These results indicate that there are important similarities between murine and primate ES cell-cycle characteristics. Like their murine counterpart, rhesus monkey ES cells show a reduced G1 phase, a predominance of hyperphosphorylated RB, an absence of DNA-damage checkpoint in G1, non cell-cycle dependent expression of cyclin E, and lack of dependency on persistent serum stimulation and active MEK signaling during cell-cycle progression.
Using the BrdUrd cumulative labelling technique, we estimated that the total cell-cycle duration of ORMES-1 cells was 20 hours. This technique, when applied to heterogeneous cell populations cycling at different rates, returns predominantly the cell-cycle duration of the slowest cycling cells [20
]. Time-lapse videomicroscopy was therefore used to provide the range of the cell-cycle duration values of individual ORMES-1 cells. This revealed that the population of ORMES-1 cells is heterogeneous with cells progressing through a complete cell-cycle in a time-window ranging from 12 to 21 hrs. The reason why ORMES-1 cells are heterogeneous in their cell-cycle duration is unclear. It is unlikely to result from mutations which would heritably influence the cell-cycle duration since careful examination of the cell-cycle durations of mother and daughter cells failed to reveal any positive correlation (data not shown). Interestingly, examination of the position of eGFP-expressing mother and daughter cells revealed extensive movements within the colony. These movements are likely to modify the microenvironment such as the accessibility of ES cells to extracellular matrix-associated cytokines provided by feeder cells. This could result in randomly influencing the cell-cycle duration of individual ES cells.
The most striking feature of the murine ES cell cycle, apart from it being unusually rapid, is a short G1 phase which represents approximately 15% of the total cell-cycle duration, or 2 hrs. The duration of the G1 phase of mouse ES cells was calculated by measuring the delay between release from mitotic block and onset of [3
H]-thymidine incorporation in synchronized cells [7
]. Conditions for propagating primate ES cells precluded large-scale amplification which would be necessary to synchronize them by mitotic block. Therefore, we used an indirect method based on the combination of cumulative BrdUrd labelling and PLM to calculate the durations of the individual phases of the cell-cycle in asynchronously growing cell populations. Despite monkey ES cells having a total cell-cycle duration up to twice as long as that of mouse ES cells, the relative duration of the G1 phase is similar in the two species. In both species, the hyperphosphorylated form of pRB is predominent [7
], further indicating that the vast majority of the cells are in the S and the G2/M phases of the cell-cycle. Taken together, these results indicate that a short G1-phase duration might be a common, and possibly universal, characteristic of ES cells, reflecting unique mechanisms of cell-cycle control which have been conserved throughout vertebrate evolution. Cells of the embryonal carcinoma (EC) stem cell line P19 also display a unusually short G1 phase [30
]. Pluripotent EC stem cells that can be induced to differentiate into mesodermal derivatives following treatment with retinoic acid (RA). Interestingly, upon synchronization by exposure to the reversible mitotic blocker, P19 EC cells were particularly vulnerable to RA while they progressed from mitosis to the next S-phase and, by contrast, became resistant to RA-induced differentiation when they reached S-phase [30
]. These observations suggest that the G1 phase corresponds to a window of increased sensitivity to differentiation signals. It is thus tempting to speculate that shortening of G1 phase might shield pluripotent cells from activities that induce differentiation [22
Which mechanism, therefore, underlies the rapid transit through the G1 phase in monkey ES cells? In mouse ES cells, there is compelling evidence that the G1 → S transition is not dependent on a functional cyclin D:Cdk4/6 → RB:E2F pathway [1
] and that rapid progression through the cell-cycle largely, if not exclusively, relies on constitutively active cyclin E:Cdk2 and cyclin A:Cdk2 complexes [6
]. Monkey ES cells also express cyclin E during all phases of the cell-cycle suggesting that ectopic cyclin E-Cdk2 kinase activity may also be a characteristic feature of primate ES cells. Interestingly, ORMES-1 cells did not express cyclin A in all phases of their cell-cycle, as opposed to mouse ES cells [6
]. Thus, mouse and monkey ES cells are likely to differ on the basis of the cell-cycle dependent regulation of cyclin A. The heterogenous cell-cycle duration that characterizes rhesus ES cells could result from discontinuous expression of cyclin A in a subset, or in all, Oct4+
Mouse ES cells fail to undergo cell-cycle arrest in the G1 phase in response to DNA damage [13
]. Both mechanisms involved in DNA-damage-dependent cell-cycle arrest – the chk1/chk2 kinase-dependent degradation of the Cdc25A phosphatase [27
] and the activation of the p53-p21 axis [31
] – are not functional in mouse ES cells [14
]. Here, we show that rhesus monkey ES cells are similarly impaired in their ability to respond to growth-arrest in G1 after treatment with ionizing radiation. Then the question arises as to how these cells maintain genomic integrity in the absence of a G1 checkpoint. Following DNA-damage both mouse and rhesus ES cells undergo apoptosis ([13
] and our unpublished data), suggesting that the function of p53 in ES cells is to trigger apoptosis in order to efficiently eliminate cells with damaged DNA. An alternative mechanism was recently suggested in mouse ES cells where, following DNA damage, transcriptionally active p53 repressed the activity of the nanog
promoter and irreversibly induced differentiation, thus eliminating DNA-damaged cells from the pool of undifferentiated stem cells [32
]. Whether a similar mechanism operates in primate ES cells remains to be determined.
We show that rhesus monkey ES cells, like their murine counterpart [8
], do not rely on persistent serum stimulation for cell-cycle progression. Noteworthy, serum was withdrawn for 24 hours, thus allowing all cells to progress through any serum-dependent checkpoint, regardless of their position in the cell-cycle at the onset of serum withdrawal. These data indicate that rhesus monkey ES cells can efficiently progress through the G1 to S phase transition in the absence of mitogenic signals. Rhesus monkey ES cells also appear not sensitive to pharmacological inhibition of MEK, a property shared with mouse ES cells [10
]. In the mouse, inhibition of MEK-dependent signalling facilitates self-renewal [33
] by sustaining expression of the expression of Oct-4 [34
]. It remains to be determined whether a similar mechanism operates in primate ES cells.