Isolation and In Vitro expansion of CSPCs
Fibronectin (FN, 10 mg/ml) (Gene Operation, 10-50-1101) in PBS was used to coat culture plate overnight at 4 degree for cell transplant. Rats were killed by overdose of chloral hydrate, and separated the joints and intercepted the joint part (keep the upper and lower femur and tibia about 1-2 cm) into the 50 ml Falcon tube containing PBS. The cartilage tissue scissors are about the size of 1-3mm fragments as shown in Figure 1A, transferred to a 15 ml centrifuge tube, adding 3 times volume of the amount of 0.25% trypsin, digest it at 37 degree for 30 min. Following complete medium termination of digestion, adding 300unit/ml type II collagenase (Worthington, 42B13253) dissolved in supernatant for overnight. The supernatant was removed and the complete medium containing 1% gentamicin was suspended and counted. The cell number was adjusted to 1×104/ml, and then transplanted into the FN covered culture plate and then separated cells can be observed on the surface of culture dish as shown in left panel of Figure 1B. After 2-3 days, replace fresh culture DF12 medium. On the day 7, cell colonies can be identified as shown in right panel of Figure 1B.
Growth and proliferation assay
After 72 hours of cell culture, cells were counted and collected with cold ethanol solution at 4 degree for overnight. Cells were finally suspended with PBST (containing a TritonX-100 of 0.2% PBS) by adding a 50 μg/ml RNA enzyme (RNase A) at 37 degree for 30-minute incubation. 50 μg/ml PI was added to cell suspension and incubated for 15 mins. After centrifuge above cells at 800 rpm for 5min, and then discard supernatant. Re-centrifugate and remove excess supernatant, afterwards using PBST to suspense cells and transfer to the flow tube before loading to BD FACS Calibur.
To characterize the CSPC identity, positive (CD73, CD105 and CD90) and negative (CD45 and CD34) cell-surface markers were detected by flow cytometry. All antibodies are listed as following: CD105-PE (Biolegend, 800504), CD90-FITC (Biolegend, 328108), CD73-APC (Biolegend, 344006), CD34-PE (Biogems, 06421-60-100), CD45-PE (BD, 555483). Harvest primary CSPC cells, prepare a single cell suspension in Cell Staining Buffer (Cat. No. 420201; BioLegend). One million of cells were pre-incubated with 5µL of Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301) per 100 µL of cell suspension for 5-10 minutes at room temperature. The tubes were centrifuged for 5 minutes at 350 g and supernatant discarded. Appropriately conjugated fluorescent antibodies were added and incubated on ice for 15-20 minutes in the dark. The cells were then washed 2 times with 2 mL of Cell Staining Buffer by centrifugation at 350 g for 5 minutes, and analysed by flow cytometry (Becton, Dickinson and Company, USA). Cell cycle analysis by quantitation of DNA content was one of the earliest applications of flow cytometry. For G2-M phase detection, cells were stained using DNA-binding dyes include propidium iodide (PI), and cells in G2-M will be approximately twice as bright as cells in G1.
RNA-sequencing and bioinformatics analysis
One million of CSPCs were seeded in T175 bottle and treated with 10 μM KGN for 3 days. Then, total RNA from CSPCs was extracted using TRIzol reagent (Thermo, 15596-026), according to the manufacturer’s instructions. RNA-sequencing and bioinformatics analyses were performed as previously described.
IL-6 ELISA assay of rat CSPC in culture
IL-6 secretion in culture supernatants were detected with ELISA kit (eBioscience, San Diego, CA) according to the manufacturer’s directions. Briefly, ELISA plates were coated with the capture antibody and incubated overnight at 4°C. Then, washed wells thoroughly and blocked for 1 h. Wells were incubated for 2 hrs with human IL-6 standards and supernatants from CSPC with or without KGN medium, which were diluted 100 times. After extensive wash steps, wells were incubated with detection antibody for 2 hrs at 4°C and then washed several times. Wells were incubated with Avidin-HRP for 30 min, washed thoroughly, incubated with substrate solution for 15 min, and then added stop solution. Finally, A450 values were measured using a microplate reader.
