Monitoring Pluripotency Dynamics in ESCs

The Rex1GFPd2 ESCs used in the current study^{10 (link)} were used between passage 15 and passage 25, and were cultured on 0.1% gelatin at all times in 2i medium, described in^{12 (link)}, which is N2B27 medium (Invitrogen) supplemented with MEK inhibitor (1 μM PD0325901) and GSK3 inhibitor (3 μM CHIR99021) plus Leukemia inhibitory factor (LIF). For priming/differentiation protocol, ESCs were passaged and seeded directly into N2B27 medium at a concentration of 4E5 cells/mL. The GFP signal was regularly analysed by flow cytometry using a CyAn ADP analyser (Beckman Coulter, Brea, CA, USA) to monitor Rex1 expression in the cells, and we titrated the time point of 24h after seeding in N2B27 to be T ESCs, and 48h after seeding in N2B27 to be P ESCs (Fig. 1a and Fig. S1).