Stem xvivo osteogenic adipogenic base medium

hMSCs were provided by the cell therapy department. They were obtained from residues of quality controls of bone marrow for transplantation harvested from adult donors after signing an informed consent. A fibroblast colony-forming unit (CFU-F) was used to optimize culture and expansion of hMSCs. Cells were cultured at 37°C in α-MEM (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS-Hyclone, Thermo scientific, Saint Aubin, France), 2 mM l-glutamine, 100 U/ml penicillin, streptomycin. Cells were used between passage 3 and 6 at which cells were >99% stained negative for CD34 and CD45 and positive for CD73 and CD90 (antibodies obtained from PharMingen). For osteogenic differentiation, hMSCs were plated at a density of 5 × 10³ cells/cm² and cultured in stem Xvivo Osteogenic/adipogenic base Medium (R&D systems, Lille, France), supplemented with 100 nM dexamethasone (Sigma, saint Quentin-Fallavier, France), 10 mM β-glycerophosphate (Sigma), and 50 μM ascorbic acid (Sigma). hMSCs were differentiated for 21 days in the absence or presence of 1 ng/ml TNF-α (R&D systems, Lille, France) and/or 50 ng/ml IL-17A (R&D systems) (23 (link), 24 (link), 31 (link)). Half of the medium was changed every 3 days.

Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington). Cells were digested for 4 h at 37°C and afterwards cultured in DMEM supplemented with 10% FCS (Biowest), 2 mM L-glutamine (Life Technologies), 0.5 mg/ml penicillin–streptomycin (Life Technologies), 50 μg/ml gentamycin (Life Technologies), and 20 uM β-mercaptoethanol (Sigma-Aldrich). After expansion of the cells, cells were seeded 30,000 cells/well in a 24-well plate for Alizarin red staining or 150,000 cells/well in a 6-well plate for mRNA analyses. To induce differentiation, cells were cultured in StemXvivo osteogenic/adipogenic base medium (R&D Systems) supplemented with 2 mM L-glutamine, 0.5 mg/ml penicillin–streptomycin, 20 μM β-mercaptoehtanol, 50 μM ascorbic acid (Sigma-Aldrich), 10 mM β-glycerophosphatase (Sigma-Aldrich), and 50 ng/µl IL-17A (R&D Systems). No samples were excluded from analyses.