Mononuclear cells from BM aspirates were isolated by density Ficoll-Paque gradient separation. BM was placed in a 50 mL syringe containing 5,000 units of preservative-free heparin, diluted 1:1 with phosphate buffered saline (PBS), resuspended in PBS to a final volume of 10 mL, and layered over an equal volume of Histopaque-1,077 (Sigma Chemical Co., St. Louis, MO, USA). After centrifugation at 2,000 rpm for 30 min, mononuclear cells were recovered from the gradient interface, rinsed twice in PBS, adjusted to a concentration of 1.5 × 10⁷ cells/10 mL, and seeded onto 100-mm culture plates in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG; 1 g/L glucose, JBI, Seoul, Korea) containing 1% penicillin-streptomycin (P/S; 10,000 units/mL, Gibco/BRL, New York, NY, USA) and 10% (v/v) heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Total numbers of nucleated and viable cells were determined using a hemocytometer and trypan blue (Gibco/BRL, Gaithersburg, MD, USA) staining. Cells were incubated at 37℃ in a humidified 5% CO₂ atmosphere and allowed to adhere for 72 hr. Non-adherent cells were then removed. The medium was changed twice a week. When cells were 80%-90% confluent, adherent cells were trypsinized (0.05% trypsin, Gibco/BRL) at 37℃ for 5 min and replated in 100-mm culture plates. After passage 3, a morphologically homogenous population of adherent cells was obtained. During this expansion, medium was changed every 4-5 days.

MSCs that adhered to spot slide bottoms were fixed with -20℃ methanol (100%) for 5 min. Cells were then rehydrated in PBS for 5 min at room temperature, washed three times with PBS, blocked with 3% bovine serum albumin in PBS, and incubated overnight at 4℃ with SH2 (American Type Culture Collection [ATCC], Rockville, VA, USA) as a positive control. Primary antibody (SH2) was removed by washing three times with PBS, and cells were then incubated with fluorescein isothiocyanate (FITC)-labeled affinity-purified antibody to mouse IgG + IgM (H + L) (DiNonA Inc., Seoul, Korea) for 1 hr at room temperature. Secondary antibodies were removed by washing three times with PBS. Coverslips were mounted onto slides with a solution containing 50% PBS and 50% glycerol. Labeled cells were observed under an Axiovert 200 (Zeiss, Thornwood, NY, USA).

Flow cytometry was performed to determine MSCs positive for SH2. Cells were permeabilized with ice cold 75% methanol in PBS for 30 min at 4℃, and washed three times. A FITC-conjugated SH2 antibody (DiNonA), diluted 1:500 in PBS, was then added, and cells were incubated for 1 hr at 4℃. Cells were analyzed within 2 hr of staining using a flow cytometer (FACScalibur, Becton Dickinson, Bedford, MA, USA). A total of 1 × 10⁶ cells were collected for each measurement. Negative control samples were stained with an isotype-matched irrelevant mAb.

To induce chondrogenic differentiation, 200,000 MSCs were placed in a 15-mL polypropylene tube and centrifuged at 1,000 rpm for 5 min. Pellets were then cultured at 37℃ in 5% CO₂ and 300 µL of serum-free chondrogenic medium consisting of Dulbecco's modified Eagle medium-high glucose (DMEM-HG, JBI) supplemented with 10 ng/mL of transforming growth factor-β₃ (TGF-β₃, R&D Systems, Minneapolis, MN, USA), 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL ascorbate-2-phosphate, 100 µg/mL pyruvate, and 50 mg/mL ITS + Premix (Becton Dickinson Biosciences, Bedford, MA, USA; 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/mL bovine serum albumin [BSA], and 5.35 mg/mL linolenic acid); the medium was replaced every 2-3 days for 3, 7, 14, or 21 days.

Total RNA was extracted from undifferentiated MSCs and from pellets after 3, 7, 14, or 21 days of differentiation using RNeasy kits (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. And 1,200 µL of RLT buffer supplemented with beta-mercaptoethanol (10 µL/mL) was added to the washed cells. RNA integrity was assessed by gel electrophoresis and RT-PCR and concentrations were determined by measuring absorbance at 260 nm.

