Human bfgf

Spheroid formation assays were performed to evaluate the regulation of cancer stem-like cells (CSCs). OSCC cells were dissociated and suspended in spheroid medium consisting of serum-free DMEM/F12 (Gibco, USA), human bFGF (20 ng/ml, Sino Biological Inc., China), human EGF (20 ng/ml, Sino Biological Inc., China), and B-27 supplement (Life Technologies, USA) in 6-well ultra-low attachment dishes (Corning, USA) under different treatment for 10 days [15 (link)]. The spheroid morphology was observed microscopically, and the number of spheroids was counted and compared.

All PSC culture and differentiation were performed as previously described (Zhao et al., 2018 (link)). Briefly, human embryonic stem cell line H1 (WiCell) was maintained in chemically defined Essential 8 medium (Stem Cell Technologies). Cells were passaged every 5–7 days and dissociated by 1 mg/ml Collagenase IV (Millipore). ESCs were induced when reaching 80–90% confluence in differentiation medium [α-MEM (Gibco) containing 2 mM l-glutamine (Gibco), 1× Insulin-Transferrin-Selenium-X (Gibco), 0.2% KnockOut SR XenoFree CTS (Gibco), 1 ng/ml human b-FGF, 20 ng/ml human GDNF (Sino Biological), 0.2% chemically defined lipid concentrate, and 200 μg/ml vitamin C (Sigma)] (Zhao et al., 2018 (link)). Medium was changed every day. No passage of cells was performed during differentiation. By day 12, most cells were PLZF+ SLCs. SLCs were collected for analyses or sorted for transplantation on day 12 of ESC differentiation.