Excess human adipose tissue was obtained from two female patients, undergoing elective surgery at Grymer Privat Hospital, Skejby, Denmark in accordance with protocols approved by the regional Committee on Biomedical Research Ethics in Northern Jutland. The two donors were 56 and 44 years of age and had BMIs of 28 and 27 respectively. The ASCs were isolated from abdominal adipose tissue, and were induced to become osteocytes, chondrocytes, and adipocytes. The histochemical stainings (Fig. 1) and the gene expression studies demonstrated that the cells could differentiate towards chondrogenic, osteogenic, and adiopogenic lineages, thus confirming their identity as mesenchymal stem cells.

In addition to the differentiation procedures, the cells were grown for up to five passages, with RNA being harvested from each passage. Also, cells were exposed to hypoxia (1% oxygen) for up to two weeks.

The yield of RNA varied between the samples from 2.5 μg to 13 μg, reflecting larger cell numbers as the cells were cultured for longer periods. By spectrophotometric analysis, we ascertained that the purity of the samples were very high with A₂₆₀/A₂₈₀ ratios ranging from 1.93 to 2.06 (mean 2.03). The integrity of the RNA was determined by electrophoretic analysis. Using an algorithm of the Agilent software, the samples were assigned RNA integrity numbers (RIN) which describes the integrity of the RNA on a scale from 1 to 10 [15]. The integrity of the RNA was high in all samples, with RINs above 9.7 for all samples (mean 9.96). The RNA samples were thus suitable for the sensitive analysis by real-time RT-PCR.

For the selection of the reference genes, we analyzed the genes tubulin, beta-polypeptide (TUBB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-2-microtubulin (B2M), ubiquitin C (UBC), 60S acidic ribosomal protein P0 (RPLP), cyclophilin A (PPIA), tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB), hypoxanthine-guanine phosphoribosyltransferase (HPRT1) as well as rRNA 18S (RRN18S), all included in the Human Endogenous Control Panel from the TATAA Biocenter (DNA-technology, Aarhus, Denmark). In order to identify the most stable reference genes across all the tested conditions, the expression levels of all reference genes were analyzed using the geNorm software. The analysis showed that the expression levels of TBP, GUSB, YMHAZ, and the PPIA varied the least across all the tested conditions, and TUBB, HPRT1, GAPDH, and ACTB varied the most (Figure 2A). Secondly, the optimal number of reference genes was assessed calculating the pairwise variation between a given number of reference genes and the inclusion of an additional gene (Fig. 2B). A large variation indicates the added gene has a significant effect and should be included. A cut-off value of 0.15 has been proposed, where the inclusion of an extra gene has little effect on the normalization [4]. As seen in figure 2B, the inclusion of three genes as opposed to two (V2/3 is 0.085) has a small effect. In the interest of an optimal determination of the variation of the other genes, we opted for the use of three reference genes (TBP, GUSB, and YMHAZ) in the further analysis.

In order to demonstrate the differences between the highly and little variable reference genes in our study, we normalized three of the most commonly used reference genes namely 18S RNA, beta-actin and GAPDH as well as the four least variable genes to the geometric mean of TBP, GUSB, and YMHAZ (Geom mean). As shown in figure 3, the levels of 18S RNA increase up to 2-fold with increasing passages and during differentiation. Contrary to this, the levels of beta-actin decrease more than 2-fold during passaging. The levels mRNA of GAPDH, which has previously been identify as a suitable reference gene for normalization in the study of preadipocytes [13], appear to be relatively stable during passaging, however, there is a decrease of transcript during chondrogenesis and a 2-fold induction during hypoxic culture. This hypoxic upregulation is not surprising, as GAPDH has previously been shown to be highly regulated by the oxygen tension in numerous other cell types [16-18].

To illustrate the effect of normalizing to reference genes, whose levels are not constant under the experimental condition, we have chosen to determine the expression levels of the chondrocyte-specific transcription factor Sox9 and the hypoxia-inducible growth factor IGF-1, after chondrogenic induction or hypoxic treatment, respectively. The levels of expression are normalized to either the geometric mean of TBP, GUSB, and YMHAZ, to beta-actin and to either 18S RNA for Sox9 or GAPDH for IGF-1. As can be seen in Fig. 4, when normalizing to the Geom mean, the expression of Sox9 is approximately 8-fold up-regulated after three weeks of chondrogenic induction when compared to levels at passage 1. Normalizing to 18S rRNA or beta-actin leads to a 2-fold under- or overestimation of Sox9 transcript levels, respectively. More dramatic results were obtained in the analysis of IGF-1 induction by hypoxia. As seen in Figure 4, when the cells are exposed to mildly hypoxic conditions for a week, the levels of IGF-1 message increase almost 2-fold. However, when normalizing to expression levels of GAPDH, which is strongly regulated by hypoxia, it erroneously appears that IGF-1 is down-regulated by hypoxic treatment. Conversely, when normalizing to beta-actin, an overestimation of IGF-1 induction occurs.

