Chemicals and Ethics used in experiments
All chemicals and media were purchased from Thermo (Waltham, MA, USA) or Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise specified. Collections of SF specimens and PBMCs were authorized by ‘GNUH 2012-05-009’ after obtaining informed consent from patients and volunteers. The protocol for animal experiments in CIA mice was approved by the Animal Center for Biomedical Experimentation at Gyeongsang National University (GNU-131209-M068).
Collection of SFs and establishment of SF-MSCs from RA patients
The control SFs were obtained from donors without evidence of inflammatory joint disease. The SFs for RA groups were obtained from the joints of RA patients who were divided into E-RA (disease duration < 2 years) or L-RA (disease duration > 10 years) groups, depending on how long they had been diagnosed with RA. Therefore, the SF-MSC were divided into three groups: CTL-SF-MSC (n = 10), E-SF-MSCs (n = 9) and L-SF-MSCs (n = 12). The clinical histories of RA patients are presented in Table 1. Cell isolation from the aspirated and cultured SFs were processed as previously described [3]. The SF specimens were filtered through a 40 μm nylon cell strainer (BD Falcon, NJ, USA) to remove debris and then cell pellets were isolated by centrifugation at 400 × g for 10 min; the supernatants were stored at -80 °C until the inflammatory cytokine analysis. The resuspended cells were explanted onto 35 mm dishes (Nunc, Roskilde, Denmark) and allowed to adhere for 2 d in culture medium before non-adherent cells were discarded. The adherent cells were cultured with advanced Dulbecco’s modified Eagle’s medium (ADMEM) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMaxTM, 10 ng/mL bFGF and 1% penicillin and streptomycin (10,000 IU and 10,000 μg/ml) at 36.5 °C in a humidified incubator with 5% CO2. The expanded cells were taken through four passages before used for further analysis.
Characterization of SF-MSCs
Expression of MSC-specific cell surface molecules in SF-MSCs were validated with flow cytometry (BD FACS Calibur, NJ, USA) in triplicate. A total of 1 × 104 cells were harvested and were then fixed with 4% paraformaldehyde at 4 °C. All antibodies were diluted with 1% bovine serum albumin (1:200) (Table S1). The fluorescein isothiocyanate (FITC)-conjugated primary antibodies were incubated with the harvested cells for 1 h, with mouse IgG1-FITC used as an isotype control. Approximately ~80% of confluent SF-MSCs differentiated into adipocytes and osteoblasts after 3 w. Adipogenesis was induced with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 mM indomethacin, 10 mM insulin and 1 mM dexamethasone, and confirmed by intracellular lipid vacuoles staining with 0.5% Oil red O solution as well as gene expression (FABP4 and PPARγ). Osteogenesis was induced with DMEM supplemented with 10% FBS, 200 mM ascorbic acid, 10 mM β-glycerophosphate, and 0.1 mM dexamethasone, and determined by the accumulation of calcium deposits visualized with 5% sliver nitrate solution (Von Kossa staining) and gene expression (ON and OCN). For chondrogenesis, 1 x 106 SF-MSCs were cultured for 3 w in 15 mL tubes containg STEMPRO Osteocyte/Chondrocyte basal medium supplemented with 10% chondrogenesis supplement. Cell pellets were then embedded in paraffin, cut into 5-mm sections and stained with 1% Alcian blue and 0.1% nuclear fast red solution counterstain to confirm synthesis of proteoglycans, as well as gene expression (COL2 and COL10A1). The protocol for the gene expression analysis was described in a later section and primer information is displayed in Table S2.
Gene expression by quantitative PCR (q-PCR)
The q-PCR was used for gene expression studies to determine pluripotency (Oct3/4, Sox2 and Nanog), apoptosis (Bax, Bak, p53, Bcl2 and Birc), differentiation (FABP4, PPARγ, ON, OCN, COL2 and COL10A1) and hypoxia-related genes (GLUT1, LDHA, LOX and PGK1). The q-PCR have three replicates of each sample and relevant primer information is displayed in Table S2. Total RNA was extracted using an RNeasy Minikit (Qiagen, CA, USA) and quantified using an OPTIZEN 3220 UV BIO spectrophotometer (Mecasys, Sungnam, Korea). Then, cDNA synthesis was performed from 1 mg total RNA using an Omniscript Reverse Transcription Kit (Qiagen) with an oligo dT primer at 60oC for 1 h. The qRT-PCR was performed using a Rotor Gene Q qRT-PCR machine (Qiagen) with Rotor-Gene 2× SYBR Green mix (Qiagen) including 2 mL cDNA per reaction and 0.5 mM forward and reverse primers. The qPCR program settings included of pre-denaturation (95°C for 10 min), 45 PCR cycles (95°C for 10 s, 60°C for 6 s and 72°C for 4 s), melting curve analysis (60°C to 95°C by 1°C per 1 s) and cooling (40°C for 30 s). All transcriptional levels of target genes were normalized against TBP expression, which is known as a stable reference gene in human MSCs [12].
