2012 Published by Elsevier Ltd.

1. Introduction
Stem cells are characterized by their ability to differentiate
into lineage-specic cell types. Bone mesenchymal stromal cells
(BMSCs) have multipotent properties suitable for tissue engineering and regenerative medical applications. However, BMSCs are
not abundant, cell harvesting requires an invasive technique, and
the cells are easily contaminated. Adipose tissue is abundant,
accessible, and easy to obtain from the body, increasing interest
in adipose-derived stem cells (ADSCs) for tissue engineering. Adipose-derived stem cells are particularly interesting because of
their rapid proliferation and multidirectional differentiation potential. They are expected to become seed cells for improving tendon
healing, such as tissue engineering, cell therapy, and gene therapy
(Butler et al., 2000). Zuk et al. found that adipose tissue can differentiate into osteoblasts, chondrocytes, lipocytes, sarcoblasts and
other cell types, and have a strong growth capacity and multiple.
ADSCs possess similar surface markers and differentiation potential as BMSCs, and can differentiate into cell types of the three germ
layers under appropriate conditions (Lin et al., 2004). However,
they lack specic surface markers, although CD9, CD10, CD13,
CD29, CD44, CD54, CD55, CD71, CD90, CD91, CD105, CD146,
CD34, CD49d, CD106, and vimentin (Strem et al., 2005; Deans
and Moseley, 2000; Mauneya et al., 2007) are frequently used to
Corresponding authors. Tel.: +86 0471 6619236 (Y. Ma), tel.: +86 0471 4995071
(D. Liu).
E-mail addresses: nmliudongjun@sima.com (D. Liu), mayz@imnu.edu.cn (Y. Ma).
0034-5288/$ - see front matter 2012 Published by Elsevier Ltd.
doi:10.1016/j.rvsc.2011.08.014

characterize them for research purposes. ADSCs have been obtained from humans (Ramasamy et al., 2008), mice (Yoshimura
and Muneta, 2007), rats (Nishida et al., 2005), and bovines
(Bosnakovski et al., 2005), but few studies have used goats. Methods for the isolation, expansion and differentiation of ADSCs have
been described for human, rat and mouse (Tropel et al., 2004).
However, no method has been described for livestock. In this study,
we describe a method to isolate ADSCs from goat adipose tissue
and compared it to the rat isolation method.
For the purposes of this review, ADSCs from goat will be dened
as post-embryonic, adipose-derived cells, naturally capable of
multipotent differentiation into connective tissue of nonhaematopoietic lineage; in particular bone, ligaments, tendons,
bers, cartilage, and adipose tissue. A hypothesis is that goat ADSCs
like rat ADSCs can more easily culture and are pluripotent stem
cells.

2. Materials and methods

2.1. Reagents
DMEM/F12 (Dulbeccos modied Eagles medium/nutrient
mixture F-12 Ham), rabbit serum, and fetal bovine serum (FBS)
were from Hyclone (Logan, UT, USA). Mouse anti-rat vimentin antibody; rabbit anti-CD49d, CD34, CD106, and CD13 antibodies; and
goat anti-mouse and uorescein isothiocyanate (FITC)-coupled
antibodies were from Boster Biological Technology Ltd. (Wuhan,

