Cells were seeded as a single cell suspension in 6-well ultra-low attachment plates (Corning) at a density of 5000 cells/well in serum-free DMEM/F12 media (1:1) (Invitrogen), supplemented with basic fibroblast growth factor (10 ng/mL; Sigma-Aldrich), human epidermal growth factor (20 ng/mL; Sigma-Aldrich), and insulin (5 μg/mL; Sigma-Aldrich). Fresh media was added to each well every two days without removing the old media [46 (link)].
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