| Summary: | Summary: Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures. : Schroeder and colleagues show that GSK3 and MEK inhibition (2i) has several simultaneous effects on murine ESC cultures. They applied long-term time-lapse imaging to quantify cell death, proliferation, and Nanog expression dynamics in single ESCs. This demonstrated that 2i treatment of ESCs leads to more uniform and high Nanog expression due to both selective and inductive effects. Keywords: 2i, Gsk3, Mek, embryonic stem cells, pluripotency, Nanog, cell heterogeneity, continuous time-lapse imaging, single-cell tracking, cell fate
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