Y. Ren et al. / Research in Veterinary Science 93 (2012) 404411

China). Sodium alginate was from Wako (Tokyo, Japan). An alkaline

phosphatase (ALP) detection kit was from BioSino Bio-technology
and Science Inc. (Beijing, China) and 200 bp Marker (Takara). All
other reagents were from Sigma (St. Louis, MO).
2.2. Animals and tissues
All experiments were approved by the Animal Ethics Committee of Inner Mongolia University before experiments began. Male
Wistar rats (200250 g, 1214 weeks) and Aerbasi Cashmere goats
(2025 kg, 120140 days) were provided by the Experimental
Animal Center, Inner Mongolia University. Animals were fed under
pathogen-free conditions. Immediately under anesthesia, adipose
tissue from rat epididymis and goat inguinal areas were surgically
obtained using sterile techniques. Five to six grams of fat was get
and used from the goats and rats.
2.3. ADSCs isolation, culture and passage
Rat ADSCs were isolated from rat fat as described previously
(Nishida et al., 2005). Goat ADSC isolation was performed according to the method for rats, with minor modications. Briey, fat
was minced in PBS, and digested with same volume of PBS containing 1% bovine serum albumin (BSA) and 0.2% collagenase type I at
37 C in a water-bath shaker (which can improve the efciency of
cell separation and is different from others) for 60 min followed by
centrifugation at 100g for 5 min to remove undigested fat tissues.
The pellet was resuspended in the same volume of erythrocytes
lysis buffer (160 mM/L NH4CL in PBS) and incubated for 20 min
at room temperature. After centrifugation, the supernatant
was discarded and the ADSC pellet was resuspended in DMEM/
F12 with 10% and 20% FBS, respectively. Cells were seeded in a
60-mm Petri dish at a density of 2  105cells/cm2 and cultured at
37 C under 5% CO2, and fed every 48 h with fresh medium. At
80% conuence, cells were trypsinized, centrifuged at 50g for
5 min and re-seeded at 2  105cells/cm2.
2.4. ADSC freezethaw and growth curve
Rat and goat ADSCs at different passage numbers were mixed
with freezing protective agent (10% DMEM/F12 + 10% DMSO + 80%
FBS) at a 0.5  106 cells/mL at 80 C for 24 h, and stocked in liquid
nitrogen. The cells were quickly thawed at 37 C. Cells at passage 5
were used for growth curves. Cells were adjusted to 1  104 cells/
mL and seeded in 24-well plates. Beginning the next day, cells were
harvested from three wells for cell counting, continuing each day
to generate a growth curve. After eight days, this growth curve
was generated.
2.5. Chromosomal analysis of ADSCs
Cells at passages 5, 15, and 20 were used for chromosomal
analysis. Cells were treated with 0.1 lg/mL colchicine for 4 h
and trypsinized. Harvested cells were incubated in 5 mL prewarmed hypotonic solution (0.075 M KCl) at 37 C for 20 min.
Then a new low permeability prepared xative (methanol:acetic
acid = 3:1) 1 mL was added, pre-xed 1 min; 1000 rpm/min centrifuged 10 min, and the supernatant was discarded. After centrifugation, 1 mL xative was added into cell suspension. The cell
suspension was dropped on frozen at 20 C glass slides with a
pipette, and the chromosomal were spread on clean glass slides
by the gradual xation/air-drying method. Preparations were
stained with Giemsa (1:9) for 15 min for conventional chromosome analysis.

405

2.6. Verifying stem cell isolation

ADSCs were veried by immunostaining and reverse transcription polymerase chain reaction (RT-PCR) methods as described
previously (Elabd et al., 2007). ADSCs at passage 5 were grown
on glass coverslips in 24-well dishes. At 80% conuence, cells were
xed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% (vol/vol) Triton X-100, and incubated sequentially
with blocking buffer (PBS + 2% BSA + 2% goat serum + 2% skim
milk + 0.15 M glycine) at 37 C for 2 h. Cells were incubated with
primary antibodies (1:200) to vimentin, CD34, CD106, CD49d and
CD13 at 7 C for 2 h. After washing in PBS, cells were covered with
FITC-labeled goat anti-rabbit or mouse IgG antibodies and stained
with propidium iodide (PI) for 15 min. Rat BMSCs was used as
positive controls and rst antibodies were replaced with PBS as
negative controls. Immunouorescence images were observed
with an Olympus BX61 confocal microscope (Olympus, Tokyo,
Japan). Reverse transcription of RNA from cells followed by PCR
was used to detect CD44 expression as described (Cao et al.,
2005). RT-PCR was performed using an RT-PCR kit according to
the manufacturers instructions (TaKaRa). Primers were: CD44:
sense 50 -CAGACCTGCCCAATGCCTTTGATGGACC-30 , anti-sense 50 CAAAGCCAAGGCCAAGAGGGATGCC-30 ; GAPDH:sense 50 -TGAACGGGAAGCTCACTGG-30 anti-sense 50 -TCCACCACCCTGTTGCTGTA-30 .
PCR conditions were 94 C for 4 min, then 35 cycles of 94 C for
1 min, 56 C (CD44) for 30 s, 60 C (GAPDH) for 30 s, 72 C for 30 s
and nal extension at 72 C for 10 min. PCR products were
examined on a 1% agarose gel. Amplied products were conrmed
under UV light after ethidium bromide (EB) staining.
2.7. Osteogenic differentiation and conrmation
For osteogenic differentiation, cells at passage 5 were incubated
in DMEM/F12 containing 10% FBS, 20 nM dexamethasone, 100 U/
mL penicillin, 100 lg/mL streptomycin, 2.5 lg/mL amphotericin,
10 mM b-glycerophosphate, and 0.05 mM L-ascorbic acid-2phosphate. Control cultures were fed only DMEM/F12 containing
10% FBS and antibiotics. After 14 and 21 days of culture, osteogenic
differentiation of stem cells was conrmed by positive alizarin red
staining of mineralised matrix (Im et al., 2005), ALP staining, and
osteocalcin expression (Caplan and Goldberg, 1999).
2.8. Adipogenic differentiation and conrmation
Cells were incubated with DMEM/F12 containing 3% FBS, antibiotics, 33 lM biotin, 17 lM pantothenic acid, 1 lM insulin, 1 lM
dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX),
5 lM rosiglitazone and 5% rabbit serum for 3 days, then fed
inducing medium without rosiglitazone and IBMX. Control cultures were fed only DMEM/F12 containing 10% FBS and antibiotics
(common ADSCs medium). After 21 days of culture, cells were
xed with 10% formalin and incubated for 20 min with Oil-Red O
to visualize lipid droplets. Adipogenic differentiation was conrmed by Nile Red staining of lipid droplets. The adipogenic determination gene PPARc2 was detected by PCR (Rodriguez et al.,
2004).
2.9. Chondrogenic differentiation and conrmation
For chondrogenic differentiation, trypsinized cells (2.5  105)
were pelleted and resuspended in 0.5 mL high-glucose DMEM containing 100 nM dexamethasone, 0.05 mM L-ascorbic acid-2-phosphate, 1.25 lg/mL BSA, 6.25 lg/mL bovine insulin, 6.25 ng/mL
seleninic acid, 5.35 lg/mL linoleic acid, 1.25 lg/mL transferrin,
10 ng/mL recombinant human bone morphogenetic protein-6
(BMP-6) and 10 ng/mL recombinant human transforming growth

