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All mESC lines were grown without feeder cells on gelatin-coated flasks (Millipore, 0.1%) in serum-containing ES cell medium (DMEM (Sigma), 15% FBS (PanBiotech), 0.1 mM β-Mercaptoethanol (Sigma), 1000 U/ml leukemia inhibitory factor (LIF, Merck)). mESCs were passaged every second day at a density of 4 × 104 cells/cm2 and medium was changed daily. Cells were differentiated by LIF withdrawal in DMEM supplemented with 10% FBS and 0.1 mM β-Mercaptoethanol at a density of 2 × 104 cells/cm2 on fibronectin-coated dishes (Merck, 10 μg/ml).

For the differentiation of mutant cell lines (Fig. 4g), cells were first adapted to 2i + LIF medium (ES cell medium with addition of 3 μM Gsk3 inhibitor CT-99021 (Axon Medchem) and 1 μM Mek inhibitor PD0325901 (Axon Medchem)) for at least five passages before undergoing differentiation via LIF withdrawal (see above). TX1072 XX and XO cells were grown in ES cell medium supplemented with 2i and differentiated by 2i/LIF withdrawal. Hek293T cells were cultured in DMEM supplemented with 10% FBS and passaged every 2 to 3 days.

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