For sphere formation efficiency assay, a total of 5000 cells were seeded into ultra-low attachment six-well plates (BD Biosciences, San Jose, CA, USA). Cells were cultured in DMEM/F12 supplemented with B27 (Gibco), N2 (Gibco), 20 ng/mL EGF and 20 ng/mL basic fibroblast growth factor (bFGF), 10 ng/mL hepatocyte growth factor (HGF) and 1% methyl cellulose (Sigma-Aldrich) was added to prevent cell aggregation. Cells were cultured in 37°C f incubator with 5% CO2 for 2 weeks, and the numbers of spheres (diameter >75μm) were counted.

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