Research in Veterinary Science 93 (2012) 404–411

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

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 February 2011Accepted 8 August 2011

Keywords:Adipose-derived stem cellIsolation and culture in vitroCell identificationDirected differentiation culture

0034-5288/$ - see front matter � 2012 Published bydoi:10.1016/j.rvsc.2011.08.014

⇑ Corresponding authors. Tel.: +86 0471 6619236 (Y(D. Liu).

E-mail addresses: nmliudongjun@sima.com (D. Liu

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cellshad normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the sameconditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stainednegatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive afterosteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining.After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALPstaining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor(PPARc2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. Morecartilage lacunae with better morphology were seen following differentiation of goat ADSC’s using thehang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop inducedcultures were better than for three-dimensional cultures.

� 2012 Published by Elsevier Ltd.

1. Introduction

Stem cells are characterized by their ability to differentiateinto lineage-specific cell types. Bone mesenchymal stromal cells(BMSCs) have multipotent properties suitable for tissue engineer-ing and regenerative medical applications. However, BMSCs arenot abundant, cell harvesting requires an invasive technique, andthe cells are easily contaminated. Adipose tissue is abundant,accessible, and easy to obtain from the body, increasing interestin adipose-derived stem cells (ADSCs) for tissue engineering. Adi-pose-derived stem cells are particularly interesting because oftheir rapid proliferation and multidirectional differentiation poten-tial. They are expected to become seed cells for improving tendonhealing, such as tissue engineering, cell therapy, and gene therapy(Butler et al., 2000). Zuk et al. found that adipose tissue can differ-entiate into osteoblasts, chondrocytes, lipocytes, sarcoblasts andother cell types, and have a strong growth capacity and multiple.ADSCs possess similar surface markers and differentiation poten-tial as BMSCs, and can differentiate into cell types of the three germlayers under appropriate conditions (Lin et al., 2004). However,they lack specific surface markers, although CD9, CD10, CD13,CD29, CD44, CD54, CD55, CD71, CD90, CD91, CD105, CD146,CD34, CD49d, CD106, and vimentin (Strem et al., 2005; Deansand Moseley, 2000; Mauneya et al., 2007) are frequently used to

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. Ma), tel.: +86 0471 4995071

), mayz@imnu.edu.cn (Y. Ma).

characterize them for research purposes. ADSCs have been ob-tained from humans (Ramasamy et al., 2008), mice (Yoshimuraand Muneta, 2007), rats (Nishida et al., 2005), and bovines(Bosnakovski et al., 2005), but few studies have used goats. Meth-ods for the isolation, expansion and differentiation of ADSCs havebeen 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 tissueand compared it to the rat isolation method.

For the purposes of this review, ADSCs from goat will be definedas post-embryonic, adipose-derived cells, naturally capable ofmultipotent differentiation into connective tissue of non-haematopoietic lineage; in particular bone, ligaments, tendons,fibers, cartilage, and adipose tissue. A hypothesis is that goat ADSCslike rat ADSCs can more easily culture and are pluripotent stemcells.

2. Materials and methods

2.1. Reagents

DMEM/F12 (Dulbecco’s modified Eagle’s medium/nutrientmixture F-12 Ham), rabbit serum, and fetal bovine serum (FBS)were from Hyclone (Logan, UT, USA). Mouse anti-rat vimentin anti-body; rabbit anti-CD49d, CD34, CD106, and CD13 antibodies; andgoat anti-mouse and fluorescein isothiocyanate (FITC)-coupledantibodies were from Boster Biological Technology Ltd. (Wuhan,

Y. Ren et al. / Research in Veterinary Science 93 (2012) 404–411 405

China). Sodium alginate was from Wako (Tokyo, Japan). An alkalinephosphatase (ALP) detection kit was from BioSino Bio-technologyand Science Inc. (Beijing, China) and 200 bp Marker (Takara). Allother reagents were from Sigma (St. Louis, MO).

