Recombinant human egf

HEK293T cells were obtained from the RIKEN Cell Bank (Saitama, Japan) and cultured in DMEM (FUJIFILM Wako Pure Chemical) supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO₂ (42 (link)). The patient-derived OS cell line 143B was obtained from the ATCC (Manassas, USA) and cultured in adherent medium containing DMEM supplemented with 10% FBS, 110 μg/mL sodium pyruvate (FUJIFILM Wako Pure Chemical), and 1% penicillin/streptomycin. Both cell types were cultured in tissue culture dishes (SARSTEDT) to ensure optimal adherence and expansion. To enrich stem-like cells, 143B cells were harvested using trypsin (BD Bioscience) and EDTA (FUJIFILM Wako Pure Chemical), then cultured in osteosphere medium containing DMEM/F12 (FUJIFILM Wako Pure Chemical) supplemented with 20 ng/mL recombinant human EGF (FUJIFILM Wako Pure Chemical), 20 ng/mL recombinant human basic FGF (FUJIFILM Wako Pure Chemical), B27 supplement without vitamin A (Gibco), GlutaMAX (Thermo Fisher Scientific), and 1% penicillin/streptomycin. Under these conditions, the cells were incubated in Ultra-Low Attachment Surface culture dishes (Corning). To assess the differentiation potential of OSCs, the cells were transferred from osteosphere to adherent medium, and from Ultra-Low Attachment Surface to tissue culture dishes, to promote adherence and differentiation.

HEK293T cells and HEK293GP cells were purchased from the RIKEN Cell Bank and Takara Bio, respectively. These cells were cultured at 37°C in a 5% CO₂ incubator and maintained in DMEM supplemented with FBS. Human patient-derived GBM cell lines TGS-01 and TGS-04 were established as described previously (23 (link)). The use of these human materials and protocols were approved by the Ethics Committees of Gifu Pharmaceutical University (Gifu, Japan) and the University of Tokyo (Tokyo, Japan). These cells were confirmed as GSCs and cultured in neurosphere medium containing DMEM/F12 (FUJIFILM Wako Pure Chemical) supplemented with recombinant human EGF at 20 ng/mL (FUJIFILM Wako Pure Chemical), recombinant human basic FGF at 20 ng/mL (FUJIFILM Wako Pure Chemical), B27 supplement without vitamin A (Gibco), and GlutaMAX (Gibco).

Glucose, recombinant human EGF, and erlotinib were purchased from FUJIFILM Wako (Osaka, Japan). Recombinant human PRSS8 was created in our laboratory as described previously^{11 (link)}. Cycloheximide (CHX) was purchased from Nacalai Tesque (Kyoto, Japan). MG132 was purchased from ChemScence (NJ, USA).