Adult outbred Sprague Dawley (SD) rats that weighing 180 to 210 g were served as bone marrow donors. Bone marrow was obtained from the femurs and tibia of adult SD rats [16]. Rat bone marrow cells were cultured in basic media (Dulbecco's modified Eagle's medium [DMEM], Sigma Chemical Co., St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Sigma). The effluent was collected in sterile tubes. Gentle pipetting resulted in obtaining of a single cell suspension. Bone marrow cells were counted and plated with a concentration of 10×10⁶/ml in T-75 flasks. The cells were then cultured in DMEM containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37℃ in a humidified atmosphere that contained 5% CO₂. Medium was changed after 4 days and every 3 days thereafter. Nonadherent hematopoietic cells were removed when medium was changed. After a mean of 7 days, cells reached subconfluence and was detached with trypsin/EDTA, reseeded at 4×10³ cells/cm², and used for experiments after the third passage. MSC features were demonstrated by typical spindle-shaped morphology.

Fibroblast like colony growth was evaluated on primary cells grown on tissue culture six-well dishes [17]. Total bone marrow-derived cells were plated at the density of 25×10⁶ cells/well. After 7 days, the capability of MSC to form fibroblast-like colonies was assessed. Images that showed MSC morphology were acquired by contrast-phase microscope.

The cells were centrifuged at 1,200 rpm for 5 minutes. and then solved in phosphate buffered saline (PBS) at the concentration of (1×10⁶/ml). The cells were stained with different fluorescently labeled monoclonal antibodies (mAb). In brief, 100 µl of cell suspension was mixed with 10 µl of the fluorescently labeled mAb and incubated in the dark at room temperature for 30 minutes. Washing with PBS containing 2% bovine serum albumin was done twice and the pellet was resuspended in PBS and analyzed immediately on flow cytometry. The mAbs were used in different combinations of fluorochromes; namely fluorescein isothiocyanate, phycoerythrin and phycoerythrin-cyanine 5. Different combinations of mAb were used against various antigens. The immunophenotyping was performed on EPICS-XL flow cytometry (Coulter, Miami, FL, USA). The cells were analyzed with the most appropriate gate FBS using the combination of forward and side scatters.

Adipogenic differentiation was performed in monolayer culture in six-well-plates as described by Pittenger et al. [9]. Briefly, 20,000 cells were seeded per well and cultured until confluency. The cells were then treated for 72 hours with the adipogenic induction medium consisting of DMEM base medium, 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml), 1 µM dexamethasone (Sigma), 0.2 mM indomethacin (Sigma), 0.1 mg/ml insulin (Sigma), 1 mM 3-isobutyl-1-methylxanthin (Sigma) and afterwards for 24 hours with adipogenic maintenance medium consisting of DMEM base medium, 10% FBS, penicillin/streptomycin solution penicillin (100 U/ml) and streptomycin (100 µg/ml) and 0.1 mg/ml insulin. This cycle was repeated four times. Afterwards the cells were cultured for one more week in adipogenic maintenance medium. For the negative control cells were cultured in cell culture medium containing 10% FBS. Samples were taken from adipogenic differentiated stem cell at different time intervals, after 4, 7, 14, and 21 days for light microscopy and reverse transcription polymerase chain reaction (RT-PCR) study.

Cells were cultured in petri dishes for 4, 7, 14, and 21 days in DMEM plus 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, 10 µM insulin, and 200 µM indomethacin [9, 18]. Dishes were washed in PBS and cells were fixed in 3.7% formaldehyde for 30 minutes, followed by staining with oil-red O for 30 minutes oil-red O was prepared by diluting a stock solution (0.5 g of oil-red O [Sigma-Aldrich] in 100 ml of isopropanol) with water (6:4) followed by filtration. After staining, plates were washed twice in water and photographed [19]. Adipogenic differentiation were quantified by counting adipocytes in five randomly selected visual fields at magnification (×100) for each specimen (n=5) and calculating the percentage of adipogenic cells (number of cells with oil-red O positive intracellular vesicles/total number of cells).

