title: cR_S6EOS_p18_R2
organism: Homo sapiens
description: Protocols: Conventional hPSC culture: Human pluripotent stem cells, iPS cells and re-primed human naive stem cells were routinely maintained in KSR/FGF medium: DMEM/F-12 (Sigma-Aldrich, D6421) supplemented with 10 ng/ml FGF2 (prepared in-house), 20% KnockOut Serum Replacement (KSR) (Thermo Fisher Scientific), 100 mM 2-mercaptoethanol (2ME) (Sigma-Aldrich, M7522), 1X MEM nonessential amino acids (Thermo Fisher Scientific, 11140050) and 2 mM L-glutamine (Thermo Fisher Scientific, 25030024) on irradiated mouse embryonic fibroblasts (MEFs). Cells were passaged as clusters by detachment with dispase (Sigma-Aldrich, 11097113001). To establish PB-Oct4EOS stable transfectants, 1 ug/ml of puromycin was applied for two passages (10 days) to transfected cultures on Matrigel (Roche). Naïve stem cell culture: Chemically reset and embryo-derived (HNES) human naive stem cells were propagated in N2B27 supplemented with t2iLGo (1μM CHIR99021, 1μM PDO325901, 10ng/ml human LIF and 2uM Go6983) with or without Rho-associated protein kinase (ROCK) inhibitor (Y-27632), and plated either on MEFs or tissue laminin (Sigma). Where indicated as tt2iLGo, CHIR99021 was used at 0.3 uM. For feeder-free culture, Geltrex (growth factor-reduced) or laminin were added to the medium at time of plating. Cells were cultured in 5% O2, 7% CO2 in a humidified incubator at 37C and passaged by dissociation with Accutase (Thermo Fisher Scientific, A1110501) or TrypLE (Thermo Fisher Scientific, 12605028) every 3-5 days. Cells were cryopreserved in CryoStem medium (Biological Industries, K1-0640).