2.2. Animals and tissues

All experiments were approved by the Animal Ethics Commit-tee of Inner Mongolia University before experiments began. MaleWistar rats (200–250 g, 12–14 weeks) and Aerbasi Cashmere goats(20–25 kg, 120–140 days) were provided by the ExperimentalAnimal Center, Inner Mongolia University. Animals were fed underpathogen-free conditions. Immediately under anesthesia, adiposetissue from rat epididymis and goat inguinal areas were surgicallyobtained using sterile techniques. Five to six grams of fat was getand 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 accord-ing to the method for rats, with minor modifications. Briefly, fatwas minced in PBS, and digested with same volume of PBS contain-ing 1% bovine serum albumin (BSA) and 0.2% collagenase type I at37 �C in a water-bath shaker (which can improve the efficiency ofcell separation and is different from others) for 60 min followed bycentrifugation at 100g for 5 min to remove undigested fat tissues.The pellet was resuspended in the same volume of erythrocyteslysis buffer (160 mM/L NH4CL in PBS) and incubated for 20 minat room temperature. After centrifugation, the supernatantwas discarded and the ADSC pellet was resuspended in DMEM/F12 with 10% and 20% FBS, respectively. Cells were seeded in a60-mm Petri dish at a density of 2 � 105cells/cm2 and cultured at37 �C under 5% CO2, and fed every 48 h with fresh medium. At80% confluence, cells were trypsinized, centrifuged at 50g for5 min and re-seeded at 2 � 105cells/cm2.

2.4. ADSC freeze–thaw and growth curve

Rat and goat ADSCs at different passage numbers were mixedwith 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 liquidnitrogen. The cells were quickly thawed at 37 �C. Cells at passage 5were used for growth curves. Cells were adjusted to 1 � 104 cells/mL and seeded in 24-well plates. Beginning the next day, cells wereharvested from three wells for cell counting, continuing each dayto generate a growth curve. After eight days, this growth curvewas generated.

2.5. Chromosomal analysis of ADSCs

Cells at passages 5, 15, and 20 were used for chromosomalanalysis. Cells were treated with 0.1 lg/mL colchicine for 4 hand trypsinized. Harvested cells were incubated in 5 mL pre-warmed hypotonic solution (0.075 M KCl) at 37 �C for 20 min.Then a new low permeability prepared fixative (methanol:aceticacid = 3:1) 1 mL was added, pre-fixed 1 min; 1000 rpm/min cen-trifuged 10 min, and the supernatant was discarded. After centri-fugation, 1 mL fixative was added into cell suspension. The cellsuspension was dropped on frozen at �20 �C glass slides with apipette, and the chromosomal were spread on clean glass slidesby the gradual fixation/air-drying method. Preparations werestained with Giemsa (1:9) for 15 min for conventional chromo-some analysis.

2.6. Verifying stem cell isolation

ADSCs were verified by immunostaining and reverse transcrip-tion polymerase chain reaction (RT-PCR) methods as describedpreviously (Elabd et al., 2007). ADSCs at passage 5 were grownon glass coverslips in 24-well dishes. At 80% confluence, cells werefixed with 4% paraformaldehyde and permeabilized with PBS con-taining 0.1% (vol/vol) Triton X-100, and incubated sequentiallywith blocking buffer (PBS + 2% BSA + 2% goat serum + 2% skimmilk + 0.15 M glycine) at 37 �C for 2 h. Cells were incubated withprimary antibodies (1:200) to vimentin, CD34, CD106, CD49d andCD13 at 7 �C for 2 h. After washing in PBS, cells were covered withFITC-labeled goat anti-rabbit or mouse IgG antibodies and stainedwith propidium iodide (PI) for 15 min. Rat BMSCs was used aspositive controls and first antibodies were replaced with PBS asnegative controls. Immunofluorescence images were observedwith an Olympus BX61 confocal microscope (Olympus, Tokyo,Japan). Reverse transcription of RNA from cells followed by PCRwas used to detect CD44 expression as described (Cao et al.,2005). RT-PCR was performed using an RT-PCR kit according tothe manufacturer’s instructions (TaKaRa). Primers were: CD44:sense 50-CAGACCTGCCCAATGCCTTTGATGGACC-30, anti-sense 50-CAAAGCCAAGGCCAAGAGGGATGCC-30; GAPDH:sense 50-TGAACGG-GAAGCTCACTGG-30 anti-sense 50-TCCACCACCCTGTTGCTGTA-30.PCR conditions were 94 �C for 4 min, then 35 cycles of 94 �C for1 min, 56 �C (CD44) for 30 s, 60 �C (GAPDH) for 30 s, 72 �C for 30 sand final extension at 72 �C for 10 min. PCR products wereexamined on a 1% agarose gel. Amplified products were confirmedunder UV light after ethidium bromide (EB) staining.