Total RNA was extracted from adipogenic differentiated MSCs at 4, 7, 14, and 21 days by using RNeasy Mini kit (Qiagen, Valencia, CA, USA) and treated with deoxyribonuclease to remove contaminating genomic DNA, following the manufacturer's instructions. Reverse transcription was performed with 1 µg RNA in a total volume of 20 µl per reaction. In order to display adiponectin expression, we used primers forward 5'-GGGATTACTGCAACCGAAGG-3' and reverse 5'-CCATCCAACCTGCACAAGTTT-3'. For adipocyte lipid-binding protein (ALBP), primers were forward 5'-CATACATAAAGTCCTTCCCGCTG-3' and reverse 5'-TTGTCTGTTGTCTTTCCTGTCAAGA-3'. For leptin, primers were forward 5'-TTCACACACGCAGTCGGTATC-3' and reverse 5'-GTGAAGCCCGGGAATGAAG-3'. For lipoprotein lipase (LPL) forward primers were 5'-GTACAGTCTTGGAGCCCATGC-3' and reverse 5'-GCCAGTAATTCTATTGACCTTCTTGTT-3'. The RT-PCR procedure was performed using the One Step RT-PCR kit (Qiagen), beginning at 50℃ for 30 minutes and 95℃ for 15 minutes for reverse transcription, then followed by 35 cycles, with each cycle consisting of denaturion at 94℃ for 1 minute, annealing at 57℃ for 1 minute, elongation at 72℃ for 1 minute, and the final extension at 72℃ for 10 minutes. The amplified DNA fragments were visualized through 2% agarose gel electrophoreses and photographed under UV light [20]. In addition real-time PCR was performed with 25 µl amplification reactions contained primers at 0.5 µm, deoxynucleotide triphosphates (0.2 mm each) in PCR buffer, and 0.03 U Taq polymerase along with SYBR-green (Molecular Probes, Inc., Eugene, OR, USA) at 1:150,000. Aliquots of cDNA were diluted 5- to 5,000-fold to generate relative standard curves with which sample cDNA was compared. Standards and samples were run in triplicate, and use glyceraldehyde-3-phosphatedehydrogenase was also included as an internal control. PCR products from all species were normalized for the amount of 18S amplicons in the reverse transcription sample, which was standardized on a dilution curve. Reactions were performed on a 7000 Real-Time PCR System (ABI PRISM, Applied Biosystem, Foster City, CA, USA). A model introduced by Pfaffl was used for calculation [21, 22].

Statistical analysis was performed using SPSS version 16 (SPSS Inc., Chicago, IL, USA). All data were presented as mean±SD. One-way ANOVA and Duncan's post-hoc test were used to compare the groups. P<0.05 was considered to be statistically significant.

We harvested the MSCs from the donor rat bone marrow; purify them in culture media with different passages. MSCs were generated by stander procedures and grown for at least two passages in culture. Contaminating hematopoietic cells were depleted during passage 1 and MSCs were morphologically defined by a fibroblast-like appearance (Fig. 1A, B) and were able to form fibroblast like colonies. Colony formation was observed after 3-5 days in culture (Fig. 2). Cells actively searched for cell-to-cell contacts. After 6-7 days in culture, the colonies became confluent and were passaged for the first time.

Immunophenotypes of MSC surface markers was tested by flow cytometric analysis (Fig. 3). All isolated MSC were negative for CD45 and CD34 while they were positive for CD29, CD44, and CD90. These results as shown in Fig. 4 indicate that, the majority of the bone marrow derived cells were mesenchymal.

Adipogenic cells isolated from MSCs of rat bone marrow were shown to be able to proliferate in culture. During propagation, the cell morphology changed markedly, from spindle-shaped cells to flat ones. This morphological change was accompanied by a change in cell proliferation ability and lipid vesicles could be observed. Adipocyte differentiation from rat bone marrow was markedly flourished at 7 days post in vitro culturing then slightly increased. The MSCs derived earlier from rat bone marrow were firstly appeared dedifferentiated and spindle-shaped semi-like fibroblast cells. However, at 4 days, where adipocyte was still less differentiated, a population of both fibroblastoid and non-fibroblastoid cell types were identified. Fibroblastoid cell population was still existed after enzymatic digestion (Fig. 5A-C). Oil-red O staining of adipocyte revealed discrete spots of cytoplasmic oil droplete within adipocytes derived from rat bone marrow (Fig. 6).

Gene expression profiles throughout the differentiation process were examined in adipogenic differentiated genes at 4, 7, 14, and 21 days. Markers for the transcription factors for ALBP gene was expressed at 90 bp, however LPL, leptin, and adiponectin genes was expressed at 80 bp, 250 bp, and 85 bp respectively. Moreover, gel electrophoresis for rat mRNA expression of adiponectin at day 4 showed that, band was absent (Fig. 7). In addition to that, real time-PCR data for some genes in cells from adipogenic differentiated MSCs demonstrated that, at 4, 7, 14, and 21 days after differentiation, LPL gene expression was between 0.12% and 2.3% (Fig. 8), while ALBP gene expression was between 0.1% and 1.2% of rat adipocyte (Fig. 9).