β mercaptoethanol

HEK293 and HeLa cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMED; Gibco) supplemented with 10% FBS (Hyclone) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco). For MEFs, the cell culture medium consists of 10% FBS (Hyclone), 1 mM sodium pyruvate (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco). For iPSC generation cell culture media, 1000 U/ml LIF (Millipore) were added based on MEF media. iPSC stable clones were maintained on mitomycin C-treated MEF feeder cells in knockout DMEM medium (Gibco) supplemented with 15% KSR, 1 mM sodium pyruvate (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 1000 U/ml LIF (Millipore). CGR8 ES cells were cultured on gelatin-coated dishes, in GMEM medium supplemented with 10% FBS (Millipore), 1 mM sodium pyruvate (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 1000 U/ml LIF (Millipore). P19 EC cells were maintained on gelatin-coated dishes, in α-MEM medium supplemented with 10% FBS (Hyclone) and 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco).

Primed-hiPSCs of both patients were maintained on irradiated mouse embryonic fibroblasts (MEFs) in DMEM/F12 GlutaMAX supplemented with 20% KSR (thermofisher), 1X non-essential amino acids, 50 µM β-mercaptoethanol (thermofisher), and 10 ng/ml of bFGF (thermofisher). Media were replaced every day. Cells were passaged as clumps every 4 to 6 days using trypsin and 10 µM of ROCK inhibitor (TEBU-Bio). Primed-like hiPSCs were converted into a naive state by cultivating them on MEFs feeders cells in knock-out DMEM medium (thermofisher) supplemented with 20% KSR (thermofisher), 2 mM L-Glutamine (thermofisher), 0.1 mM MEM NEAA (thermofisher) and 50 µM β-mercaptoethanol (thermofisher) and containing basic fibroblast growth factor (b-FGF, thermofisher), human leukemia inhibitory factor (LIF, Peprotech), transforming growth factor-β1 (TGFβ, TEBU-Bio), and four small inhibitory molecules (4i), 3 μM CHIR99021 (R&D systems), 1 μM PD0325901 (R&D systems), 5 μM SB203580 (R&D systems), and 5 μM SP600125 (R&D systems)^{27 (link)}. Cells were maintained in 4i hiPSC medium for two weeks replacing daily with fresh medium. The growing naive-hiPSCs colonies were picked up and passaged every 2–3 days as single cells with accutase. For each passage, 10 μM of ROCK inhibitor (TEBU-Bio) were used.

ES cells were cultured on gelatin-coated in ES-Medium consisting of DMEM + GlutaMAX (Gibco: 31966-021) supplemented with 2 mM L-glutamine (Gibco: 25030-024), 50 U/ml penicillin/streptomycin (Gibco: 15140-122), 1× nonessential amino acids (Gibco: 11140-035), 1× essential amino acids (Gibco: 11130-036), 0.1 mM β-Mercaptoethanol (Gibco: 31350-010), 15% FCS (Gibco: 10270-106), LIF (1000 U/ml; Sigma–Aldrich: ESG1107), and 2i (3 µM CHIR-99021, Cayman Chemical: 13122; 1 µM PD0325901, BioGems: 3911091). Induced TS cells reprogrammed from 5 Factor ESCs were cultured in TSC Medium consisting of advanced RPMI 1640 (Thermo Fisher Scientific: 11875-093), 20% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate (Thermo Fisher Scientific: 11360070), 50 U/ml penicillin/streptomycin, and 0.1 mM β-Mercaptoethanol. A mixture consisting of 70% of feeder-MEF conditioned TSC medium and 30% fresh, unconditioned TSC medium was prepared and supplemented with FGF4 (Reliatech: #100-017 L; 25 ng/ml), Heparin (Sigma–Aldrich: #H3149-10KU; 1 µg/ml) for the culture of iTSCs. Induced XEN cells reprogrammed from iGATA6 ESCs were cultured on gelatin-coated culture dishes in XEN cell culture medium, consisting of RPMI 1640, 15% FCS, 2 mM L-glutamine, and 50 U/ml penicillin/streptomycin.