Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

Abstract

Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs.

Figure 1

Effects of Olo II on cell proliferation, cell cycle progression, and cell morphology. DBA252 or D3 mESCs were seeded in 6-well plates (6.5 ×10⁴/well). After incubating for 24 h, cells were treated with different concentrations of Olo II for 24 h. A, DBA252 cell proliferation was determined by the absorbance at 630 nm, which correlates with the number of cells. The results are mean ± SD from three independent experiments. Similar results were obtained with D3 cells. B, Cell cycle profile analysis by flow cytometry. C, Microscopic analysis of cell and colony morphology of DBA 252 cells under a phase contrast microscope.

Figure 2

Effects of Olo II on cell cycle progression. A, The effect of Olo II on the cell cycle profiles at different time points. DBA252 cells were treated with 10 μM Olo II for the time points indicated and the cell cycle profile was analyzed by flow cytometry. B & C, DBA252 cells were synchronized by incubation with 50 ng/ml nocodazole for 16 h to arrest the cells at the G2M phases (Noc). Nocodazole was washed out with fresh medium and incubated for 1 h to allow cells to reenter the cell cycle (designated as 0 h). Afterward, the cells were cultured in the absence of (B) or in the presence of 10 μM Olo II (C) for the indicated time courses. The cells were collected at different time points and analyzed for cell cycle progression by flow cytometry.

Figure 3

Effects of Olo II on the expression of cycle regulators. DBA 252 cells were treated under the conditions described in Fig.1. A, The mRNA levels of cyclins E, A, and B were determined by RT-qPCR. The mRNA level of tested gene in control cells (0 μM) is designated as 100%. The results are means ± SD of three independent experiments. Statistical analysis was performed with Student t test. The difference was considered statistically significant when p<0.05 (*). B, Graph, the protein levels of cyclin B determined by flow cytometry. The protein levels were determined by fluorescence intensity from a flow cytometer. The value determined from control cells (0 μM, control) is designated as 1. Inset, the protein levels of cyclin B was analyzed by Western blot. β-actin were used as a loading control.

Figure 4

Rb phosphorylation and the effect of Olo II. A, Detection of hyperphosphorylated Rb (pRb-S780) by fluorescence microscopic analysis of DBA 252 cells labeled with anti-pRb antibodies and DAPI. The arrow indicates a single cell with intensive fluorescence staining of pRb, which is at the mitotic phase identified by its condensed mitotic chromosomes (DAPI). B, Flow cytometry analysis of pRb. Control (Con) represents the cells without first antibody incubation. pRb-S780 is detected as a shoulder peak (the cell population with the strongest fluorescence intensity, indicated by arrow). C, Olo II-induced dephosphorylation of pRb. Cells were treated with Olo II at different concentrations for 24 h. Dephosphorylation was indicated by the disappearance of the shoulder peak in the flow cytometry profiles. D, Dual analysis of Rb phosphorylation and cell cycle by flow cytometry. DBA 252 cells treated with Olo II for 24 h or control cells were doubly labeled with anti-Rb-S780 or anti-Rb-T807/811 antibodies to detect pRb and PI to detect DNA. Nearly all pRb positive cells are in G2/M phases and shown in the boxed areas.

Figure 5

Effect of Olo II on the expression of pluripotency markers. A, DBA 252 cells were treated with Olo II at different concentrations for 24 h. The mRNA levels of Oct4, Sox 2 and Nanog were determined by RT-qPCR. The results are means ± SD of three independent experiments. The mRNA level of tested gene in control cells (0 μM) is designated as 100%. Statistical analysis was performed with Student t test. The p<0.05 (*) was considered statistically significant. B&C, Determination of protein levels of Oct4 (B) and Nanog (C) were determined by flow cytometry. Oct4 was confirmed by Westernblot (B, inset. β-actin was used as a loading control). The fluorescence intensity determined from control cells (0 μM, control) is designated as 1.

Figure 6

Recovery of mESCs from Olo II treatment. A, DBA 252 cells were treated with 10 μM or 40 μM Olo II. The cells were then cultured in the fresh medium for 5 and 10 days, respectively, and photographed under a phase contrast microscope. B, Five day old EBs formed from control cells and cells recovered from Olo II treatment as shown in A. C, The cell cycle profiles of the cells shown in A were determined by flow cytometry. D, The differentiation capacities of EBs shown in B were determined by the expression of three differentiation markers. The mRNA level of each gene was determined by qRT-PCR and compared with its mRNA level in undifferentiated ESCs (designated as 100%). The results are means ± SD of three independent experiments. E, The same experiments were performed with D3 cells. EB and EB+Olo are EBs formed from control cells and cells recovered from Olo II treatment (10 μM), respectively. In both D and E, the differences in the expression of tested genes between ESCs and EBs are statistically significant (* p<0.05, with the exception of SMA in D), but the differences between control EBs and Olo II treated EBs are not.

Figure 7

A, cycloheximide (Chx) inhibits Olo II-induced de novo synthesis of Oct4 and Nanog. DBA 252 cells were treated with 20 μM Olo II and 20 μg/ml Chx alone or in combination as indicated for 24 h. B, Effect of MG-132 on Olo II-induced Oct4 and Nanog protein synthesis. DBA 252 cells were treated with 20 μM Olo II (Olo) and 3 μM MG-132 (MG) alone or in combination for the times indicated. Con represents the control experiments. The protein levels of Oct4 and Nanog were indicated by fluorescence (Flu) intensity via flow cytometry analysis.