Subcutaneous adipose tissue was obtained from patients undergoing elective surgical procedures at Grymer Private Hospital, Aarhus, Denmark. The protocols were approved by the regional Committee on Biomedical Research Ethics in Northern Jutland. Cells were isolated from the adipose tissue essentially using methods previously described [20]. Briefly, harvested tissue was washed repeatedly with phosphate-buffered saline (PBS) and then enzymatically dissociated with 65 units/mL crude collagenase (Wako, Richmond, VA) in PBS supplemented with 20 mg/mL bovine serum albumin (BSA) at 37°C for 60 min. After incubation, the stromal vascular fraction of cells was pelleted by centrifugation at 400 g for 10 min. To remove contaminating erythrocytes, the pellet was resuspended in distilled water for 10 s after which the salt balance and pH were restored to physiological levels. The suspension was filtered through a 100-μm nylon mesh to remove cellular debris, where after the cells were collected by centrifugation, filtered through a 70-μm nylon mesh, and incubated overnight at 37°C/5% CO2 in control medium (DMEM/F12, 10% FBS, 100 u/ml penicillin, 0.1 mg/ml streptomycin, and 0.05 mg/ml gentamicin). Following incubation for 24 hours, all medium was changed to remove residual nonadherent mononuclear cells. The cells were maintained at 37°C/5% CO2 with medium changes twice a week. When cells were confluent, they were passaged at a ratio of 1:3. All subsequent studies on cell growth and differentiation were carried out twice and in duplicate.

For studies of changes in gene expression during passaging of the cells, RNA was isolated from cells at passages 0, 1, 2, 3, 4 and 5. Briefly, during the subculturing procedure, aliquots of the cells were seeded into 6-well plates (Corning, Costar, Acton, MA) at a concentration of 2 × 10⁵ pr well in two mL of media and cultured for two days, after which RNA was harvested.

Prior to hypoxic treatment, the ASCs (passage 1) were seeded in 6-wall plates at a concentration of 2 × 10⁵ cells pr well in two mL of media. One day after seeding, the cells were transferred to a hypoxic workstation (Xvivo, BioSpherix, Redfield, NY), and incubated in an atmosphere with 1% oxygen and 5% CO₂ balanced with nitrogen for up to two weeks.

The ASCs (passage 1) were seeded in 6-well plates at a concentration of 2 × 10⁵ cells per well in two mL of media and cultured in control medium until confluence. To induce chondrogenesis, the medium was switched to chondrogenic induction medium, consisting of high-glucose (4.5 g/l) Dulbecco's modified Eagle's medium (Invitrogen, Taastrup, Denmark) supplemented with 10 ng/ml transforming growth factor β3 (TGFβ3) (R&D Systems, Oxon, UK), 10^-7 M dexamethazone (Sigma-Aldrich, Broendby, Denmark), 50 μg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), 40 μg/ml L-proline (Sigma-Aldrich), 100 μg/ml sodium pyruvate, 1× ITS⁺ Premix (BD Bioscience, Brøndby, Denmark). After three weeks, RNA was harvested from duplicate wells and other wells were used for histochemical staining.

ASCs were seeded in 6-well plates at a concentration of 2 × 10⁵ pr well in two mL of control medium. After 24 hours, osteogenic differentiation was induced by culturing ASCs in osteogenic medium (control medium supplemented with 0.1 μM dexamethasone, 50 μM L-ascorbic acid 2-phosphate, 0.5 μM calcitriol, and 10 mM glycerol 2-phosphate, all from Sigma-Aldrich). After three weeks, RNA was harvested from duplicate wells and other wells were used for histochemical staining.

ASCs in passage 1 were seeded in 6-well plates at a concentration of 2 × 10⁵ pr well in two mL of control medium. After 48 hours, adipogenesis was induced by culturing ASCs in adipogenic medium (control medium supplemented with 0.45 mM isobutyl methylxanthine (Sigma-Aldrich), 170 nM insulin (Invitrogen), 0.2 mM indomethacin (Sigma-Aldrich). After two weeks, histochemical stainings were carried out.

