Abstract
SummaryMouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.
Highlights
One characteristic of embryonic stem cells (ESCs) is that they proliferate at an unusually rapid pace, with a doubling time of 12–14 hr
Recent studies have indicated that ESCs cultured in defined medium in the presence of two small-molecule inhibitors, PD0325901 and CHIR99021, more closely reflect pluripotent ESCs from the inner cell mass, whereas ESCs cultured in serum are more similar to pluripotent cells from post-implantation embryonic stages (Marks et al, 2012; Nichols and Smith, 2009; Weinberger et al, 2016)
To assess the global effect of the 2i culture conditions on the mouse ESC cell cycle, we used bromodeoxyuridine (BrdU) incorporation and propidium iodide (PI) staining in combination with flow cytometry to determine the distribution of cells over the different phases of the cell cycle
Summary
One characteristic of embryonic stem cells (ESCs) is that they proliferate at an unusually rapid pace, with a doubling time of 12–14 hr. Active kinases and the absence of CDK-inhibitor proteins ensure rapid progression into S-phase, resulting in an extremely short G1-phase. This model is, based on studies performed in ESCs grown in serum (hereinafter termed serum ESCs). Recent studies have indicated that ESCs cultured in defined medium in the presence of two small-molecule inhibitors, PD0325901 and CHIR99021, (hereinafter termed 2i ESCs) more closely reflect pluripotent ESCs from the inner cell mass (often referred to as ground-state), whereas ESCs cultured in serum are more similar to pluripotent cells from post-implantation embryonic stages (Marks et al, 2012; Nichols and Smith, 2009; Weinberger et al, 2016). Our analysis of the cell cycle in 2i ESCs indicates that G1 control in ground-state pluripotent ESCs is distinct from that in serum ESCs
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