A total of 60 20-day-old male broiler embryos (weight, ~40 g) were provided by the Poultry Experimental Base of the Institute of Animal Sciences (Chinese Academy of Agricultural Sciences, Beijing, China). All procedures conformed to the guidelines established by the Institutional Animal Care and Use Committee at Chinese Academy of Agriculture Sciences (Beijing, China).

The following reagents were used in the present study: Dulbecco's modified Eagle's medium (DMEM)/F12, fetal bovine serum (FBS), L-glutamine, and penicillin and streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) trypsin (dilution, 1:250) and 0.02% (w/v) EDTA (Amresco, Inc., Framingham, MA, USA), rabbit anti-chicken CD29 (bs-0486R), CD44 (bs-2507R), CD71 (bs-1782R) and CD73 (bs-4834R) polyclonal primary antibody, goat serum, EDTA, fluorescein isothiocyanate (FITC)-conjugated goat-anti-rabbit immunoglobulin (Ig)G (all from Bioss, Beijing, China), ascorbic acid sodium salt, dexamethasone, hepatocyte growth factor, indometacin, insulin transferrin-selenium, 3-isobutyl-1-methylxanthine (IBMX), β-glycerophosphate, oil red O, collagenase I (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), basic fibroblast growth factor (bFGF), fibroblast growth factor-4 (FGF-4; both from Peprotech, Inc., Rocky Hill, NJ, USA), alizarin red (Boster, Wuhan, China) and TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.).

Adipose tissue samples were collected from 20-day-old broiler embryos under aseptic conditions. The adipose pads were washed with PBS supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin to remove any other cells, including hemocytes and endothelial cells. The adipose pats were fully chopped into small pieces and subsequently incubated with 0.2% (m/v) collagenase I in PBS at 37°C for 40 min. The enzymatic activity was neutralized by adding an equal volume of DMEM/F12 containing 5% (v/v) FBS. The cell suspension was filtered through a 74-mm-mesh sieve and then centrifuged at 200 × g for 8 min at room temperature. The precipitate was resuspended in complete growth medium composed of DMEM/F12, 10% (v/v) FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 ng/ml bFGF. Cells were seeded into petri dishes at a density of 1.0×10⁵/ml, and cultured at 37°C with 5% CO₂. After 24 h, the dishes were washed with PBS to clean out any non-adherent cells, including pericytes, blood cells, endothelial cells and preadipocytes (14,17–19).

The cells were referred to as ‘passage 0’ (P0) when their confluence reached 80%. Subsequently, the cells were sub-cultured at the ratio of 1:2 after standard trypsin digestion. This generation was referred to as P1. The cells became purified with increasing passages, and were then harvested for other relevant trials.

The morphology and adhesion of ADSCs prior to and after culture was observed under an inverted microscope.

Total RNA was extracted from ADSCs at P5 by using TRIzol and then subjected to RT using an RNA PCR kit (version 3.0; Takara Biotechnology, Co., Ltd., Dalian, China) according to the manufacturer's instructions. Primers were designed in accordance with the sequences of GAPDH (internal control) CD71, CD29, CD31, CD73 and CD44 from GenBank. The template complementary DNA was amplified by PCR using the gene-specific primers listed in Table I. The PCR reaction was performed using the PCR Master Mix kit (Takara Biotechnology, Co., Ltd.), according to the manufacturer's instructions. PCR analyses were performed in 50 µl reactions, containing 25 µl 2X PCR mix, 15 µl ddH₂O, 5 µl template cDNA and 2.5 µl forward and reverse primers. Cycling conditions were as follows: Initialization at 94°C for 5 min, then 35 cycles of a denaturation at 94°C for 30 sec, annealing at 50–60°C for 30 sec, elongation at 72°C for 30 sec and final elongation at 72°C for 5 min. PCR products were assessed by 2% agarose gel electrophoresis and bands were visualized with an ultraviolet transilluminator.