Total RNA was processed using the Genesys Applied Biosystem Facility (Genesys, Munster, Germany), according to manufacturer's recommendations. Each RNA pool (2 µg) was labeled with Digoxigenin-UTP using the ABI Chemiluminescent RT-IVT Labeling Kit v 1.0. Double-stranded cDNA was prepared from total RNA. UTP-digoxigenin-labeled complementary RNA (cRNA) was synthesized by in vitro transcription. Labeled cRNA (10 µg) was hybridized to ABI Human Genome Survey Microarray v 2.0, which was then incubated with alkaline phosphatase-linked digoxigenin antibody. Phosphatase activity was then initiated to produce the chemiluminescent signal. Chemiluminescent (cRNA) and fluorescent (spot background) signals of the cRNA and standard control spots were then scanned. Chemiluminescent detection and image acquisition was performed using an Applied Biosystems 1,700 (AB1,700) Chemiluminescent Microarrays Analyzer, according to the manufacturer's instructions. Using the software supplied with the AB1,700 apparatus, the spot chemiluminescent signal was normalized over the fluorescent signal of the same spot to obtain normalized signal value. For inter-array normalization, global median normalization was applied across all microarrays (7).

Data analysis and data normalization were performed using the method described by Quackenbush (8). For background correction, the mean intensities of areas surrounding spots were subtracted from spot intensities (local area background). Data sets were normalized by dividing the mean intensity value of every spot (in duplicate) by sum of all spot intensities within a sample to eliminate experimental or data acquisition variations. Normalized data were used to calculate the gene expression level ratios of different culture stages. A two-fold expression cut-off was applied. For hierarchical gene cluster analysis, expression ratios were calculated for all genes as described by Eisen et al.(9).

First strand cDNA was synthesized using reverse transcriptase (RT) and 1 µg of total RNA. Reactions were conducted in 20 µL of buffer containing; 0.5 µL oligo (dT)_12-18 primer (Gibco/BRL, Grand Island, NY, USA), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 40 mM DTT, 0.5 mM deoxynucleotide triphosphate (dNTP) mixture (Invitrogen, Carlsbad, CA, USA), 10 unit RNase inhibitor (Gibco/BRL), and 200 units of MMLV reverse transcriptase (Invitrogen). After incubation at 37℃ for 60 min, reactions were stopped by heating at 70℃ for 15 min. To remove remaining RNA, 1 µL of E. coli RNase H (4 mg/mL) was added to reaction mixtures and incubated at 37℃ for 30 min. cDNAs obtained were used as a template for PCR amplification using gene-specific primers for target genes and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are listed in Table 1.

PCR amplification of cDNA was performed in an automated thermal cycler (GeneAmp PCR System 2,400, Applied Biosystems, Foster City, CA, USA), in a final volume of 25 µL containing; 4 µL of cDNA solution, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 0.2 mM dNTP mixture, 0.4 pM of each primer, and 5 units of Taq DNA polymerase (Promega, Madison, WI, USA). After PCR, amplified products were analyzed by electrophoresis in 1.5% agarose gel and visualized by ethidium bromide staining under UV light illumination.

The in vitro growth pattern of MSC is shown in Fig. 1. Human bone marrow-derived MSCs were cultured and expanded. During the log phase of growth, cells proliferated with a population doubling time of 30 hr, and this growth period was followed by a confluent growth-arrested phase.

Colonies were examined approximately 7 days after initial plating. A morphologically homogeneous population of 90% confluent fibroblast-like cells was obtained after 2 weeks. The cells were replated into culture dishes and cultured for 2 weeks. The replated cells were used for subsequent experiments. The cultured MSCs were positive for SH2 by flow cytometry (Fig. 2).

MSCs were pelleted into micromasses and differentiated in serum-free medium in the presence of TGF-β₃ and dexamethasone. Immediately after centrifugation, the cells appeared as flattened pellets at the bottom of tubes. One day later, pellets had a thickened lip, and between days 2 and 3, pellet became spherical without any increase in size. Pellets then grew in size and pellet diameters increased to about 2-fold on days 7 and 14 (Fig. 3A).

Using normalized microarray data, we identified 1,486 differentially expressed genes (Fig. 4A), which included 1,303, 121, 40, 20, and 2 genes exhibiting minimum 2 to < 5, 5 to < 10, 10 to < 20, 20 to < 70 and > 70-fold changes, respectively. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes (2-48 fold changes, Table 2) were confirmed by RT-PCR.

The expression levels of the 10 genes selected (Hrad 6B, annexin A2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl, and IL-15) were analyzed by RT-PCR, by using total RNAs obtained from 5 samples (Fig. 4B). The expression levels of 9 genes (Hrad 6B, annexin A2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) were low in undifferentiated cells and increased in differentiated cells by RT-PCR and microarray, but the expression pattern of IL-15 was different. Expression level of IL-15 tended to be decreased in microarray, but increased in RT-PCR (Fig. 5).