Mesencult acf

Early (P3-15) passage cells were seeded in standard MSC medium in 24-well plates (1.25x10⁴ cells/well) and grown to 90% confluence. The medium was subsequently replenished with either control (MesenCult-ACF Chondrogenic Differentiation Basal Medium (STEMCELL Technologies, Vancouver, Canada), 2mM L-glutamine) or differentiation (MesenCult-ACF Chondrogenic Differentiation Basal Medium, 2mM L-glutamine, MesenCult-ACF 20X Chondrogenic Differentiation Supplement) medium and incubated at 37°C/5% CO₂ for 18 days. On day 18, the cells were fixed in 10% neutral buffered formalin and stained with Alcian blue solution (8x, pH2.5) (Sigma-Aldrich, Sydney, Australia). Chondrogenesis was visualised by Alcian blue staining of filamentous glycosaminoglycans.

Characterization of colony forming unit-fibroblasts (CFU-Fs) and multi-differentiation capacity of GMSCs were performed as per previously described methods [28 (link), 31 (link)]. Isolated mononuclear cells were seeded at a density of 1 × 10³ per dish in 100-mm culture dishes containing the regular medium. Adherent cells were cultured for 18 days. The cultures were subsequently treated with PBS containing 0.1% Crystal violet and 4% paraformaldehyde (PFA). Cell clusters containing > 50 cells were counted as single colonies (CFU-F) under a Primovert microscope (Zeiss). GMSCs at the 3rd-5th passages were used for multi-differentiation capacity into adipocytes, osteoblasts, and chondrocytes. For adipogenic differentiation, GMSCs at 70%–80% confluence were cultured in an osteogenic medium containing α-MEM supplemented with 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μg/mL insulin (Cell Science & Technology Institute, Sendai, Japan), and 2 mM glutamine. For osteogenic differentiation, GMSCs at 70%–80% confluence were cultured in an osteogenic medium containing α-MEM supplemented with 2 mM β-glycerophosphate (Wako, Osaka, Japan) and 50 μg/mL ascorbic acid (Wako). For chondrogenic differentiation, GMSCs (5 × 10⁵ cells) were cultured in MesenCult™ -ACF, a chondrogenic differentiation medium (STEMCELL™ Technologies, Canada), according to the manufacturer’s instructions.

HA (M_w=20 kDa) was purchased from Lifecore Biomedical (Minnesota, USA). EGCG was purchased from DSM Nutritional Products Ltd (Heerlen, the Netherlands). Sorafenib tosylate was obtained from AbMole BioScience (Houston, USA). Amicon Ultra-15 centrifugal filters were purchased from Merck Millipore Corporation (Darmstadt, Germany). MesenCult-ACF and MethoCult H4434 Classic media were obtained from Stemcell Technologies (Vancouver, Canada). Dextran (M_w=70 kDa) and radioimmunoprecipitation assay (RIPA) buffer was purchased from Sigma-Aldrich (Minnesota, USA). Anti-HARE monoclonal antibody (mAb, MBL International, clone #34 − 2), anti-CD44 mAb (Thermo Fisher, clone Hermes-1) and control rat IgG (Thermo Fisher) were used as received. The following primary antibodies used for Western blotting were rabbit polyclonal unless otherwise stated: phospho-S6 (Ser235/236) and S6 (GeneTex, USA); phospho-STAT5a (Tyr694), STAT5a (C-term), phospho-ERK1/2 (Thr202/Tyr204) and ERK1/2 (Abcepta, USA); β-actin (mouse monoclonal, Proteintech, USA). Secondary antibodies (anti-rabbit/anti-mouse IgG-horseradish peroxidase conjugates) and CellTiter-Glo cell viability assay reagent were purchased from Promega (Madison, USA). RNeasy Mini kit (Qiagen, Germany), QuantiTect Reverse Transcription kit (Qiagen, Germany) and Micro BCA protein assay kit (Thermo Scientific, USA) were used per the manufacturer’s protocol.