Patient recruitment and isolation of synovial cells
We obtained synovial tissues from surgeries of joint replacement for patients with OA and RA. We consecutively collected all the available OA and RA samples for this study. Written informed consent for this study and the ethics approval from medical research ethics committee of Tokyo Medical and Dental University (Approval number: M2000-979) were obtained. The synovial tissues were collected from 70 patients (RA:18, OA: 52). All the patients with RA have been received treatment with disease modified anti-rheumatic drugs (DMARD)s including biologics. Ten patients were administered steroids (average dose; 2.9 mg/day). Other characteristics are described in Table 1.
Tissue samples were collected consecutively from joint replacement surgeries to eliminate any bias as previously reported (13). Briefly, joint tissues were obtained immediately after the surgeries, followed by removal of bone and adipose tissues with scissors. Synovial tissues were minced into small pieces, and then subjected to enzymatic digestion. For cell culture, we digested tissues with 2 mg/mL collagenase type 4 (Worthington, NJ, USA), 0.8 mg/mL Dispase II, 0.1 mg/mL DNase I (Roche, Basel, Switzerland) in Dulbecco’s modified Eagle Roche’s medium (DMEM) at 37 °C. After 15 min, we collected the supernatant and replaced with fresh enzyme mix. These procedures were repeated every 15 min for total 1 h. After lysing red blood cells with ACK-lysing buffer, obtained cells were treated with antibodies as described below then sorted by FACS Aria II and III (BD Biosciences, CA, USA) with 100 μm nozzle.
The following antibodies and reagents were used for the analysis of synovial cells with flow cytometry and cell sorting: anti-CD45-APC-H7 (2D1, BD Biosciences, CA, USA), anti-CD235a-APC-Alexa Fluor750 (11E4B-7-6, Beckman Coulter, FL, USA), anti-CD31-PE-Cyanine7 (WM-59, eBioscience, CA, USA), anti-CD146-APC (P1H12, eBioscience), anti-CD34-PE (4H11, eBioscience), anti-PDPN-PerCP-eFluor710 (NZ-1.3, eBioscience), anti-THY1-FITC (5E10, BD Bioscience), anti-CD73-PE-CF594 (AD2, BD Bioscience), anti-CD271-APC (ME20.4, eBioscience), anti-CD54-PE-CF594 (HA58 BioLegend, CA, USA), anti-CD44-APC (G44-26 BD Bioscience), anti-CD29-APC (TS2/16 BioLegend), human TruStain FcX (BioLegend) and Live/Dead fixable aqua dead cell stain kits (Molecular Probes, Thermo Fisher Scientific, MA, USA).
Flow cytometry analysis
The gating strategy of SF subsets was as shown (Supplementary Figure 1). While the mean ratios of CD34-THY1-, CD34-THY1+ and CD34+THY1+ subsets in RA were 35.5%, 24.1% and 24.8%, respectively, the mean ratios of these subsets in OA were 56.6%, 10.2% and 17.2%. These results were compatible as previously reported (13).
We evaluated the expression of MSC surface markers (THY1, CD73, CD271, CD54, CD29 and CD44) in the SF subsets. Considering the expression of MSC markers in the freshly-isolated synovial cells, we evaluated the mean fluorescence intensity (MFI) as expression levels of these markers in the individual subsets by flow cytometry in the 12 consecutive samples (RA: 3, OA: 9) (Supplementary Figure 2).
We sorted CD34–THY1– fibroblasts, CD34–THY1+ fibroblasts and CD34+THY1+ fibroblasts and cultured them in DMEM supplemented with 10% FBS (Gemini Bio, CA, USA), 2 mM L-glutamine, antibiotics (penicillin and streptomycin), and essential and nonessential amino acids (Life Technologies, CA, USA). The cells were expanded for 20-30 days for assays.
Osteoblast, chondrogenic and adipocyte differentiation
Osteoblastic induction was performed as previously reported (6). 3.0 × 103 cells/cm2 were plated in a 12-well plate in osteogenic differentiation medium containing L-ascorbic acid-2-phosphate (0.2 mM; Wako Pure Chemical Industries, Osaka, Japan), beta-glycerophosphate (5 mM; Wako Pure Chemical Industries), dexamethasone (1 nM; Wako Pure Chemical Industries) and incubated at 37 °C in 5 % CO2. All media were changed twice per week. Each SF subset was cultured for 3-4 weeks. Histological staining was performed with alizarin red (Merk Millipore, MA, USA) and alkaline phosphatase (ALP) staining for osteoblast differentiation
For chondrogenic differentiation, 1.25-2.5 × 105 cells were placed in a 15-mL polypropylene tube (AGC Techno Glass Co., Ltd, Shizuoka, Japan) and centrifuged at 1,500 × g for 5 min. The cells were cultured in chondrogenic induction medium containing 1,000 ng/mL of BMP-2 (PeproTech, NJ, USA) and 10 ng/mL of transforming growth factor-β3 (PeproTech), incubated at 37 °C in 5% CO2 for 3 weeks. All media were changed twice per week. Histological staining was performed with safranin O staining for chondrogenesis.
For Adipogenesis, 7.0 × 103 cells/cm2 are plated and cultured in StemPro™ Adipogenesis Differentiation Kit (Gibco, Thermo Fisher Scientific, MA, USA) for 3 weeks. All media were changed twice per week. Oil-red staining was used to evaluate adipogenesis.
For chondrogenesis and adipocyte differentiation, we referred to the previous reports with some modifications (20-21). Briefly, we performed some pellet culture at a density of 1.25 × 105 cells due to imbalance in the number of each subset.
Quantitative real-time Polymerase chain reaction (qPCR)
cDNA was synthesized with QuantiTect Reverse Transcription kit (Qiagen, Hilden, German). Quantitative polymerase chain reaction (qPCR) was performed with Brilliant III Ultra-Fast SYBR Green qPCR master mix (Agilent Technologies, CA, USA) on a LightCycler96® (Roche). The following primers were used as shown in Table 2.
Knockdown of gene expression by small interfering (si) RNA
SFs were seeded at 1.2 × 104 into 12-well cell culture plates and subsequently transiently transfected with 20 pM of THY1 or control small interfering (si) RNA (Thermo Fisher Scientific) using Lipofectamine RNAiMax (Thermo Fisher Scientific) according to the manufacturer's protocol. Cells were incubated with siRNA for 3 day and subjected to osteogenic differentiation as described above.