2.7. Osteogenic differentiation and confirmation

For osteogenic differentiation, cells at passage 5 were incubatedin 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-2-phosphate. Control cultures were fed only DMEM/F12 containing10% FBS and antibiotics. After 14 and 21 days of culture, osteogenicdifferentiation of stem cells was confirmed by positive alizarin redstaining of mineralised matrix (Im et al., 2005), ALP staining, andosteocalcin expression (Caplan and Goldberg, 1999).

2.8. Adipogenic differentiation and confirmation

Cells were incubated with DMEM/F12 containing 3% FBS, antibi-otics, 33 lM biotin, 17 lM pantothenic acid, 1 lM insulin, 1 lMdexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX),5 lM rosiglitazone and 5% rabbit serum for 3 days, then fedinducing medium without rosiglitazone and IBMX. Control cul-tures were fed only DMEM/F12 containing 10% FBS and antibiotics(common ADSCs medium). After 21 days of culture, cells werefixed with 10% formalin and incubated for 20 min with Oil-Red Oto visualize lipid droplets. Adipogenic differentiation was con-firmed by Nile Red staining of lipid droplets. The adipogenic deter-mination gene PPARc2 was detected by PCR (Rodriguez et al.,2004).

2.9. Chondrogenic differentiation and confirmation

For chondrogenic differentiation, trypsinized cells (2.5 � 105)were pelleted and resuspended in 0.5 mL high-glucose DMEM con-taining 100 nM dexamethasone, 0.05 mM L-ascorbic acid-2-phos-phate, 1.25 lg/mL BSA, 6.25 lg/mL bovine insulin, 6.25 ng/mLseleninic 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

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 (originalmagnification � 100).

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factor-beta 3 (TGF-b3). Cells were cultured for 2–4 weeks to obtainadherent cultures. For three-dimensional (3D) cultures, the cellpellet was resuspended in a solution of 1% low viscosity sodiumalginate at 5 � 1010 cells/mL. The alginate-cell suspension wasdropped into 200 mM CaCl2, causing the instantaneous formationof semisolid microspheric beads. Beads were cured for 5 min inCaCl2. To induce differentiation, beads were washed with PBSand maintained in differentiation medium as described above for2–4 weeks. For hanging-drop cultures, aliquots of 20 lL DMEMmedium containing cells at 3 � 105 cells/mL were cultivated inhanging drops on the lids of 90-mm dishes filled with PBS for6 days and transferred into dishes containing DMEM medium for4 days. Medium was replaced with differentiation medium andmaintained for 2–4 weeks. Control cells were fed with only med-ium without differentiating supplements. Chondrogenic differenti-ation was confirmed by positive staining for Alcian blue andCOL2A1 expression (Awada et al., 2004). Number of cartilage lacu-nae after chondrogenic-induced differentiation of rat and goatADSCs was counted, each result was average.

Above all, three goat and rat cell lines were investigated andthree repeats of each group were done. At the same time, parallelexperiments were with similar results.