Proliferation and cell cycling in SF-MSCs
The Vybrant® MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Cell Proliferation Assay (Molecular Probes, Eugene, OR, USA) was used to evaluate the proliferation of SF-MSC cultures following the manufacture’s protocol. Cell proliferation was quantified with an MTT colorimetric assay on a microplate reader (Molecular Devices) and absorbance of 540 nm. For the analysis of changes in the cell cycle, SF-MSCs were fixed with 70% ethanol, stained with 10 µg/ml propidium iodide (PI) solution and were analyzed using flow cytometry.
Senescence-associated β-galactosidase activity staining
Cellular senescence was evaluated using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA). SF-MSCs were fixed for 15 min in fixation solution at room temperature and stained with β-Galactosidase staining solution, then incubated at 37°C overnight. For measurement of β-galactosidase activity, Mammalian β-Galactosidase Assay Kit (Thermo, Rockford, IL, USA) was used. SF-MSCs were harvested, added M-PER reagent for 10 min and centrifuged for 10 min at 27,000 x g. The supernatant was transferred into 96-well plate and treated the β-Galactosidase reagent for 30 minutes at 37°C. The optical density was determined at a wavelength of 405 nm using a microplate reader (Molecular Devices).
Evaluation of telomere length and telomerase activity
Telomere lengths of SF-MSCs were investigated using a nonradioactive chemiluminescent TeloTAGGG telomere restriction fragment (TRF) length assay kit (Roche, Indianapolis, IN, USA) following the manufacturer’s instructions.
Suppression of PBMC proliferation by SF-MSCs
Human PBMCs were isolated from healthy donors (n = 6) with density gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare, Uppsala, Sweden). PBMCs were then resuspended in RPMI 1640 complete medium supplemented with 10% FBS and 1% penicillin and streptomycin (10,000 IU and 10,000 μg/ml); cultures were stimulated with 1 μg/mL to activate T-cell proliferation. The PHAL-activated PBMCs (1 × 105 cells/well) were co-cultured for 5 d in a 96-well plate with three differentially conditioned pre-seeded SF-MSCs at PBMC:MSC ratios of 1:1, 1:2, and 1:4 before the addition of 5-bromo-2-deoxyuridine (BrdU). PBMC proliferation levels were performed using a Cell Proliferation ELISA, BrdU (colorimetric) Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
Analysis of cytokine levels in SFs and SF-MSCs
The frozen SF supernatant samples were thawed and used to evaluate inflammatory cytokine levels. The levels of TNF-α and IL-1β in the SFs were determined with a Quantikine® ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol. Briefly, standards and samples were incubated in wells pre-coated with the respective human primary antibody. The resulting antigen-antibody complex was detected using human TNF-α or IL-1β conjugated to horseradish peroxidase with preservatives, and the conjugate was quantified by a colorimetric reaction with a 3,3',5,5'-tetramethylbenzidine substrate. The resultant color intensity was read at 450 nm using a microplate reader (Molecular Devices). For SF-MSCs, 1 × 105 cells/well were cultured in 96 well plates in the serum-starvation medium (1% FBS ADMEM), followed by supplementation of human recombinant TNF-α (50 ng/mL; R&D Systems) for 2 d to activate inflammatory cytokine production. After collecting the supernatant, the levels of MMPs (MMP-1, MMP-3 and MMP-13) and other cytokines (IL-6 and IDO) were analyzed in the same manner as SFs. All samples were assayed in duplicate and the concentration of target proteins in each sample was determined by interpolation from the standard curve.