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Y. Ren et al. / Research in Veterinary Science 93 (2012) 404411

Fig. 1. Phase contrast photomicrograph showing morphological characteristics of goat (A) and rat (B) ADSCs of 5th-passage cultures. (I) 6 h; (II) 4 d; (III) 10 d (original
magnication  100).

factor-beta 3 (TGF-b3). Cells were cultured for 24 weeks to obtain

adherent cultures. For three-dimensional (3D) cultures, the cell
pellet was resuspended in a solution of 1% low viscosity sodium
alginate at 5  1010 cells/mL. The alginate-cell suspension was
dropped into 200 mM CaCl2, causing the instantaneous formation
of semisolid microspheric beads. Beads were cured for 5 min in
CaCl2. To induce differentiation, beads were washed with PBS
and maintained in differentiation medium as described above for
24 weeks. For hanging-drop cultures, aliquots of 20 lL DMEM
medium containing cells at 3  105 cells/mL were cultivated in
hanging drops on the lids of 90-mm dishes lled with PBS for
6 days and transferred into dishes containing DMEM medium for
4 days. Medium was replaced with differentiation medium and
maintained for 24 weeks. Control cells were fed with only medium without differentiating supplements. Chondrogenic differentiation was conrmed by positive staining for Alcian blue and
COL2A1 expression (Awada et al., 2004). Number of cartilage lacunae after chondrogenic-induced differentiation of rat and goat
ADSCs was counted, each result was average.
Above all, three goat and rat cell lines were investigated and
three repeats of each group were done. At the same time, parallel
experiments were with similar results.

3. Results

growth and proliferation activity reduced with aging and cell cycle
was prolonged in ADSCs from rats and goats.

3.2. Growth, chromosome analysis and markers

Rat ADSCs entered exponential growth phase between day 2
and 3, and reached a growth plateau between day 6 and 7. Goat
ADSCs entered exponential growth phase at 2 d, and reached a
growth plateau a 6 d. Compared to rat ADSCs, the growth rate of
goat ADSCs was signicantly higher, starting at 4 d (P < 0.05)
(Fig. 2).
Rat and goat ADSCs at passage 5 was used for chromosome
analysis. The normal diploid chromosome ploidy which did not
exists the phenomenon of broken or missing of ADSCs was 88%
(26/30) and in rat cells (Fig. 3A) and 93% (28/30) in goat cells
(Fig. 3B). The abnormal ones were produced during operation at
random. Rat and goat ADSCs at passage 15 and 20 were similar
normal proportion.
After 5 passages, cells were stained with antibodies against
vimentin, CD34, CD13, CD106, and CD49d. As shown in Fig. 4, cells
isolated from goats and rats stained positively for vimentin, CD49d,
and CD13 and negatively for CD34 and CD106. RT-PCR showed that
the isolated cells from both rats and goats expressed CD44. These
ndings indicated that the isolated cells were ADSCs.

