Esgro complete plus serum free clonal grade 1i medium

The wild-type hybrid ESC line JB1, which is (JF1 × C57BL/6) F1, and the JB1-derived Zfp57^-/- ESC line were described previously^{24 (link),57 (link)}. ESCs were cultured under serum-free conditions on gelatinized tissue culture dishes and maintained in ESGRO Complete Plus Serum-free Clonal Grade 1i Medium (Merck-Millipore) in the presence of the Gsk3 inhibitor CHIR99021 (at 3 μM). The wild-type inbred 129 ESC line E14 and its derived Zfp57^-/- ESC line were cultured under standard feeder-free conditions as previously described^{24 (link)}. The wild-type and Zfp57^-/- JB1 and E14 ESCs were differentiated toward NPCs following the protocol described by Gaspard and collaborators (Fig. 1a)^{58 (link)}. In vitro corticogenesis of ESCs was started with plating at the optimal density of 7.500 ES cells/cm² and culturing in chemically defined default medium (DDM). From day 2 to 10 of culture, a Sonic hedgehog inhibitor (cyclopamine) was added at 1 μM concentration. From day 10 to 12, the medium was replaced with DDM only. The cells were cultured at 37 °C under an atmosphere of 5% CO₂.

Mouse wild-type, Zfp57-/- and Msh2-/- E14 ESCs were cultured under standard feeder-free conditions on gelatinized tissue culture dishes and maintained in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 100 uM 2-mercaptoethanol (Sigma), 1 mM sodium pyruvate, 2 mM l-glutamine, 1 × penicillin–streptomycin, 15% fetal calf serum (HyClone) and 10³ U/ml leukemia inhibitory factor (LIF, Millipore). The wild-type hybrid ESC line JB1, which is (JF1 × C57BL/6) F1, and the JB1-derived Zfp57-/- ESC line were described previously [7 (link), 36 (link)]. Wild-type and Zfp57-/- JB1 ESCs were cultured under standard feeder-free conditions on gelatinized tissue culture dishes with ESGRO Complete™ Plus Serum-free Clonal Grade 1i Medium (Merck Millipore) in the presence of 3 μM Gsk3 inhibitor CHIR99021. For H₂O₂ exposure, 0.5 mM H₂O₂ was diluted in PBS immediately before adding it to the media and cells were collected 30 min later. Cells were cultured at 37 °C under an atmosphere of 5% CO₂.