ADSCs of P5 were fixed in 4% (m/v) paraformaldehyde for 15 min at room temperature, subsequently washed with PBS. The cells were permeabilized using 0.2% (v/v) Triton X-100 for 20 min at room temperature and washed with PBS. The samples were blocked with 10% (v/v) goat serum for 30 min at room temperature, and then incubated in 1% bovine serum albumin (BSA) in PBS containing the polyclonal rabbit anti-chicken antibodies to CD29, CD44, CD71 and CD73 (all at 1:100 dilution) for 12 h at 4°C. Primary antibody was replaced with PBS for the negative control. The samples were washed with PBS and incubated in PBS containing FITC-conjugated goat anti-rabbit IgG as the secondary antibody for 1 h at room temperature. The samples were washed with PBS after the incubation. They were counterstained with DAPI and were visualized by using a Nikon TE-2000-E confocal microscope with a digital camera system (Nikon, Tokyo, Japan).

The expression of cell-associated surface markers was measured by flow cytometric analysis. In brief, ADSCs of P5 were collected by standard trypsin digestion, and the cells were fixed and permeabilized in 70% (v/v) ice-cold ethanol for 12 h. The samples were blocked with 10% (v/v) goat serum for 30 min at room temperature and were incubated in 1% BSA containing rabbit anti-chicken polyclonal antibodies to CD29, CD44, CD71 and CD73 (all at 1:100 dilution) for 1 h at room temperature. Primary antibody was replaced with PBS for the negative control. Subsequently, the samples were washed with PBS and incubated in PBS containing FITC-conjugated goat anti-rabbit IgG as the secondary antibody for 1 h at room temperature. After washing with PBS, the labeled cells were detected with a flow cytometer. Flow cytometric data were analyzed using CXP software version 2.0 (Beckman Coulter, Brea, CA, USA).

ADSCs at P3, P16 and P30 were collected and seeded in triplicate in 24-well plates at 1×10⁴ cells/well. Cell viability assessment was performed following trypan blue staining at room temperature for 5 min and cells were counted following detachment over 9 successive days. The population doubling time (PDT) was calculated using the following formula: PDT=(t-t0)xlg2/(lgNt-lgN0), with t0 representing the starting time of culture, t the termination time of culture, N0 the initial number of cultured cells and Nt the final number of cells.

ADSCs were divided into a control group and an induction group. When the confluence of cells reached 70–80%, the experimental group was treated with adipogenic medium consisting of DMEM/F12 containing 10% FBS, 200 µM indomethacin, 1 mM dexamethasone, 10 µM insulin transferrin and 0.5 mM IBMX. The control group was cultured in complete growth medium without the factors mentioned above. The medium was replaced every 2 days. After 1 week, the two groups were evaluated for accumulation of intracellular lipid using oil red O staining for 40 min at room temperature and expression of adipogenic cell-specific genes by RT-PCR.

The cells were divided into two groups as above. Upon reaching 60–70% confluence, the cells in the experimental group were cultured in osteogenic medium containing DMEM/F12 supplemented with 10% FBS, 0.1 mM dexamethasone, 10 mM β-glycerophosphate and 50 µg/ml ascorbate. In the control group, cells were cultured with normal growth medium. The medium was replaced every 2 days. After 3 weeks, the formation of calcium nodes was assessed by alizarin red staining for 25 min at room temperature and osteoblast-specific genes were detected by RT-PCR.

ADSCs were divided into two groups as described above. When the confluence reached 60–70%, the induction group was cultured in hepatocyte medium containing DMEM/F12 supplemented with 10% FBS, 20 ng/ml FGF-4, 20 ng/ml hepatocyte growth factor and 1% insulin transferrin. The control group was cultured in normal growth medium. The medium was exchanged every 2 days. After 2 weeks, the capacity of the cells to secrete glycogen was detected by periodic acid Schiff staining for 30 min at 37°C, and hepatocyte-like cell-specific genes were detected by RT-PCR.

Values are expressed as the mean ± standard error of the mean. Comparisons between multiple groups were evaluated by Dunnett's post-hoc test after one-way analysis of variance. SPSS version 19 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

The primary ADSCs obtained from subcutaneous adipose tissue were seeded into 60-mm Petri dishes, and after 24 h cells had begun to stretch into various shapes. After 48 h of culturing, a small number of polygonal cells were also observed under the inverted microscope, and a minority of cells had irregular shapes (Fig. 1A). The cells expanded rapidly and reached 80–90% confluence after 5 days of seeding, while being arranged in a whirl pattern (Fig. 1B). In primary culture, numerous hemocytes were admixed with the ADSCs (Fig. 1B). However, from P3, the ADSCs were fully purified, and the shape was also homogenous; the ADSCs exhibited a fibroblast-like morphology (Fig. 1C). No difference in morphology was apparent among the cells of different passages, and the biological properties had remained constant after several cycles of sub-culture (Fig. 1D-F). The cells were cultured to P36 and P37, at which they had a typical senescent appearance, including vacuoles, tabular shapes and karyopyknosis in the majority of cells (Fig. 1G and H). With increasing time, they even started to detach from the plates.