3.1. Morphological characteristics of goat and rat ADSCs

3.3. Osteogenic differentiation
ADSCs from rats and goats began to adhere after 46 h innoculation, rst with a small, round and nonuniform cell size, with
some mononuclear blood cells. Gradually they extended into short
or long spindles, and formed polygons 48 h later, with broblastlike morphology 4 d later. After 810 d, cells had grown to
8085% conuence. ADSCs of both rats and goats showed a typical
broblast-like morphology after passage. The growth of rate ADSCs
were stable (qualitative statement) and were reached to 8085%
conuence after 34 d culturing after the rst passage (Fig. 1). Cells
cultured in 20% FBS reached to 8085% conuence faster about 12
24 h than those in 10% FBS in the same innoculation density. The

Osteogenic differentiation of goat and rat ADSCs at passage 5

was conrmed by alizarin red and ALP staining (Fig. 5A-I, B-II).
After feeding ADSCs with osteogenic-inducing media, dark red
mineralised bone matrix (bone nodules) was seen in alizarin
red-stained goat and rat sections.1 Cells stained positively for
ALP. Bone nodules were larger in goat cells than in rat cells, and
ALP staining was more intense (measure). The expression of
1
For interpretation of color in Fig. 5, the reader is referred to the web version of
this article.

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407

osteocalcin mRNA was detected (Fig. 5A,B-III), indicating that both

goat and rat ADSCs had differentiated into osteoblasts.
3.4. Adipogenic differentiation
Adipogenic differentiation of goat and rat ADSCs was conrmed
by Oil Red-O staining. After feeding ADSCs with adipogenicinducing media for 21 d, oil droplets were present in the cytoplasm
(Fig. 6A-I, B-I). The oil droplets appeared to be larger in goat cells
than in rat cells, and the droplets in goat cells and in rat cells
had a rounder shape (measure). The expression of peroxisome
proliferator-activated receptor (PPARc2) was seen in both goat
and rat adipo-induced cells (Fig. 6A-II, B-II), indicating that both
types of ADSC cell had differentiated into fat cells following
adipogenic induction.
3.5. Chondrogenic differentiation
After 21 d induction in adherent, 3D and hanging-drop culture,
chondrogenic differentiation was conrmed by Alcian blue
staining. All cells subjected to different induction methods showed
the formation of the distinct lacuna structure of cartilage (Fig. 7A,B
IIII). Comparing the three methods of chondrogenic induction in
rat ADSCs, more cartilage lacunae and a more representative morphology were found after hanging-drop culture. (Table 1). The cell
morphology in the adherent-induced culture group changed from

Fig. 2. Growth curves of goat (A) and rat (B) ADSCs. Rat ADSCs entered exponential
growth phase between days 2 and 3, and reached a growth plateau between days 6
and 7. Goat ADSCs entered exponential growth phase at 2 d, and reached a growth
plateau at 6 d. Compared to rat ADSCs, the growth rate of goat ADSCs was
signicantly higher, starting at 4 d. Fifth-passage ADSCs were harvested daily for
cell counting. P < 0.05.

spindle-shaped to polygonal, and a few cells appeared to be weakly

positive after alcian blue staining. For induced differentiation of
goat ADSCs, both the adherent-induced culture and the hanging-

Fig. 3. Normal chromosome morphology and structure of goat (A) and rat (B) ADSCs.

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Y. Ren et al. / Research in Veterinary Science 93 (2012) 404411

Fig. 4. Identication of goat (A) and rat (B) ADSCs. ADSCs at 10th day of the 5th passage labeled with anti-CD34 (I), anti-CD49d (II), anti-CD106 (III), anti-CD13 (IV) (original
magnication  100). CD44 mRNA was detected by RT-PCR (V). M: 200 bp Marker; 1: GAPDH (360 bp); 2: CD44 (420 bp); 3: negative control.

drop induced culture were more representative than the 3D

induced culture. For cells in adherent and hanging-drop cultures,
the number of cartilage lacuna was higher (Table 1) and the
morphology better, but no signicant differences were observed
between them. COL2A1 expression was seen in both goat and rat
induced cells (Fig. 7A-IV, B-IV), indicating that both goat and rat
ADSCs had differentiated into chondrocytes following chondrogenic induction.

