Mouse E14 ESCs (male) (Hooper et al., 1987 (link)) were cultured in naïve or primed conditions as described below. Dnmt3a/b knock-out ESCs (male) (von Meyenn et al., 2016 (link)), Tet1-3 knock-out ESCs (male) (Hu et al., 2014 (link)) and Tdg knock-out ESCs (male) (Kunz et al., 2009 (link)) were grown in primed conditions as described below.
Naïve ESCs were cultured in serum free media with 2i inhibitors (Neurobasal, N2, B27, 103 U/ml LIF, 1 μM Mek inhibitor PD0325901 and 3 μM Gsk-3β inhibitor CHIR99021) without feeders at 37°C and 5% CO2.
Primed ESCs were cultured in serum containing media (DMEM 4,500 mg/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 15% fetal bovine serum, 1 U/ml penicillin, 1 μg/ml streptomycin, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, and 103 U/ml LIF ESGRO) without feeders at 37°C and 5% CO2.
For the 2i release experiment, ESCs in naïve culture conditions were washed with PBS before addition of media for primed ESC growth.
EB differentiation was performed by seeding 1000 primed ESCs per well in ultra-low attachment 96-well plates (Sigma-Aldrich) in primed ESC culture media without Lif.
Naïve ESCs were cultured in serum free media with 2i inhibitors (Neurobasal, N2, B27, 103 U/ml LIF, 1 μM Mek inhibitor PD0325901 and 3 μM Gsk-3β inhibitor CHIR99021) without feeders at 37°C and 5% CO2.
Primed ESCs were cultured in serum containing media (DMEM 4,500 mg/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 15% fetal bovine serum, 1 U/ml penicillin, 1 μg/ml streptomycin, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, and 103 U/ml LIF ESGRO) without feeders at 37°C and 5% CO2.
For the 2i release experiment, ESCs in naïve culture conditions were washed with PBS before addition of media for primed ESC growth.
EB differentiation was performed by seeding 1000 primed ESCs per well in ultra-low attachment 96-well plates (Sigma-Aldrich) in primed ESC culture media without Lif.
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