Ascorbic acid 3 phosphate

mOct4⁻ BM-MAPCs were obtained from the Stem Cell Institute at KU Leuven. Cells were cultured as described previously [29 (link)]. In short, dishes coated with fibronectin were used when splitting the cells every 48 hours. Medium contained 60 % low-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Sigma-Aldrich, St. Louis, MO, USA), 40 % MCDB-201 (Sigma-Aldrich), 1 × selenium-insulin-transferrin-ethanolamine (ITS; Sigma-Aldrich), 0.2 mg/ml LA-BSA and 0.8 mg/mL powdered bovine serum albumin (BSA; Sigma-Aldrich), 10⁻⁴ M ascorbic acid 3-phosphate (Sigma-Aldrich), 100 units of penicillin, 1000 units of streptomycin (Gibco®, Invitrogen, Carlsbad, CA, USA), 2 % fetal bovine serum (Gibco®, Invitrogen), 10 ng/mL human platelet-derived growth factor (R&D systems, Minneapolis, MN, USA), 10 ng/mL mouse epidermal growth factor (Sigma-Aldrich), 1000 units/ml mouse leukemia inhibitory factor (Esgro®, Millipore, Billerica, MA, USA) and 1 × chemically defined lipid concentrate (Gibco®, Invitrogen). Finally, β-mercaptoethanol (1 ×; Gibco®, Invitrogen) was added freshly to the media before sterilization with a 22-mm filter (Millipore).
The GL261 cell line, extensively used in mouse models of glioblastoma, was obtained from Dr S. Van Gool, KU Leuven. GL261 cells were cultured as described earlier [30 (link)].

Cells were cultured in twelve-well plates at a density of 1 × 10⁵ cells per well. To induce adipogenic differentiation, the medium was replaced with basal α-modification of Eagle's medium plus 100 nM dexamethasone, 50 μg/mL ascorbic acid 3-phosphate, and 50 μg/mL indomethacin (Sigma-Aldrich, St. Louis, MO, USA) for 2 weeks. The differentiated cells were fixed with 4% polyoxymethylene for 15 min before staining with 0.3% Oil Red O (Sigma-Aldrich) solution to evaluate adipogenesis. To induce chondrogenic differentiation, the cells were incubated in the presence of chondrogenic differentiation medium (Lonza, Basel, Switzerland) with recombinant transforming growth factor beta-3 protein (R&D Systems, Minneapolis, MN, USA) for 2 weeks. The induced cells were fixed with 4% polyoxymethylene for 15 min, followed by staining with 1% Alcian blue (Sigma-Aldrich). To induce osteogenic differentiation, the cells were incubated in the presence of 10 nM dexamethasone, 50 mg/mL ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate (all from Sigma-Aldrich) for 5 days. Then, the differentiated cells were fixed with 4% polyoxymethylene for 15 min, followed by alkaline phosphatase staining (Beyotime) to assess mineral deposition according to the manufacturer's instructions.

Cells were planked into plates containing basal complete medium and cultured to 80%–90% confluency. The basal medium was then replaced with the corresponding differentiation-inducing mixture. The medium was replaced with a DMEM with low glucose (Hyclone, New York, United States) complete medium containing 10 nM dexamethasone (Sigma, United States), 50 mg/mL ascorbic acid 2-phosphate (Sigma, United States), and 10 mM β-glycerophosphate (Sigma, United States) to induce osteogenic differentiation. Adipogenic differentiation induction medium contained 100 nM dexamethasone (Sigma, United States), 50 μg/mL ascorbic acid 3-phosphate (Sigma, United States), and 50 μg/mL indomethacin (Sigma, United States). Chondrogenic differentiation was induced using a chondrogenic differentiation medium (Lonza, Basel, Switzerland) supplemented with recombinant TGFb3 protein (R&D Systems, Minneapolis, MN, United States) (Li et al., 2020 (link)).