Epidermal growth factor (egf)

Human brain tumor cell lines (U87, U138, U251, and T98G) were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and antibiotics under a humidified atmosphere containing 5% CO₂ at 37 °C. Patient glioblastoma cells were first cultured in DMEM/F-12 medium (Thermo Fisher Scientific) containing B27 supplement minus vitamin A (Thermo Fisher Scientific), epidermal growth factor, and basic fibroblast growth factor (20 ng/ml each; Wako Pure Chemical Industries, Osaka, Japan) to generate glioblastoma stem-like cells as neurospheres. These glioblastoma stem-like cells were then cultured in DMEM supplemented with 10% fetal bovine serum (FBS) to generate patient-derived glioblastoma cell lines or with all-trans retinoic acid (RA) for differentiation. To generate CD1d-transfected U87 cells, pCMV6-XLA4/hCD1d (OriGene Technologies, Rockville, MD, USA) was transfected into U87 cells using a cationic lipid-based transfection reagents, Lipofectamine 3000 (Thermo Fisher Scientific), according to the respective manufacturer’s instructions. Mock group cells were mock-transfected with Lipofectamine 3000 reagent only.

5 × 10³ EPCs under a dextran‐free and exposed to 10% dextran for 24 h were applied in methylcellulose‐containing M3236 medium (StemCell Technologies, Vancouver, Canada) with 20 ng/mL stem cell factor (Kirin), 50 ng/mL VEGF (R&D Systems), 20 ng/mL interleukin‐3 (Kirin), 50 ng/mL basic fibroblast growth factor (Wako), 50 ng/mL epidermal growth factor (Wako), 50 ng/mL insulin‐like growth factor‐1 (Wako), and 2 U/mL heparin (Ajinomoto) in a 3 cm‐dish. After 15 days in culture, the number of small or large type EPC colonies in a dish was counted under a phase contrast microscope.

Single cells were seeded in 96-well ultra low attachment plates (Corning Inc, Corning, NY, USA) at 100–1,000 cells/100 µl medium in each well. Cells were grown in serum-free DMEM/F12 medium (Gibco) supplemented with 20 ng/ml basic fibroblast growth factor (Wako), 20 ng/ml epidermal growth factor (Wako), and B27 supplement (Gibco). Cultures were supplemented with 25 µl of fresh medium every 3–4 days, and the number of spheres was counted on days 7 and 14. Microscopic images were obtained with a CKX41 inverted microscope and DP21 digital camera (Olympus, Tokyo, Japan).

The suitable duration of maturation in vitro in mouse GV oocytes was evaluated. The fresh COCs were cultured in 100 μL droplets of MEMα supplemented with 5% FCS, 10 ng/mL epidermal growth factor (Wako, Osaka, Japan), 2.1 mg/mL sodium bicarbonate, 75 mg/mL penicillin G salts, and 50 mg/mL streptomycin sulfate under liquid paraffin oil (Kanto Chemical, Tokyo, Japan) at 37.5°C in an atmosphere of 5% CO₂ in air for 8, 10, 12, 14, or 16 h. After the culture, the fresh COCs were used for IVF to evaluate fertilization and developmental ability.