1 For interpretation of color in Fig. 5, the reader is referred to the web version ofthis article.

3. Results

3.1. Morphological characteristics of goat and rat ADSCs

ADSCs from rats and goats began to adhere after 4–6 h innocu-lation, first with a small, round and nonuniform cell size, withsome mononuclear blood cells. Gradually they extended into shortor long spindles, and formed polygons 48 h later, with fibroblast-like morphology 4 d later. After 8–10 d, cells had grown to80–85% confluence. ADSCs of both rats and goats showed a typicalfibroblast-like morphology after passage. The growth of rate ADSCswere stable (qualitative statement) and were reached to 80–85%confluence after 3–4 d culturing after the first passage (Fig. 1). Cellscultured in 20% FBS reached to 80–85% confluence faster about 12–24 h than those in 10% FBS in the same innoculation density. The

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

3.2. Growth, chromosome analysis and markers

Rat ADSCs entered exponential growth phase between day 2and 3, and reached a growth plateau between day 6 and 7. GoatADSCs entered exponential growth phase at 2 d, and reached agrowth plateau a 6 d. Compared to rat ADSCs, the growth rate ofgoat ADSCs was significantly higher, starting at 4 d (P < 0.05)(Fig. 2).

Rat and goat ADSCs at passage 5 was used for chromosomeanalysis. The normal diploid chromosome ploidy which did notexists 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 atrandom. Rat and goat ADSCs at passage 15 and 20 were similarnormal proportion.

After 5 passages, cells were stained with antibodies againstvimentin, CD34, CD13, CD106, and CD49d. As shown in Fig. 4, cellsisolated from goats and rats stained positively for vimentin, CD49d,and CD13 and negatively for CD34 and CD106. RT-PCR showed thatthe isolated cells from both rats and goats expressed CD44. Thesefindings indicated that the isolated cells were ADSCs.

3.3. Osteogenic differentiation

Osteogenic differentiation of goat and rat ADSCs at passage 5was confirmed by alizarin red and ALP staining (Fig. 5A-I, B-II).After feeding ADSCs with osteogenic-inducing media, dark redmineralised bone matrix (bone nodules) was seen in alizarinred-stained goat and rat sections.1 Cells stained positively forALP. Bone nodules were larger in goat cells than in rat cells, andALP staining was more intense (measure). The expression of

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

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osteocalcin mRNA was detected (Fig. 5A,B-III), indicating that bothgoat and rat ADSCs had differentiated into osteoblasts.

3.4. Adipogenic differentiation

Adipogenic differentiation of goat and rat ADSCs was confirmedby Oil Red-O staining. After feeding ADSCs with adipogenic-inducing 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 cellsthan in rat cells, and the droplets in goat cells and in rat cellshad a rounder shape (measure). The expression of peroxisomeproliferator-activated receptor (PPARc2) was seen in both goatand rat adipo-induced cells (Fig. 6A-II, B-II), indicating that bothtypes of ADSC cell had differentiated into fat cells followingadipogenic induction.

3.5. Chondrogenic differentiation

After 21 d induction in adherent, 3D and hanging-drop culture,chondrogenic differentiation was confirmed by Alcian bluestaining. All cells subjected to different induction methods showedthe formation of the distinct lacuna structure of cartilage (Fig. 7A,BI–III). Comparing the three methods of chondrogenic induction inrat ADSCs, more cartilage lacunae and a more representative mor-phology were found after hanging-drop culture. (Table 1). The cellmorphology in the adherent-induced culture group changed from

Fig. 3. Normal chromosome morphology and

spindle-shaped to polygonal, and a few cells appeared to be weaklypositive after alcian blue staining. For induced differentiation ofgoat ADSCs, both the adherent-induced culture and the hanging-

structure of goat (A) and rat (B) ADSCs.

Fig. 4. Identification 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) (originalmagnification � 100). CD44 mRNA was detected by RT-PCR (V). M: 200 bp Marker; 1: GAPDH (360 bp); 2: CD44 (420 bp); 3: negative control.

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drop induced culture were more representative than the 3Dinduced culture. For cells in adherent and hanging-drop cultures,the number of cartilage lacuna was higher (Table 1) and themorphology better, but no significant differences were observedbetween them. COL2A1 expression was seen in both goat and ratinduced cells (Fig. 7A-IV, B-IV), indicating that both goat and ratADSCs had differentiated into chondrocytes following chondro-genic induction.