Chondrogenesis was confirmed using the stain Alcian blue (1 g/L in 0.1 M HCl) (Sigma-Aldrich) for 30 min at room temperature. Before staining, the chondrogenic cultures were fixed in 4% formaldehyde for 15 min and washed with several changes of PBS. The calcium deposits in cells undergoing osteogenesis were stained with Alizarin red S (Sigma-Aldrich). The cells were rinsed in PBS, fixed in ice-cold 70% ethanol and incubated with Alizarin red solution (2 g/100 mL in distilled water, pH 4.2) for 15 min, after which the wells were rinsed repeatedly with water. Adipogenesis was assessed by staining the accumulation of intracellular triglycerides with Oil red O essentially as described previously [21]. Briefly, the cells were fixed in 4% formaldehyde for 1 hour, washed repeatedly with PBS, after with they were incubated the Oil red O working solution for 15 min, and then washed with water. The Oil red O working solution was freshly prepared from 6 mL of 0.5% (w/v) Oil red O in isopropanol mixed with 4 mL of water.

Total RNA was isolated using the Aurum Total RNA mini kit (Bio-Rad, Copenhagen, Denmark) according to manufacturers instructions. Briefly, the cells were lyzed in 250 μL of lysis buffer, after which the RNA was immobilized on a silica membrane, washed, DNAse I treated and eluted in 80 μl RNase-free water by centrifugation. The purity of the RNA was determined by measuring the absorbance A₂₆₀/A₂₈₀ in a Nanodrop spectrophotometer. The quality of the RNA was assessed using lab-on-a-chip electrophoresis technology (Agilent 2100 Bioanalyzer, Naerum, Denmark). On the basis of 1 μg of RNA, cDNA was synthesized using the iScript cDNA synthesis kit (Biorad) according the manufacturer's instructions.

The reference genes, tubulin, beta-polypeptide (TUBB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-2-microtubulin (B2M), ubuiquitin C (UBC), 60S acidic ribosomal protein P0 (RPLP), cyclophilin A (PPIA), tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB), hypoxanthine-guanine phosphoribosyltransferase (HPRT1) as well as rRNA 18S (RRN18S), were all included in the Human Endogenous Control Panel from the TATAA Biocenter (DNA-technology, Aarhus, Denmark). For the analysis of the differentiation of the stem cells, we analyzed the expression of the chondrocyte related transcription factor Sox9 and the insulin-like growth factor 1 (IGF-1). Primers for these genes were designed in-house using the PrimerSelect program of the Lasergene software package (DNA-STAR, Madison, WI) and manufactured by DNA-technology. The forward and reverse primers for Sox9 were 5'-CAC ACA GCT CAC TCG ACC TGG-3' and 5'-TTC GGT TAT TTT TAG GAT CAT CTC G-3', respectively, and the forward and reverse primers for IGF-1 were 5'-GAT GGG GTC TCG CAC TGT CCC-3' and 5'-GAG CCG AGA TCA TGC CAC TG-3'.

Quantitative PCR was performed on a My-Cycler real-time PCR system (Bio-Rad, Hercules, CA). The reactions were carried out in duplicates using the SYBR Green PCR supermix (Bio-Rad), using the manufacturer's instructions. The final reaction volume was 25 μL, and 5 pmol of primers and 0.25 μL of cDNA were used in each reaction. Each assay also included a blank. The PCR protocol consisted of an initial step at 95°C (3 min) followed by 40 cycles of 15 seconds at 95°C for DNA denaturation and 30 seconds of annealing and elongation. The annealing/elongation step was carried out at 60°C. To confirm product specificity, a melting curve analysis was performed after each amplification. The relative expression of each gene in the samples was calculated on the basis of a four-fold serially diluted standard curve derived from a pool of all the cDNA samples.

To find the most stable housekeeping genes, we used the geNorm software package [4], which uses an algorithm to calculate the relative variation (M) of a given housekeeping genes to the remaining control genes. During this process, the most variable gene is excluded and the relative variation of the remaining genes is calculated. Secondly, also using the geNorm software, we determined how many reference genes should minimally be used for normalization purposes. This determination is based on the stepwise inclusion of housekeeping genes in the calculation of the geometric mean of the genes. For a more detailed description of the geNorm software, please refer to the excellent article by Vandesompele et al, 2002[4].

For the comparison of the data normalized to the geometric mean of TBP, GUSB, and YMHAZ, to beta-actin and to either 18S RNA for Sox9 or GAPDH for IGF-1, first the data was confirmed to have a normal distribution, after which the data sets were compared using Student's t-test. Differences were deemed statistically significant with p < 0.05. All calculations were done with the aid of the exact tests package of SPSS 11.0 software (SPSS, Chicago, ).