Feeder-free Culture and Differentiation of Mouse Embryonic Stem Cells

E14 mES cells were cultured under feeder-free conditions on 0.5% gelatin-coated dishes with mouse embryonic stem cell (ESC) medium consisting of knockout DMEM (Gibco), 10% FBS (Gibco), 2 mM L-glutamine (Merck Millipore), 1% nonessential amino acids (Merck Millipore), 0.3 mM β-mercaptoethanol (Nacalai Tesque), and 1000 U/ml LIF (Merck Millipore) at 37 °C and 5% CO₂. For the differentiation assay, mES cells were cultured in mouse ESC medium without LIF and supplemented with 0.5 μM retinoic acid25 (link). To change from serum + LIF to 2i + LIF conditions, E14 mES cells cultured with serum + LIF were subcultured in 2i + LIF medium (N2B27 medium [Life Technologies] with 1 μM PD0325901 and 3 μM CHIR99021 [Stemgent], together known as 2i medium, and 1000 U/ml LIF). After several passages, the adapted cells were used for study. For the differentiation assay under 2i + LIF conditions, mES cells were cultured without 2i + LIF20 (link)21 (link).