Gap 1 phase length and mouse embryonic stem cell self-renewal

Abstract

In somatic cells, the length of the G1 phase of the cell cycle is tightly linked to differentiation, and its elongation can drive differentiation in many cases. Although it has been suggested that the situation is very similar in embryonic stem cells (ESCs), where a rapid cell cycle and a short G1 phase maintain the pluripotent state, evidence has been contradictory. Here we show that, in murine ESCs, elongation of the cell cycle and elongation of G1 are compatible with their pluripotent state. Multiple methods that lengthen the cell cycle and that target cyclin-dependent kinase, retinoblastoma protein, and E2F activity all fail to induce differentiation on their own or even to facilitate differentiation. The resistance of murine ESCs to differentiation induced by lengthening G1 and/or the cell cycle could allow for separate control of these events and provide new opportunities for investigation and application.

Fig. 1.

Overexpression of p21 and p27 elongate the cell cycle in G1 but are insufficient to induce loss of pluripotency. (A) Flow cytometry analysis of p21 and p27 overexpression showing cell-cycle stage distribution of cells expressing high levels of cotransfected EGFP. Quantification of the cell-cycle elongation in G1 is shown below the histograms. (B) Real-time time-lapse microscopy measurements of cell-cycle times of WT cells vs. cells with G1 lengthened by CDK inhibitor overexpression. (C) Day 4 Oct4 reporter, Nanog reporter, and SSEA-1 levels remain constant in p21- and p27-overexpressing cells. The mCherry, p21, and p27 distributions are gated for expression of the fusion protein (i.e., mCherry⁺, p21⁺, p27⁺). The ESC lines used are Oct4-GiP, Nanog-GFP, and J1. In the combined retinoic acid (RA) plus p21/p27 (RA + p21 and RA + p27) treatments, RA was added 24 h after the addition of p21/p27. (D) Quantitative RT-PCR analysis of lineage markers for FACS-sorted cells expressing mCherry, p21, or p27. For reference, samples differentiated by LIF withdrawal for 4 d are provided.

Fig. 2.

Effects of CDK2 and cyclin A knockdown on the cell cycle and pluripotency. (A) Effect of knockdown on proliferation rate and cell-cycle structure by DNA content. (B) Flow cytometry analysis of Oct4 reporter, Nanog reporter, and SSEA-1 levels at 4 d after siRNA treatment.

Fig. 3.

Treatment of ESCs with mCherry-tagged dnCDK2, dnE2F1, and Rb constructs induce cell-cycle effects but not differentiation. (A) Effect of overexpressing mutants on cell-cycle structure by DNA content. (B) Growth curves of treated cells over a 3-d period. (C) Analysis of Oct4 reporter, Nanog reporter, and SSEA-1 levels by flow cytometry after 4 d of overexpression.

Fig. 4.

Pharmacological CDK inhibitors can induce multiple effects at sublethal concentrations. (A Left) Proliferative dose–response curves of two small-molecule inhibitors: CVT-313, a CDK2 inhibitor, and roscovitine, a broader CDK inhibitor. (Right) Percentage of cells in G1, as determined by FACS cell-cycle profile. (B) Flow cytometry analysis of pluripotency marker levels after 10 d of treatment with sublethal concentrations of inhibitors. The CDK inhibitors induced a minor decrease in fluorescence marker levels (demarcated by a shift in arrow position over the major peak). Reductions in marker levels of magnitude suggestive of differentiation could be found only in a fraction of cells in roscovitine-treated samples. (C) Morphologies of ESCs treated with sublethal concentrations of inhibitors for an extended period (10 d). CVT-313 did not induce noticeable changes, but roscovitine did in some cells.

Fig. 5.

Rb dephosphorylation generated by p21 and p27 overexpression. (A) Schematic of Rb inactivation during the G1/S transition in somatic cells. In mouse ESCs, CDK activity is high, and Rb appears to be largely in the hyperphosphorylated form. (B) Gel-shift analysis shows that the active, hypophosphorylated form of Rb accumulates from p21/p27 overexpression.

Fig. 6.

Effects of modulating G1 length on the kinetics of Nanog reporter loss during LIF withdrawal. Nanog-GFP reporter ESCs were first transfected for 24 h, then LIF was removed to begin kinetics measurement. Values indicated are the means of GFP fluorescence in the Nanog-GFP line for the construct-expressing population after background correction. The mCherry curve is reproduced in light blue in each subsequent graph for reference.