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发表于 2009-3-5 00:10 |只看该作者 |倒序浏览 |打印

作者：Anne-Catherine Fluckigera,b, Guillaume Marcya,b, Mlanie Marchanda,b, Didier Ngrec,d,e, Franois-Loc Cossetc,d,e, Shoukhrat Mitalipovf, Don Wolff, Pierre Savatiera,b,g, Colette Dehaya,b,g作者单位：a Institut National de la Sant et de la Recherche Mdicale (INSERM), U, Cerveau et Vision, Department of Stem Cells and Cortical Development, Bron, France;b Universit Claude Bernard Lyon I, IFR Institut Fdratif des Neurosciences, Bron, France;
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      【摘要】5 u7 o0 x! ^& U9 e7 M2 C% ^" M" z- J
   Using flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non-phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth-arrest in G1 after -irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent. ' X3 v3 P4 a8 W3 c8 V0 Z6 L
      【关键词】 Rhesus embryonic stem cells Cell cycle Stemness
   INTRODUCTION

There is accumulating evidence that cell cycle control in mouse embryonic stem cells (ESCs) and somatic cells differs in that the mouse ESC cell cycle is not dependent on a functional p16ink4a/cyclin D:cyclin-dependent kinase (Cdk) 4/retinoblastoma protein (pRB):E2F pathway . Taken together, these findings indicate that the mitotic cycle of ESCs largely escapes from external mitogenic stimuli and instead relies largely on intrinsic factors.
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A crucial question is whether the unusual cell cycle characteristics of mouse ESCs are shared by primate ESCs. Here, we show that rhesus monkey ESCs display most of the cell cycle characteristics of mouse ESCs, including shortening of G1 phase, non-phasic expression of cyclin E, predominance of hyperphosphorylated RB, lack of dependence on serum stimulation and MEK signaling, and absence of a DNA damage checkpoint in G1. We thus propose that these unique cell cycle characteristics are critical, and possibly universal, features of ESCs.. x' t; ~4 O, V+ k2 A: q
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MATERIALS AND METHODS! |# }3 w. T: e1 V
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Rhesus Monkey ESC Culture

Procedures for rhesus ESC line Oregon Monkey ES (ORMES)-1 and ORMES-6 cell cultures have been described previously . Briefly, ORMES ESCs were grown at 37¡ãC in 5% CO2 atmosphere on mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 20% fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com), 0.1 mM ß-mercaptoethanol (Sigma, St. Louis, http://www.sigmaaldrich.com), 1% nonessential amino acids (Invitrogen), and 2 mM L-glutamine (Invitrogen). ESC colonies were passaged every 4¨C5 days by treatment with 1 mg/ml collagenase IV (20¨C30 minutes at 37¡ãC), followed by mechanical dissociation.
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PD98059 and U0126 (Sigma) were dissolved in dimethyl sulfoxide (Sigma) and added to the medium at a concentration of 25 and 10 µM, respectively., k# i3 X, f. U) h) ~4 A

Lentiviral Vector Production and Infection of ESCs* n( H) S( M0 w% N$ h5 f
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R4SA-EFs-EGFP-W, a simian immunodeficiency virus (SIV)¨Cbased vector, harbors the sequence encoding the enhanced Green Fluorescent Protein (eGFP) driven by the minimal version of the human elongation factor 1 promoter (EFs). It was generated by replacing the ClaI/XhoI restriction fragment containing the cytomegalovirus (CMV)-GFP cassette in pSIV-gaMES4 . Briefly, 293T cells were transfected with a mixture of DNAs containing 10 µg of the pGRev plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G) envelope, 10 µg of pSIV3  plasmid encoding the gag, pol, tat, and rev proteins, and 13 µg of the R4SA-EFs-EGFP-W plasmid, using the calcium phosphate precipitation technique. The following day, cells were refed with 7 ml of DMEM and further cultured for 24 hours. The supernatant was then collected, cleared by centrifugation (3000 RPM, 15 minutes), and passed through a 0.8-µM filter. Prior to infection, ORMES-1 cells were treated with 1 mg/ml collagenase IV for 3¨C5 minutes at 37¡ãC. Clumps of undifferentiated ORMES-1 cells were isolated by mechanical dissociation and transferred to fresh medium (500 µl) containing SIV-eGFP. Cells were incubated for 4 hours at 37¡ãC before being replated on fresh feeder cells.

Time-Lapse Videomicroscopy Recording of Cell Division

ORMES-1 cells were grown in a 5% CO2 atmosphere at 37¡ãC for 4¨C5 days in an incubating chamber (PeCon, Erbach, Germany, http://www.pecon.biz/pecon/index.htm) placed on a Leica DMIRBE inverted microscope stage (Leica, Heerbrugg, Switzerland, http://www.leica.com). Observations of individual eGFP  cells were made with a 10x objective under halogen illumination. Twenty fields were scanned per coverslip per hour using Metamorph software (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com). Subsequent analysis of the movies allowed estimation of the cell cycle length of individual eGFP  cells as measured by the time elapsed between two successive mitoses .

Cell Cycle Kinetics Measurements
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S phase and G1  G2 M phase durations can be derived from bromodeoxyuridine (BrdU) cumulative labeling experiments  was used to determine the G2/M duration. Forty-eight hours after plating, cultures were pulse-labeled for 1 hour with BrdU (20 µg/ml). For both procedures, three independent experiments were performed with the ORMES-1 cell line and two experiments with ORMES-6. Each time point was repeated on two sister cover-lips. After survival periods, cultures were fixed in 2% PFA and processed for the detection of BrdU incorporation and Oct-4 expression. Cells were counterstained with Hoechst 33258 for 3 minutes to allow the identification of mitotic figures.