SF-MSCs administration to CIA mice
Injection of MSCs into collagen induced arthritis (CIA) mice was conducted following a previous publication [3]. Briefly, pathogen-free male DBA/1 mice (7-9-w-old; Orient Bio, Seoul, Korea) were immunized with 100 μl bovine type II collagen (Chondrex, Redmond, WA, USA) emulsified in complete Freund’s adjuvant (CFA, Chondrex) by injection into the intradermal region of the tail on Day 0, and boosted by an equal volume of bovine type II collagen and incomplete Freund’s adjuvant (IFA, Chondrex) on Day 21. The experiment included 4 groups (each n = 8), a PBS injection control and CTL-SF-MSCs, E-SF-MSCs, or L-SF-MSCs injected groups. SF-MSCs were intraperitoneally injected on Day 21 and repeated for five consecutive days with 200 mL PBS or the SF-MSC lines (5 × 106 cells per 200 μL PBS). The clinical arthritis scores (0-4 scale) were evaluated for each limb in accordance with a well-defined standard and had a total possible score of 16. To measure hind paw thickness, a caliper was placed across the ankle joint at the widest point to measure ankle thickness. On Day 48, CIA mice were humanely sacrificed by cervical dislocation. The hind paws were scanned by a SkyScan 1076 micro-CT apparatus (Bruker, Kontich, Belgium) and reconstructed into a three-dimensional structure with a voxel size of 18 µm using NRecon software and CT Analyzer (Bruker). Joint tissue specimens from CIA mice were fixed with 10% formalin, decalcified for 3-4 w in 10% EDTA, and embedded in a paraffin block. Joint sections (5 μm) were stained with hematoxylin and eosin (H&E), Safranin O or tartrate resistant acid phosphatase (TRAP) to evaluate articular inflammation, cartilage damage, and TRAP-positive multinucleated cells (osteoclast), respectively. The total number of TRAP-positive multinucleated cells containing three or more nuclei was counted in 10 areas of each CIA mouse ankle [3, 13].
Induction of the RA-like inflammation milieu
Because both low partial pressure of oxygen (hypoxia) and inflammation are relevant features in the synovial joints of RA patients [14], and in vitro RA-like inflammation milieu was induced in E-SF-MSCs to explore whether immunomodulation properties and senescence were altered in inflammation-exposed SF-MSCs. The E-SF-MSCs were cultured in the normal culture condition for 3 d with an alteration in gas composition: 21% O2, 5% CO2 and 74% N2 or 3% O2, 5% CO2 and 92% N2 in a 95% humidified atmosphere, maintained in multi-gas incubators (ASTEC, Fukuoka, Japan) to the reflect normoxia or hypoxia, respectively. In addition, the media for hypoxic E-SF-MSCs were supplemented with 20 ng/mL TNF-α and 20 ng/mL IL-1β (R&D Systems) as the representative inflammatory cytokines. Both E-SF-MSCs and L-SF-MSCs under normoxic conditions were used as control counterparts.
Western blot analysis
The induction of hypoxia was validated by upregulation of HIF1α expression. The cell extracts of E-SF-MSCs with or without hypoxia were prepared with RIPA buffer supplemented with a HaltTM Protease Inhibitor Cocktail Kit (Pierce Biotechnology, Rockford, IL, USA). The concentration of total protein in the cell extracts was quantified using the Bicinchoninic Acid Protein Assay Reagent Kit (Pierce Biotechnology). A 25 μg aliquot from each sample was fractionated on a 10% SDS-PAGE by gel electrophoresis and was transferred onto a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). The membranes were blocked with 0.1% bovine serum albumin (BSA), incubated with anti-HIF-1α or anti-GAPDH primary antibodies (1:100 dilution with BSA) at 4°C for overnight, incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution with BSA) at RT for 1 h, and detected using a chemiluminescence assay (Amersham Biosciences Corp, Piscataway, NJ, USA) with X-ray film for visualization.
Apoptosis assays
The proportion of apoptosis in E-SF-MSCs with or without induction of an RA-like inflammation milieu was determined using an Annexin V-FITC Apoptosis Detection Kit (Invitrogen, Eugene, OR, USA) following the manufacturer’s instructions. From these cultures, 1 × 104 cells were harvested, washed twice in PBS, suspended with 200 μL binding buffer, treated with 10 μL Annexin V stock solution, incubated at 4°C for 30 min, counterstained with propidine iodide (PI), and analyzed using flow cytometry (BD FACS Calibur).
IDO activity measurements
The indoleamine 2,3-dioxygenase (IDO) activity was measured following a previous protocol [15]. Cultures were harvested and 2.5 × 104 E-SF-MSCs with or without induction of an RA-like inflammation milieu was cultured for 4 d and then supplemented with 100 μM L-Tryptophan (Sigma) for 4 h. The supernatant was then harvested from the cultures and 30% trichloroacetic acid (Sigma) was added before an additional incubation at 50°C for 30 min. This solution was diluted 1:1 in Ehrlich reagent (Sigma, USA) and the optical density was measured at 492 nm using a microplate reader (Molecular Devices). Serially diluted L-Kynurenine (Sigma) made with fresh culture medium was used as the standard.
Statistical analysis
The statistical significance was analyzed using paried T-test, one-way analysis of variance (ANOVA), and Tukey’s multiple comparison test using SPSS 21.0 (IBM, Armonk, NY. USA) followed by Games-Howell post hoc analysis. All data were presented as mean ± Standard Deviation (SD). A value of p < 0.05 was considered as statistically significant difference.