Fig. 5. Osteogenic differentiation of goat (A) and rat (B) ADSCs. ADSCs treated with osteoproduced by osteoblasts. Cells were positively stained for ALP (II) (Original magnificatGAPDH (360 bp); 2: osteocalcin (424 bp); 3: negative control.

4. Discussion and conclusions

In this study, rat ADSCs from epididymis were compared withgost ADSCs from inguinal area. Both cell types were highly depen-dent on serum for growth. Cells cultured in 20% FBS reached to80–85% confluence faster than those in 10% FBS in the sameinnoculation density (Im et al., 2005). However, in serum-freeand low-serum conditions, the effect of induction was better than

genic media for 21 d stained with alizarin red (I). Arrows show mineralized matrixion � 100). Osteocalcin mRNA was detected by RT-PCR (III). M: 200 bp marker; 1:

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 (originalmagnification � 100). PPARc2 mRNA was detected by RT-PCR (II). M: 200 bp marker; 1: GAPDH (360 bp); 2: PPARc2 (564 bp); 3: negative control.

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

Fig. 7. Chondrogenic differentiation of goat (A) and rat (B) ADSCs. ADSCs treated with cstained with alcian blue. Arrows show lacuna structure of cartilage (original magnificatio(360 bp); 2: COL2A1 (364 bp); 3: negative control.

obtained from rat adipose tissues from the groin and epididymisand 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

hondrogenic media for 21 d in adherent (I), 3D (II) and hanging-drop (III) culturesn � 100). COL2A1 mRNA was detected by RT-PCR (IV). M: 200 bp marker; 1: GAPDH

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

Number of adherentinduced cultures

Number of 3Dinduced cultures

Number of hanging dropinduced cultures

Rat ADSCs 6 11 23Goat ADSCs 49 10 54

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

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normal and pathological mesenchymal tissues, and is an importantmarker of the mesoderm. Positive vimentin staining verified thatboth rat and goat ADSCs were derived from the mesodermal stemcells. CD49d is a surface marker that regulates the settlement andhoming of hematopoietic stem cells (HSCs) to the bone marrow.CD49d and CD106 are good markers for distinguishing ADSCsand MSC, respectively (Wagner et al., 2005). CD106 is expressedin BMSCs of rats while CD49d is not (Barry and Murphy, 2004),and CD49d is expressed in rat and goat ADSCs while CD106 isnot. CD34 is a surface marker of HSCs and is expressed in lymphnodes, bone marrow HSCs, and various endothelial cells. CD34-negative staining demonstrated that ADSCs from both rats andgoats were not derived from circulating stem cells (Pittengeret al., 1999; Peng and Huard, 2004). CD44 is a hyaluronate recep-tor, which is crucial in the development of neoextracellular matrixand plays a role in numerous pathologic and physiologic events(Strem et al., 2005). In this study, PCR results of ADSCs from bothrats and goats indicated that CD44 was expressed in both. The re-sults showed a single band, verification of the ADSCs by bothimmunohistochemistry and PCR confirmed the reliability of ourconclusions, avoiding the unreliability of using a single method.These results showed a high purity of both rat and goat ADSCswas obtained. Although the ADSCs from both rats and goats wereconfirmed by the cell surface molecule detection and PCR assays,multidirectional induction was also performed to determineccessful induction.

Rat and goat ADSCs of the 5th passages from osteogenic induc-tion in vitro showed osteogenic activity in 14 d (Alizarin red stain-ing was weakly positive) and with increased osteogenic activity at21 d. In this study, goat ADSCs of the 10th, 15th, and 20th passageswent through the same osteogenic induction, with similar results.This supported the hypothesis that ADSCs obtained by separationretained multidirectional differentiation ability after subculture.Dexamethasone (Deans and Moseley, 2000; Cheng et al., 1994)vitamin C and b-glycerophosphate are reported to be requirementsfor ADSCs to differentiate into osteoblasts and for in vitro.Dexamethasone promotes the differentiation and maturation ofosteoblasts, increases ALP activity, and promotes collagen synthe-sis of the extracellular matrix. Vitamin C promotes both collagensynthesis and calcification in cultured cells. Moreover, it can alterboth ALP activity and the synthesis of noncollagen matrix protein.b-glycerophosphate provides phosphate ions for osteoblasts, andpromote the deposition and calcification of physiological calcium,and is therefore necessary for ADSC mineralization. ALP hydrolyzesorganophosphate and promotes the release of inorganic phosphate,and thus the formation of hydroxyapatite. Accordingly, it is anessential enzyme in osteogenic process. The expression of ALPindicates osteogenic status, and beginning of differentiation intoosteoblasts. Therefore, it is related to the differentiation and mat-uration of osteoblasts. Osteocalcin is a calcium-conformationaldependent noncollagen protein of bone matrix, synthesized mainlyby osteoblasts and other molecules (Rickard et al., 1996). In thisstudy, osteocalcin was expressed in ADSCs from both rats andgoats after osteogenic induction.

