hiPSCs culture
hiPSCs culture was performed as described previously (22–24). Cells were maintained in Essential 8 (E8) medium (Thermo Fisher Scientific, Waltham, MA, USA) on vitronectin-coated dishes (Thermo Fisher Scientific), and attached cells were maintained at 37°C in 5% CO2. All experiments were conducted with three cell lines.
Human OA synovial fluid preparation
Synovial fluid (SF) was obtained from the knee joints of patients with OA (n = 3) by Seoul St. Mary’s Hospital and transferred to a 15 mL conical tube. The samples were centrifuged at 1,800 × g for 10 min (25). The supernatant was collected, aliquoted, and stored at -80°C (26). After random selection, Chondrogenic pellets in the samples were treated with 5% SF in medium.
NGF treatment after chondrogenic differentiation using hiPSCs
hiPSCs were expanded and 4.5 × 106 cells were seeded into Aggrewell plates (STEMCELL Technologies, Vancouver, Canada) and cultured in T75 cell culture flasks to generate embryonic bodies (EBs). The EBs were washed from the T75 cell culture flask, and the cell clumps were removed via filtration through a 40-micrometer cell strainer. EBs were maintained in E8 medium for 5 d for expansion and then maintained in fresh E7 medium for another 5 d. EBs were harvested and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (P/S) (Gibco) and then placed in a 0.1% gelatin-coated dish to induce cell outgrowth. The medium was changed every other day.
Briefly, 3 × 105 outgrowth cells were resuspended in chondrogenic differentiation medium (CDM; DMEM containing 20% knockout serum replacement, 1× non-essential amino acids, 1 mM L-glutamine, 1% sodium pyruvate, 1% insulin-transferrin-selenium (ITS)+, 10 − 7 M dexamethasone, 50 mM ascorbic acid, and 40 µg/mL L-proline supplemented with 10 ng/mL recombinant human bone morphogenetic protein 2 and transforming growth factor beta-3) and transferred to a 15 mL conical tube. The cells were then centrifuged at 1,800 × g for 5 min. The generated pellets were maintained for 14 d, and medium was replaced with fresh CDM every other day (6, 24, 27). Recombinant human NGF protein (10 ng/mL; Abcam, Cambridge, United Kingdom) (12, 28, 29) was added to the differentiated chondrogenic pellets on day 3 and its expression level was analyzed on days 7 and 14 of differentiation.
Polymerase chain reaction assays
Samples were harvested and stored at -80°C. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). cDNA synthesis was performed using a RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific). Real-time polymerase chain reaction (RT-PCR) was performed using the StepOne™ Real-Time PCR System (Thermo Fisher Scientific). Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase and relative gene expression was calculated using the 2−ΔΔCt method. The PCR primer sequences used are listed in Supplemental Table 1 [see Additional file 1].
Western blotting
Proteins were extracted from the chondrogenic pellets using radioimmunoprecipitation assay buffer (Sigma–Aldrich, St. Louis, MO, USA) and centrifuged at 14,000 ×g for 30 min at 4°C. Protein concentration was measured using the Bradford assay. Protein samples were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.1% Tween® 20 detergent (TBST) for 1 h. The primary antibodies used were: anti-NGF (1:1000; Abcam, ab52918), anti-TrkA (1:1000; Sigma–Aldrich, 06-574), anti-tumor necrosis factor-alpha (TNF-α) (1:1000; Abcam, ab9739), anti-interleukin 1 beta (IL-1β) (1:1000; Abcam, ab972) were incubated at 4°C overnight. The membranes were washed three times with 0.1% TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies. After three washes with 0.1% TBST, proteins were detected using a Bio-Image Analysis System (Amersham Imager 600; Fuji Photo Film, Tokyo, Japan). Band intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Histological analysis of chondrogenesis pellets
Three slides containing OA cartilage samples were provided by Dr. Chan Kwon Jung. All slide samples were deparaffinized using two cycles of xylene treatment and then rehydrated. Samples were stained with toluidine blue for 10 min using 0.05% toluidine staining reagent and then washed with tap water. Alcian blue staining was performed by incubating the slides in hematoxylin for 5 min, followed by washing with tap water for 5 min. The slides were then treated with 1% Alcian blue solution for 30 min and washed with tap water. The slides were incubated with nuclear fast red dye for 1 min. Safranin O staining was performed by incubating the slides with 0.001% fast green solution (Sigma–Aldrich) for 5 min. After dipping in 1% acetic acid for 5 min, the slides were incubated with 0.1% safranin O (Sigma–Aldrich) for 5 min. Finally, after all staining procedures were performed, the slides were dehydrated and samples were mounted. The stained slides were observed under a light microscope.
Immunohistological staining
For immunohistological staining, sample slides were heated at 60°C in an oven for 10 min and deparaffinized using xylene following rehydration. Endogenous peroxidase activity was blocked via treatment with 3% hydrogen peroxide (Sigma–Aldrich) for 10 min. The slides were washed and blocked with 1× TBS containing 10% normal goat serum for 20 min at room temperature (RT). Primary antibodies were diluted in the blocking solution at the following ratios: NGF (1:250; Abcam, ab52918), TrkA (1:200; Sigma–Aldrich, 06-574), collagen Type II (1:200; Abcam, ab34712), collagen Type X (1:250; Abcam, ab58632), osteocalcin (1:100; Abcam, sc-30044), and collagen Type I (1:400; Abcam, ab138492). The samples were incubated with the diluted primary antibodies at 4°C overnight. The next day, the slides were washed in 1× TBS and incubated with a biotinylated secondary antibody for 40 min. The slides were washed and treated with ABC reagent (Vector Laboratories, Newark, CA, USA) for 30 min. The washed slides were subsequently incubated with DAB solution (Vector Laboratories) for 1 min. Finally, the slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich) for 1 min followed by dehydration and mounting.
For immunofluorescence staining, the slides were washed with 1× TBS and treated with Alexa Fluor-594 and − 488-conjugated secondary antibodies diluted in blocking buffer after primary antibody treatment. Secondary antibodies were then incubated for 40 min at RT. The slides were stained with 4′,6-diamidino-2-phenylindole (10236276001; Roche, Basel, Switzerland) for 10 min. Subsequently, the slides were washed with 1× phosphate-buffered saline (PBS) and mounted using antifade. All immunostained samples were analyzed using bright-field or fluorescence microscopy.
Alizarin Red S Staining
Paraffin sections were deparaffinized. The slides were washed with 1× PBS. Alizarin Red S solution was added to the slides at RT. The stained slides were then washed with distilled water. Calcium deposition on the samples was confirmed using light microscopy analysis.
Von Kossa staining of Chondrogenic pellets
Von Kossa staining was performed on paraffin slides. The slides were washed with tap water after deparaffinization and incubated with 5% silver nitrate solution under a UV lamp for 40 min. The slides were washed with tap water and then treated with 5% sodium thiosulfate for 20 min. Stained slides were viewed under a light microscope (25, 30, 31).
Statistical analysis
All experiments were performed identically in triplicate, and graphs of the experimental data were generated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The t-test was used to analyze non-parametric quantitative datasets, and a one-tailed p-value was calculated. Values of (p < 0.01; **, p < 0.005; and ***, p < 0.001) indicated significance.