Project description:The cell cycle of primed pluripotent mouse serum ESCs is characterised by a shortened G1-phase. Here we assessed the differences in cell cycle between serum ESCs and naive 2i ESCs.
Project description:The cell cycle of primed pluripotent mouse serum ESCs is characterised by a shortened G1-phase. Here we assessed the differences in cell cycle between serum ESCs and naive 2i ESCs.
Project description:The cell cycle of primed pluripotent mouse serum ESCs is characterised by a shortened G1-phase. Here we assessed the differences in cell cycle between serum ESCs and naive 2i ESCs.
Project description:Mouse 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.
Project description:Naive and primed pluripotent stem cells (PSCs) and germ cells express the Oct4 gene. The Oct4 gene contains two cis-regulatory elements, the distal enhancer (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type-specific and stage-specific manner. Here, we generated double transgenic mice carrying both Oct4-?PE-GFP and Oct4-?DE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. Using this dual reporter system, we successfully cultured pure populations of naive (GFP+RFP-) and primed (GFP-RFP+) PSCs. We found that GFP+RFP- cells were metastable (not naive) in serum-containing medium; stable naive pluripotent cells were observed in medium containing two inhibitors (Meki and GSKi) but lacked serum. Finally, we suggest that the activity of Oct4 DE and PE is regulated by the repressive histone marks and DNA methylation in a cell-type-specific manner.