Esgro mlif

ES cells were grown on a layer of feeder cells in DMEM-ES (Sigma D6546) medium supplemented with 15% FCS, 1 mM sodium pyruvate, 1 mM glutamine, 100 units per ml penicillin and 100 μg ml⁻¹ streptomycin, 0.15 mM MTG, 25 mM HEPES buffer, 1 × non-essential amino acids (Sigma) and 10³ units per ml LIF (ESGRO mLIF, Millipore ESG1107). Medium was changed every day. Before in vitro differentiation (IVD), the cells were grown without feeder cells for two passages and the medium used for the last passage was IMDM-ES (Sigma I3390).

E14tg2a mES were obtained from American Type Culture Collection (ATCC CRL-182) and the hybrid 129/Sv:CAST/EiJ mES were obtained from Jop Kind’s group (Hubrecht Institute). Both lines were tested for mycoplasma contamination. Cells were grown on 0.1% gelatin in ES cell culture media; DMEM (1×) high glucose + glutamax (Gibco), supplemented with 10% FCS (Greiner) 100 µM β-mercaptoethanol (Sigma), 100 µM Non-essential amino acids (Gibco), 50 µg/mL Pen/Strep (Gibco), and 1000 U/mL ESGRO mLIF (Millipore). Cells were split every 2 days and media changed every day. Cells were harvested before FACS by washing 3 times with 1× PBS with calcium and magnesium and incubated with 0.05% Trypsin (Life Technologies). Cell were resuspended in ES culture media and cell clumps were removed by passing the cells through a BD Falcon 5 mL polystyrene tube with a filter top.

Mouse ESCs (E14Tg2a; ATCC, CRL-1821) were maintained on gelatin (Sigma, G1890)-coated plates in the ESGRO complete plus clonal grade medium (Millipore), as previously described^{24 (link),60 (link)}. For experiments, ESCs were cultured on gelatin-coated plates in the M15 medium: DMEM (Thermo Fisher, 11965084) supplemented with 15% FBS (Gemini, 100–125), 10 μM 2-mercaptoethanol (Sigma, M3148), 0.1 mM nonessential amino acids (Thermo Fisher, 11140050), 1x EmbryoMax nucleosides (Millipore, ES-008-D), 1 U/ml of ESGRO mLIF (Millipore, ESG1107).

Mouse embryonic stem cells (E14Tg2a, ATCC) were maintained on 1% gelatin-coated plates in the ESGRO complete plus clonal grade medium (Millipore), as previously described^{72 (link),73 (link)}. Embryonic stem cells (ESCs) were cultured on gelatin-coated plates in DMEM (Invitrogen) supplemented with 15% FBS, 1X-Gultamax (Gibco), Na-Pyruvate (Gibco), 10 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids (Gibco), 1U/ml of ESGRO mLIF (Millipore), and 2i inhibitors (MEK inhibitor PD0325901, Gsk3b inhibitor (CHIR99021 – Stem Cell technology) in naive (or +2i) condition. In epiblast stem cells (EpiSCs) or primed condition, cells were cultured in chemically defined medium (IMDM and F12-Invitrogen) supplemented with 2%-BSA (Sigma), Insulin (Roche), Transferrin (Roche), CD-lipid concentrate (Gibco), FGF2 (R&D), and Activin-A (R&D) (Ref). For spontaneous differentiation (SD), cells were cultured in gelatin-coated plate in previously described DMEM-FBS media without LIF and 2i-inhibitors. Cell lines were continuously monitored under a microscope and confirmed to be free of mycoplasma contamination by using a MycoAlert mycoplasma detection kit (Lonza) and DAPI staining.