4. Discussion and conclusions

In this study, rat ADSCs from epididymis were compared with
gost ADSCs from inguinal area. Both cell types were highly dependent on serum for growth. Cells cultured in 20% FBS reached to
8085% conuence faster than those in 10% FBS in the same
innoculation density (Im et al., 2005). However, in serum-free
and low-serum conditions, the effect of induction was better than

Fig. 5. Osteogenic differentiation of goat (A) and rat (B) ADSCs. ADSCs treated with osteogenic media for 21 d stained with alizarin red (I). Arrows show mineralized matrix
produced by osteoblasts. Cells were positively stained for ALP (II) (Original magnication  100). Osteocalcin mRNA was detected by RT-PCR (III). M: 200 bp marker; 1:
GAPDH (360 bp); 2: osteocalcin (424 bp); 3: negative control.

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409

Fig. 6. Adipogenic differentiation of goat (A) and rat (B) ADSCs. ADSCs treated with adipogenic media for 21 d stained with Oil Red-O (I). Arrows show oil droplets (original
magnication  100). PPARc2 mRNA was detected by RT-PCR (II). M: 200 bp marker; 1: GAPDH (360 bp); 2: PPARc2 (564 bp); 3: negative control.

in cells cultured in 10% FBS, while the growth rate was slowed
(Alhadlaq and Mao, 2004). In the same culture conditions, goat
ADSCs grew faster than rat ADSCs. This may be because of differences in the adipose tissues in these diverse species. ADSCs were

obtained from rat adipose tissues from the groin and epididymis
and were found to be similar (Caplan, 2005).
In this study, ADSCs stained positive for vimentin, CD49d, CD13,
and CD44, and negative for CD34, CD106. Vimentin is present in

Fig. 7. Chondrogenic differentiation of goat (A) and rat (B) ADSCs. ADSCs treated with chondrogenic media for 21 d in adherent (I), 3D (II) and hanging-drop (III) cultures
stained with alcian blue. Arrows show lacuna structure of cartilage (original magnication  100). COL2A1 mRNA was detected by RT-PCR (IV). M: 200 bp marker; 1: GAPDH
(360 bp); 2: COL2A1 (364 bp); 3: negative control.

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Y. Ren et al. / Research in Veterinary Science 93 (2012) 404411

Table 1
Number of cartilage lacuna after chondrogenic-induced differentiation of rat and goat ADSCs.

Rat ADSCs
Goat ADSCs

Number of adherent
induced cultures

Number of 3D
induced cultures

Number of hanging drop

induced cultures

6
49

11
10

23
54

Note: Three methods of chondrogenic induction were carried out using the same cell density.

normal and pathological mesenchymal tissues, and is an important

marker of the mesoderm. Positive vimentin staining veried that
both rat and goat ADSCs were derived from the mesodermal stem
cells. CD49d is a surface marker that regulates the settlement and
homing of hematopoietic stem cells (HSCs) to the bone marrow.
CD49d and CD106 are good markers for distinguishing ADSCs
and MSC, respectively (Wagner et al., 2005). CD106 is expressed
in BMSCs of rats while CD49d is not (Barry and Murphy, 2004),
and CD49d is expressed in rat and goat ADSCs while CD106 is
not. CD34 is a surface marker of HSCs and is expressed in lymph
nodes, bone marrow HSCs, and various endothelial cells. CD34negative staining demonstrated that ADSCs from both rats and
goats were not derived from circulating stem cells (Pittenger
et al., 1999; Peng and Huard, 2004). CD44 is a hyaluronate receptor, which is crucial in the development of neoextracellular matrix
and plays a role in numerous pathologic and physiologic events
(Strem et al., 2005). In this study, PCR results of ADSCs from both
rats and goats indicated that CD44 was expressed in both. The results showed a single band, verication of the ADSCs by both
immunohistochemistry and PCR conrmed the reliability of our
conclusions, avoiding the unreliability of using a single method.
These results showed a high purity of both rat and goat ADSCs
was obtained. Although the ADSCs from both rats and goats were
conrmed by the cell surface molecule detection and PCR assays,
multidirectional induction was also performed to determine
ccessful induction.
Rat and goat ADSCs of the 5th passages from osteogenic induction in vitro showed osteogenic activity in 14 d (Alizarin red staining was weakly positive) and with increased osteogenic activity at
21 d. In this study, goat ADSCs of the 10th, 15th, and 20th passages
went through the same osteogenic induction, with similar results.
This supported the hypothesis that ADSCs obtained by separation
retained multidirectional differentiation ability after subculture.
Dexamethasone (Deans and Moseley, 2000; Cheng et al., 1994)
vitamin C and b-glycerophosphate are reported to be requirements
for ADSCs to differentiate into osteoblasts and for in vitro.
Dexamethasone promotes the differentiation and maturation of
osteoblasts, increases ALP activity, and promotes collagen synthesis of the extracellular matrix. Vitamin C promotes both collagen
synthesis and calcication in cultured cells. Moreover, it can alter
both ALP activity and the synthesis of noncollagen matrix protein.
b-glycerophosphate provides phosphate ions for osteoblasts, and
promote the deposition and calcication of physiological calcium,
and is therefore necessary for ADSC mineralization. ALP hydrolyzes
organophosphate and promotes the release of inorganic phosphate,
and thus the formation of hydroxyapatite. Accordingly, it is an
essential enzyme in osteogenic process. The expression of ALP
indicates osteogenic status, and beginning of differentiation into
osteoblasts. Therefore, it is related to the differentiation and maturation of osteoblasts. Osteocalcin is a calcium-conformational
dependent noncollagen protein of bone matrix, synthesized mainly
by osteoblasts and other molecules (Rickard et al., 1996). In this
study, osteocalcin was expressed in ADSCs from both rats and
goats after osteogenic induction.
Hong et al. (2006) used a gelatin sponge as a scaffold for 3D
culturing of human ADSCs after adipogenic differentiation in vitro,