5% Agarose gel with identical wells were casted and rat-CSPC were seeded at a density of 1×106/10 μL. Cells were cultured in chondrogenic medium with either 10µM KGN or its solvent, DMSO as control. Medium were changed every other day for 28 days before harvested. Total RNA was extracted with TRIzol reagent (Life Technologies) following manufacture’s instruction. 500ng RNA was reverse-transcribed to cDNA using PrimeScriptTM RT reagent Kit (TaKaRa, Dalian, China). Specific genes were amplified with One Step SYBR PrimeScript RT-PCR Kit II (TaKaRa) on CFX96 real-time PCR detection system (BioRad, Hercules, CA, USA). The relative gene expression was normalized to GAPDH transcript level and calculated using the 2–ΔΔCt method.
STAT-3 siRNA knockdown assay
Rat-CSPCs were seeded in 6-well plate at 1×105 per well. Serum-free medium was given 2 hours pre-transfection. We designed a STAT-3 siRNA oligonucleotide (5'-AAC AUC UGC CUA GAU CGG CUA dTdT-3'; 3'-dTdT GUA GAC GGA UCU AGC CGA U-5') and had it synthesized by GenePharma Limited (Shanghai, China). Lipofectamine™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA) was used as the transfection reagent following manufacturer’s instructions with 200–600 nmol siRNA per well. 1 mL per well culture medium with serum was added 3 hours post-transfection. The siRNA transfection were repeated after 72 hours and cells were harvested 5 days after first transfection process. The control siRNA were set as the negative control group.
The animals were exposed to 1 mg/mL BrdU (Sigma-Aldrich, Steinheim, Germany) for 15 days, which is effective and safe for label-retaining cell studies. Oral BrdU administration was combined with exercise acclimatization, and rats were killed with excessive sodium pentobarbital (Apoteket Produktion & Laboratorier AB (APL), Sweden) at 30, 60 and 90 days after the start point. Two animals, not exposed to BrdU, were killed at day 30 and 90 as negative controls for BrdU technique.
Histology and immunohistochemistry
The knee joint samples were cut vertically at the cartilage defect sites with a thickness of 5 μm and stained with Hematoxylin and Eosin (H&E), Toluidine blue, Safranin O and Fast green. For immunohistochemical (IHC) analysis, the slides were incubated with primary antibodies Phospho-Stat3 (Tyr705) (Mouse mAb #4113, Cell Signaling Technology) and Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Rabbit mAb #8828, Cell Signaling Technology) at 4 °C overnight. Tissue sections were then incubated with 5% bovine serum albumin (BSA, Sigma) and incubated overnight with mouse-anti Collagen II (Developmental Studies Hybridoma Bank, USA), rabbit-anti Aggrecan (Proteintech, USA), rabbit-anti CD44, and mouse-anti CD105 (Abcam, USA) antibidies. Then, the sections were incubated with Alexa Flour 488/546-conjugated secondary antibody (Invitrogen, USA) for immunofluorescence or HRP-conjugated secondary antibodies (KPL, USA) for immunochemistry. The images were captured with a microscope (Olympus BX51, Japan).
Animals and cartilage regeneration experiments
All animal care and experimental procedures complied with the National Research Council’s guide for the care and use of laboratory animals, and all procedures were approved by the Institutional Animal Care and Use Committee and the Ethical Committee for Medical Research of Shenzhen University (Shenzhen, China).
The destabilization of medial meniscus (DMM) model in rats was created according to the previously reported protocol. Briefly, male rats weighted at 100 ± 10 g were anesthetized with 2% isoflurane in air using an anesthesia machine (RWD Life Science Co., Ltd., Shenzhen, China). After opening the capsule of knee joint, the tibial ligament of medial meniscus was cut off to destabilize the joint. Then, the knee joint capsule was closed immediately. Thirty days after surgery, 50μL KGN dissolved in saline at the concentration of 10 mM was injected into knee capsule every 7 days for seven weeks. Two weeks later, rats were executed followed by knee joint isolation for staining as shown in Figure 5A.
All data were expressed as the means ± SD. Differences between groups were evaluated by independent-sample t test or one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test in SPSS 22.0 software. A p-value less than 0.05 was considered to be statistically significant.