Patient recruitment and synovia preparation
Four patients were used for synovia preparation. RA patients (n = 2) who satisfied the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) and OA patients (n = 2) were recruited from the outpatient clinic at the Department of Rheumatology, Seoul St. Mary’s Hospital, Seoul, South Korea. These patients had received arthroscopic synovectomy or total knee replacement surgery. Synovia samples were obtained during the operations. Eligibility for OA inclusion required that individuals had primary knee OA, which was diagnosed according to American College of Rheumatology criteria. The experimental protocol was approved by the Catholic University of Korea Human Research Ethics Committee.
Isolation and maintenance of RA and OA fibroblast-like synoviocytes
RA and OA synovia were stored in the sample bank of the Rheumatism Research Center, Seoul, South Korea. These samples were classified only by disease and stored in a blind manner. The institutional review board permitted that a patient consent form was not necessary because samples were anonymous and did not contain patient information. Tissues were homogenized and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco by Invitrogen, Carlsbad, CA, USA) containing 0.01% collagenase and were incubated for 4 hours at 37°C with vigorous shaking. Cells were washed and resuspended in DMEM supplemented with 20% fetal bovine serum (FBS) (Gibco by Invitrogen), and 1% penicillin/streptomycin solution (Gibco by Invitrogen). Cells were cultured until the adherent fibroblast cells achieved confluence.
Lentivirus production and transduction
293FT cells (Invitrogen, Carlsbad, CA, USA) were plated at 80% confluence in 100-mm dishes and transfected with 12 μg of four-in-one reprogramming plasmid (Oct4, Sox2, Klf4, and c-Myc), 9 μg packaging pPAX2 plasmids, and 3 μg pMD2G plasmids using Lipofectamine 2000 (Invitrogen). After about 48 to 72 hours of culture, the virus was harvested and mixed with Lenti-X Concentrator (Clontech Laboratories, Mountain View, CA, USA). After overnight incubation at 4°C, viruses were precipitated at 1,500 × g and resuspended in phosphate-buffered saline. For virus infection, RA or OA FLSs were seeded onto six-well plates. The lentivirus was applied with culture medium for overnight infection. The iPSC colonies were picked after 18 to 20 days of reprogramming.
Cell culture and maintenance of patient-specific iPSCs
RA or OA FLSs were maintained in DMEM containing 20% FBS at 37°C, with 95% air and 5% CO2 in a humidified incubator. All of the cells used for reprogramming were at passage 8. Patient-specific iPSCs were maintained in Matrigel-coated tissue culture dishes (BD Biosciences, San Jose, CA, USA) with E8 human ESC medium.
Quantitative real-time polymerase chain reaction
Total RNA was isolated using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcriptase polymerase chain reaction was performed using an iScript™ cDNA Synthesis Kit (BIORAD, Marnes-La-Coquette, France). Gene expression was quantified by SYBR Green real-time polymerase chain reaction using an ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The relative mRNA levels were normalized to the values of GAPDH mRNA for each reaction. The primer sequences are described in Additional file1.
The iPSC clones were fixed with 4% paraformaldehyde and immunostaining was performed using the following primary antibodies: SSEA-4, Tra-1-60 and Tra-1-80 (Millipore, Billerica, MA, USA), Oct3/4 and Nanog (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Sox2 (BioLegend, San Diego, CA, USA). Samples were incubated with Alexa Fluor 594-conjugates or 488-conjugated secondary antibody (Invitrogen) and detected by indirect immunofluorescence microscopy.
Teratoma formation was performed and analyzed with the approval of the Institutional Animal Care and Use Committee of Applied StemCell (protocol number APP-12-001-Y2; Sunnyvale, CA, USA). Briefly, undifferentiated iPSCs (1 × 106) were suspended in 10 μl Matrigel (BD Biosciences) and delivered using a 28.5 gauge syringe into the subrenal capsule of 8-week-old SCID-beige mice. Eight weeks after cell delivery, the tumors were explanted and subjected to hematoxylin and eosin staining.
For osteogenic differentiation, we cultured iPSCs in osteogenic differentiation medium (ODM) as described by Kao and colleagues. ODM is DMEM supplemented with 15% FBS, 50 μg/ml ascorbate-2-phosphate, 10 nmol/l dexamethasone, and 10 mmol/l β-glycerophosphate. At day 7 after osteogenic induction, in vitro mineralization of cells was assessed using the OsteoImage Mineralization Assay kit (Lonza, Basel, Switzerland) according to the manufacturer’s manual. Fluorescent signals from the hydroxyapatate portion were detected by fluorescence microscope (Axio observer 2.1; ZEISS, Thornwood, NY 10594, USA).
We added 30 μl Chromosome Resolution Additive (Genial Genetic Solutions Ltd, Runcorn, UK) to each six-well plate. After 1 hour of incubation, colcemid® was treated for 30 minutes. Cells were harvested using trypsin and treated by prewarmed hypotonic solution (KCl). Fixation was then performed with 1:3 acetic acid:methanol solution and the slide was prepared for chromosome analysis. The chromosome analysis was performed using a trypsin-Giemsa banding technique. At least 20 metaphases were analyzed.
This study protocol was approved by the institutional review board of The Catholic University of Korea (KC12TISI0861).