Fgf 2

Human dental pulp stem cells (hDPSCs, Lonza, PT-5025, Basel, swiss) were cultured in M-DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37 °C in a 5% CO₂ incubator. For the experiments, hDPSCs were cultured in standard odontogenic induction medium, namely, M-DMEM supplemented with 10% FBS, antibiotics, 10 nM dexamethasone (Sigma–Aldrich, D4902, St Louis, MO, USA), 50 ng/mL BMP-2 (Sigma–Aldrich, B3555, St Louis, MO, USA), 20 ng/mL TGF-β1 (Bio Legend, 580704, San Diego, CA, USA), and 5 ng/mL FGF-2 (Bio Legend, 571504, San Diego, CA, USA) for 3 or 5 days at 37 °C in a 5% CO₂ incubator. For drug exposure, MS-275 (Apexbio, A8171, Houston, TX, USA) was initially dissolved in dimethyl sulfoxide (DMSO), then the concentration was adjusted with PBS, and it was added to the media. The medium was changed every 2–3 days. The hDPSCs from passages 3 to 5 were used for the experiments. DMSO (Sigma-Aldrich, D5879, St Louis, MO, USA) was added to the control at an additional 1/5000 to offset the effect of DMSO on the experiment.

Sorted cells were seeded into 96-well plates at a density of 5–10 × 10³ cells/well and cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific) and 10% fetal bovine serum (FBS, Sigma-Aldrich) supplemented with or without 20 ng/mL epidermal growth factor (EGF, BioLegend) or 20 ng/mL fibroblast growth factor-2 (FGF2, BioLegend) or both. The medium was changed every 3–4 days. For colony formation, 5.0 × 10³ cells were seeded into 6-well plates using MethoCult (STEMCELL Technologies Inc.). Consecutively, 2 to 3 weeks after the start of culture, the number of proliferated colonies was counted.