Cell culture.
Human iPSC (hiPSC) line was obtained from the American Type Culture Collection (ATCC-BYS0113). The hiPSCs were maintained on Matrigel (Corning)-coated dishes in PeproGrow-hESC (PeproTech) medium containing 2 μM Y-27632. The medium was changed every day. All cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
The hiPSCs were then induced to form EBs. Briefly, iPSCs were digested and resuspended in EB differentiation medium (iPSC culture medium without β-FGF) and then transferred to ultra-low adhesion 6-well plate (Corning). The differentiation medium was changed every 2 days. After 10 days, the EBs were transferred into culture plates with 0.1% gelatin-coated (Corning) and cultured for another 3 days. Then cells were digested and digested solution was filtered through 100 μm nylon mesh. Cells were resuspended in the MSCs growth medium, which consisted of DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), MEM Non-Essential Amino Acids (Gibco), 2ng/ml FGF (PeproTech), 20ng/ml EGF (PeproTech).
Collection and culture of bone marrow mesenchymal stem cells (BMSCs)
The tibia and femur of SD rats were isolated under sterile conditions. Next, the tibia and femur were put into cooled PBS and washed away the attached blood. The bone marrow cavity was washed by DMEM medium containing 10% FBS repeatedly. The cells of bone marrow cavity were flushed into a sterile culture dish and cultured in an incubator containing 5% CO2 at 37 ℃. After 24h, the nonadherent cells were gently washed off with PBS, and the MSCs growth medium was changed every three days.
Flow Cytometric analysis
The hiPSC derived MSCs were washed with PBS, harvested by 0.25% trypsin/EDTA, and resuspended in 100 μl staining media (2% FBS and 2% HEPES in PBS) and stained with mouse anti-human CD29 (BD), mouse anti-human CD44 (BD), mouse anti-human CD34 (BD), mouse anti-human CD105 (BD), and mouse anti-human CD45 (BD) for 30 min at 4°C. Isotype-matched antibody (IgG2b-FITC) was used to determine nonspecific fluorescence. Samples were run on CytoFLEX (BECKMAN) instrument. A minimum of 20,000 cells was assayed for each analysis.
Chondrogenic differentiation of MSCs
For chondrogenic differentiation of MSCs[17, 18], 5 × 105 hiPSC derived MSCs or rat bone marrow MSCs cells were centrifuged at 500 g for 10 min to form a small pellet in a 15 ml centrifuge tube. The cell pellets were cultured for 21 days in the chondrogenic differentiation medium consisting of DMEM (Gibco) supplemented with 10 % FBS, 1:100 ITS-G (ThemoFisher), 100 nM dexamethasone, 10 ng/mL recombinant TGF-β3 (PeproTech), 40 μg/ml L-proline (Sigma-Aldrich). The medium was changed every 3 days.
Rat cartilage defect model
We established a rat cartilage defect model as previously described[19]. The 8-week-old SD rats were anesthetized through intraperitoneal injection of ketamine. The skin around the knee was disinfected, and the right knee of rats was exposed by surgery. Next, a defect (diameter 1.5 mm and 1.5 mm in depth) was created in the center of the groove. The rats were divided into two groups, the experimental group was implanted into hydrogel mixed with rat bone marrow MSCs which were treated with or without wedelolactone. After 6 weeks, all rats were sacrificed, and joint tissues were collected for further analyzed.
Alcian blue staining and toluidine blue staining of chondrocytes
MSCs were seeded in a 6-well plate, then cells were induced in chondrogenic differentiation medium for 3 days. Then the medium was removed and cells were washed with PBS for 3 times. The cells were stained with toluidine blue staining solution or alcian blue staining solution for 30 minutes. Next, the cells were washed with PBS for 5 minutes. Then the stained cells were observed under a microscope.