The specific surface markers of ADSCs were detected by RT-PCR and immunofluorescence. RT-PCR revealed that the ADSCs were positive for CD29, CD44, CD71 and CD73, and negative for CD31, while GAPDH served as an internal control (Fig. 2A). Immunofluorescence microscopy indicated that the ADSC surface antigens CD29, CD44, CD71 and CD73 were expressed by the cells (Fig. 2B). Flow cytometry further confirmed that ADSCs at P5 exhibited high expression of CD29, CD44, CD71 and CD73, as the positive rates were 98.77, 98.73, 98.65 and 98.57%, respectively (Fig. 3).

The proliferation of ADSCs at P3, P16 and P30 was detected by determining the cell number with a hemocytometer, and the data were used to generate a growth curve (Fig. 4). The growth curves suggested that there was a latency phase of ~24 h, followed by rapid proliferation of the cells after entering the logarithmic phase. As the density of the ADSCs increased, the proliferation was inhibited and the cells reached a growth plateau phase at 7–8 days, after which they began to degenerate. The growth curves were all typically sigmoidal and significant differences in the cell growth/kinetics were identified between the different groups (P<0.05). The PDT was 38.95, 41.27 and 45.05 h for ADSCs at P3, P16 and P30, respectively.

Oil Red O staining was used to demonstrate the adipogenic differentiation of the ADSCs (20). After culture in adipogenic differentiation medium for 7 days, the morphology of the ADSCs changed from fibroblast-like to oblate-like and numerous lipid droplets appeared in the cells/dishes (Fig. 5A). With prolonged induction, the number of lipid droplets increased gradually and they assembled into larger droplets. The adipogenic differentiation was confirmed by oil red O staining, as the induced cells were positive (Fig. 5B), while cells cultured in normal growth medium were negative on oil red O staining (Fig. 5C).

To further verify the adipogenic differentiation of the ADSCs, the expression of adipogenic markers was evaluated in the two groups by RT-PCR. In the ADSCs incubated in differentiation medium, the adipocyte-specific genes PPAR-γ and LPL were expressed (Fig. 5D), which did not occur in the control group.

The capacity of broiler ADSCs to differentiate into osteogenic cells was assessed, and the induced cells were subjected to morphological and phenotypic analysis. On the 7th day of induction, the cell shape had changed; the cells became confluent and formed mineralized nodules, whose size increased after further induction. After culture in osteogenic medium for 21 days, morphological changes of the ADSCs were evident and became polygonal (Fig. 6A). Furthermore, the nodules were positive on alizarin red staining (Fig. 6B). However, no change in morphology and no staining with alizarin red were observed in the control group (Fig. 6C).

To determine that differentiation had occurred, the expression of osteogenic markers was assessed in the two groups by RT-PCR. The osteogenic genes OPN and collagen I were expressed in the induced group, but not in the control group (Fig. 6D).

The capacity of broiler ADSCs to differentiate into hepatocyte-like cells was proven by morphological and phenotypic analysis of the induced cells. After culturing in hepatocyte differentiation medium for 7 days, the morphology of certain ADSCs in the experimental group changed to a round shape from the long spindle type. After 14 days, numerous cells in the induction group exhibited a cobblestone-like morphology (Fig. 7A) and were positive on glycogen staining in the cytoplasm (purple; Fig. 7B), indicating that the induced cells had acquired the function of synthesis and storage of liver glycogen. In the control group, no morphological changes and no glycogen staining were observed (Fig. 7C).

In addition, RT-PCR indicated that in the induction group, the genes ALB and AFP, which are specific to hepatocyte-like cells, were expressed, while they were not expressed in the control group.