Hong et al. (2006) used a gelatin sponge as a scaffold for 3Dculturing of human ADSCs after adipogenic differentiation in vitro,

confirmed by Oil Red-O staining. After a short-term culture, thegelatin sponge was transplanted into the backs of immune de-pleted rats. Four weeks later, biochemical and immunohistochem-ical methods were used to demonstrate that the transplant hadbeen transformed into adipose tissue. Hence, human ADSCs werefound to be compatible with this organism, and adipose tissueengineering with biodegradable gelatin sponge found to be feasi-ble. Effects of dexamethasone, an inducer of osteogenesis andadipogenic differentiation, depend on dosage and time. At a lowconcentration, it induces osteoblasts while at a high concentration,it triggers glucocorticoid receptor interaction with insulin, whichinduces PPARr. Then, adipocyte genes are transcribed and cellsdifferentiate into adipocytes (Hoynowski et al., 2007). In this study,the hypothesis that ADSCs of rats and goats could differentiate intoadipocytes was verified by Oil Red-O staining and PCR results.Accordingly, ADSCs might be used as filler cells for treatment orproduction of biological products.

The chondrogenic-induced differentiation and culture of ADSCshave been extensively researched (Im, 2005; Nesic et al., 2006;Miljkovic et al., 2008). Culture in 3D is considered more beneficialfor chondrogenic-induced differentiation so it was explored in thisstudy. Adherent-induced cultures are simple and the least time-consuming of culture techniques. For chondrogenic-induced differ-entiation of ADSCs, its low efficiency was adverse to the formationof cartilage lacuna. For chondrogenic-induced differentiation ofgoat ADSCs, adherent culture was simple, the inductive effectwas acceptable, and the structure of osteogenic nodules formedby the cell accumulation was clear. Adherent culture for ADSCsfrom inguinal adipose tissue in rats did not give results similar tothose with goat cells. This difference shows that the nature of cellsdiffers by species. Some cells can be induced in adherent condi-tions adjacent to each other, and form a cartilage lacuna, whileother cells do not have this property. Therefore, the adherent cul-ture is not adverse to chondrocyte induction, but rather the speciesorigin of the cells. The 3D culture is widely used in the induced dif-ferentiation of cartilage in both rats and mice, with clear effects. Inthis study, clear osteogenic nodules were formed in 3D cultures ofrat and goat cells, but the alginate gel was fragile when it was usedas a 3D structure in culturing. Although it requires a longer periodof pre-cell treatment, the hanging-drop culture formed over 95%cell clones. More ossification nodules formed more after induction,with more representative morphology. Hence, this was consideredthe optimal chondrocyte induction method for these experiments.

This study showed the distinct phenotype, morphology, andmethod of isolation of ADSCs in goats. The findings may haveimplications for defining the physiologic roles of ADSCs in arthritis,bone diseases, and joint regeneration. In animal studies, optimalbone regeneration has been achieved in defect sites by seedinggene-modified ADSCs onto suitable carriers. ADSCs maybe com-bined with impaction bone grafting may effectively restore bonestock and improve allograft incorporation.

Conflict of interest

We declare that we have no conflict of interest.

Y. Ren et al. / Research in Veterinary Science 93 (2012) 404–411 411

Acknowledgements

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

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