Histology staining of chondrogenic pellets
Chondrogenic pellets were collected after 21 days of chondrogenic differentiation. Then the pellets were fixed in 4% buffered formaldehyde for 6 hours. Next, these pellets were dehydrated in different concentrations of alcohol. After clarified in the pellet in xylene three times, the pellets were infiltrated with paraffin for 1 h in a 65 °C oven. Then, these pellets were sliced at 7 µm for histology staining. The slides were deparaffinized by moving them through 200 mL of 100%, 95%, 80%, and 75% EtOH sequentially for 4 min each. The slides were incubated in 3% H2O2 for 15 min for endogenous peroxidase blocking. Then, the slides were blocked with goat serum solution at room temperature for 10-15 minutes, Incubated with 100 µL of primary antibodies (anti-COL2A1 or anti-ACAN) at 4 °C for overnight. After slides were washed with PBS 3 times, they were incubated with an appropriate amount of biotin-labeled goat anti-mouse/rabbit IgG polymer at room temperature for 15 minutes. The slides were washed with PBS for 3 times, then incubated with an appropriate amount of horseradish enzyme-labeled streptomyces ovalbumin working solution for 10 to 15 minutes. Next slides were washed with PBS 3 times and incubated with DAB solution for 5 to 8 minutes. Last, these stained slides were observed under a microscope.
MSCs were cultured with chondrogenic differentiation medium for 3 days in confocal dishes. The cells were fixed with 4% paraformaldehyde (dissolved in PBS, pH 7.4) for 10 minutes at room temperature. Next, the cells were incubated with PBS (containing 0.1-0.25% Triton X-100) for 10 minutes. Then, the cells were incubated with goat serum at room temperature for 30 minutes to block the nonspecific binding of antibodies. After that, the cells were incubated with 100 µL of primary antibodies (anti-COL2A1, anti-ACAN, and anti-SOX9) at 4 °C for overnight. The next day, the cells were washed 3 times with PBS and then incubated with second antibodies for 1h at room temperature. DAPI was used to stain nuclei. The stained cells were observed under a confocal microscope.
Chromatin immunoprecipitation quantitative PCR
The ChIP assay was performed as previously described[20]. Briefly, the cells were seeded in a 10 cm dish, and then the cells were cross-linked with 1% paraformaldehyde for 10 min. The cells were incubated with glycine to terminate the cross-linking. The cells were lysed on ice, and were centrifugated at 12000×g. The nuclei of the cells were resuspended in the lysis solution. The chromatin DNA of the cells was sonicated. 30 μl protein A-agarose beads were added to the mixture. Then the mixture was incubated with H3K27me3 antibody, EZH2 antibody, or control immunoglobulin G (IgG) for 2 h at 4 °C. Subsequently, the antibody/protein/DNA complex was eluted from the beads. The remaining proteins and RNAs were degraded with protease K and RNase A. The DNA was extracted with phenol-chloroform and purified with a PCR purification kit.
iTRAQ (isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tags)
Chondrogenic pellets were lysed by adding an appropriate amount of lysate solution and then heated at 95 ℃ for 10min. The mixture was centrifuged at 12000×g for 10 minutes, the supernatant was transferred to a new 1.5 ml tube. DTT (dithiothreitol) was added to the supernatant. The protein solution was transferred to a 10 K ultrafiltration tube for centrifugation. Then, the collected solution was discarded, and replaced with a new one. Trypsin was added into the ultrafiltration tube. The proteins were enzymatically hydrolyzed at 37 ℃ for 16 h. The enzymatically hydrolyzed polypeptide was concentrated and then labeled by iTRAQ regent. The iTRAQ-labeled samples were analyzed using Liquid Chromatography with Tandem Mass Spectrometry (Thermo Fisher Easy-nlC 1000 Liquid Chromatograph and Thermo Fisher Q Exactive).
Statistical analysis
All statistical analyses were processed using SPSS 10.0 software. Data are presented as mean ± SD. All the data are presented of three independent experiments. Comparisons between three groups were performed using one-way analysis of variance (ANOVA). Comparisons between two groups were performed using a two-sided Student’s t-test. For all tests, Statistical significance was set at p < 0.05.