conrmed by Oil Red-O staining. After a short-term culture, the

gelatin sponge was transplanted into the backs of immune depleted rats. Four weeks later, biochemical and immunohistochemical methods were used to demonstrate that the transplant had
been transformed into adipose tissue. Hence, human ADSCs were
found to be compatible with this organism, and adipose tissue
engineering with biodegradable gelatin sponge found to be feasible. Effects of dexamethasone, an inducer of osteogenesis and
adipogenic differentiation, depend on dosage and time. At a low
concentration, it induces osteoblasts while at a high concentration,
it triggers glucocorticoid receptor interaction with insulin, which
induces PPARr. Then, adipocyte genes are transcribed and cells
differentiate into adipocytes (Hoynowski et al., 2007). In this study,
the hypothesis that ADSCs of rats and goats could differentiate into
adipocytes was veried by Oil Red-O staining and PCR results.
Accordingly, ADSCs might be used as ller cells for treatment or
production of biological products.
The chondrogenic-induced differentiation and culture of ADSCs
have been extensively researched (Im, 2005; Nesic et al., 2006;
Miljkovic et al., 2008). Culture in 3D is considered more benecial
for chondrogenic-induced differentiation so it was explored in this
study. Adherent-induced cultures are simple and the least timeconsuming of culture techniques. For chondrogenic-induced differentiation of ADSCs, its low efciency was adverse to the formation
of cartilage lacuna. For chondrogenic-induced differentiation of
goat ADSCs, adherent culture was simple, the inductive effect
was acceptable, and the structure of osteogenic nodules formed
by the cell accumulation was clear. Adherent culture for ADSCs
from inguinal adipose tissue in rats did not give results similar to
those with goat cells. This difference shows that the nature of cells
differs by species. Some cells can be induced in adherent conditions adjacent to each other, and form a cartilage lacuna, while
other cells do not have this property. Therefore, the adherent culture is not adverse to chondrocyte induction, but rather the species
origin of the cells. The 3D culture is widely used in the induced differentiation of cartilage in both rats and mice, with clear effects. In
this study, clear osteogenic nodules were formed in 3D cultures of
rat and goat cells, but the alginate gel was fragile when it was used
as a 3D structure in culturing. Although it requires a longer period
of pre-cell treatment, the hanging-drop culture formed over 95%
cell clones. More ossication nodules formed more after induction,
with more representative morphology. Hence, this was considered
the optimal chondrocyte induction method for these experiments.
This study showed the distinct phenotype, morphology, and
method of isolation of ADSCs in goats. The ndings may have
implications for dening the physiologic roles of ADSCs in arthritis,
bone diseases, and joint regeneration. In animal studies, optimal
bone regeneration has been achieved in defect sites by seeding
gene-modied ADSCs onto suitable carriers. ADSCs maybe combined with impaction bone grafting may effectively restore bone
stock and improve allograft incorporation.

Conict of interest
We declare that we have no conict of interest.

Y. Ren et al. / Research in Veterinary Science 93 (2012) 404411

Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China in Research Foundation for Function
of Gap Junction Protein during Embryonic Development of Sheep
(No. 30660123).

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