Investigating BMP2 Effects on hESCs

HESCs were grown in DMEM/F12 medium without phenol red (Sigma-Aldrich, St. Louis, MO, United States of America), added with 10% charcoal dextran-treated fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, United States of America), 1% ITS - Premix (BD Biosciences, San Jose, CA, United States of America) and 5 ng/mL puromycin (Thermo Fisher Scientific, Ottawa, ON, CAN). Every culture was kept at 37 °C in an incubator with 5% CO₂. HESCs were cultivated for a day after being seeded at a density of 4 × 10⁵ cells per plate in 60-mm tissue culture dishes with full culture media. HESCs were treated with BMP2 (0, 10, 25, or 50 ng/ml) for the concentration-dependent research or with 25 ng/mL BMP2 for the time-course study after serum deprivation in DMEM/F12 media without FBS for 18 h. For the concentration-dependent investigation, cells were taken at 24 h, while for the time-course study, cells were taken at 3, 6, 12, 24 h, and 48 h.