In Situ Immunofluorescence/ X- M- G9 N  J" T/ z; W, M
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ORMES cells were fixed in 2% PFA in phosphate-buffered saline (PBS) at 4¡ãC for 1 hour. Immunohistochemistry was performed by a two-step procedure. Cells were permeabilized in Tris Buffer Saline (TBS) 0.2% Triton X-100, 0.1% Tween-20, for 20 minutes. Non-specific binding was blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 1 hour at room temperature (RT). To detect Oct-4 protein, cells were first incubated for 1 hour at 30¡ãC with mouse monoclonal antibody anti-Oct-4 (C-10: sc5279) from Santa Cruz Biotechnology (Santa Cruz, CA, http://scbt.com) (1:1000 in DAKO-diluent). Cyclin E and cyclin A, respectively, were detected using C-19 (sc-198) and H-432 (sc-751) antibodies (1:100; Santa Cruz Biotechnology) incubated overnight at 4¡ãC. Ki67 was detected using phycoerythrine-conjugated anti-human Ki67 monoclonal antibody (ref. no. 36525; BD Biosciences, San Diego, http://www.bdbiosciences.com). After three rinses in TBS, cells were exposed either to affinity-purified goat anti-mouse or goat anti-rabbit immunoglobulin G (IgG) conjugated either to indocarbocyanine or to cyanin (Cy3 and Cy2, respectively; Jackson ImmunoResearch Laboratories) for 1 hour at RT followed by nuclear staining with 1 ng/ml Hoechst 33258 for 3 minutes. After three rinses in TBS, coverslips were mounted on slides. To detect both Oct-4 expression and bromodeoxyuridine (BrdU) incorporation, cells were first treated to reveal Oct-4 expression as described above. DNA was then denatured by incubation in 2N HCl, followed by a wash in a borate buffer (pH 8.5). Non-specific binding was blocked with 10% normal goat serum. BrdU incorporation was revealed by incubation with Alexa 488-conjugate anti-BrdU antibody (1:50 in DAKO-diluent) (Molecular Probes Inc., Eugene, OR, http://www.probes.invitrogen.com) for 2 hours at RT. Nuclei were counterstained with Hoechst 33258. Coverslips were examined using an oil objective microscope under UV light to detect fluorescein isothiocyanate (FITC) (filter 450¨C490 nm), indocarbocyanine 3 (filter 550¨C570 nm), and Hoechst 33258 (filter 355¨C425 nm). Cover-slips were scanned at regular spacing with a grid corresponding to a field of 0.128 mm2. One hundred to 150 fields were observed per coverslip. Immunopositive cells were plotted onto a chart using the Mercator software (Explora Nova, La Rochelle, France, http://www.exploranova.com).9 `# u+ F9 T1 o0 _( [. z
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Flow Cytometric Analysis of Cell Cycle Distribution
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Single-cell suspensions of ORMES cells were obtained by treatment with 1 mg/ml collagenase IV (37¡ãC for 20¨C30 minutes) followed by treatment with 0.1% trypsine (37¡ãC for 3 minutes). For DNA content analysis, cells were fixed in 70% ethanol, rehydrated in PBS, treated for 30 minutes with RNase A (1 mg/ml) and for 5 minutes with propidium iodide (PI) (1 µg/ml). Fluorescence intensity was determined by flow cytometry on a FACScan equipped with a 488-nm argon laser (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Data acquisition was performed with the CellQuest (Becton, Dickinson and Company) software, and the percentages of G1-, S-, and G2-phase cells were calculated with the MODFIT-LT software program (Verity Software House, Topsham, ME, http://www.vsh.com). To discriminate proliferating ORMES-1 cells from non-proliferating feeder cells, cultures were exposed to BrdU (20 µg/ml) for 24 hours. Fresh medium containing BrdU was changed every 12 hours. Colonies were then dispersed into a single-cell suspension, fixed in 70% ethanol, and rehydrated in PBS. DNA denaturation was subsequently performed by incubation in 2N HCl followed by three washes in PBS containing 0.5% Tween and 0.5% bovine serum albumin (BSA) (PBT). Cells were then incubated with FITC-conjugated anti-BrdU monoclonal antibody (1:50 in PBT; BD Biosciences) for 30 minutes at RT in the dark. After extensive washes in PBT, cells were incubated with 1 mg/ml RNase (20 minutes, RT) followed by PI treatment (1 µg/ml) and analyzed as indicated above.& y4 w" ~: N! j0 V. L/ c
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Western Blotting

Cells were lysed in 20 mM HEPES (pH 7.4), 100 mM NaCl, 50 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and a cocktail of protease inhibitors (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) for 1 hour at 4¡ãC. Protein lysates were then cleared by centrifugation (14,000 RPM for 20 minutes). Total proteins (30 µg) were electrophoresed on 10% SDS-polyacrylamide gel and electro-blotted onto nitrocellulose membranes. For analysis of pRB expression, 2 x 106 cells were lysed in 100 ml of 60 mM Tris-HCl (pH 6.8), 1.25% SDS, and 175 mM 2-Mercaptoethanol. Samples (30 µl) were analyzed on 5% SDS polyacrylamide gel. After overnight treatment with blocking buffer (50 mM Tris-HCl , 150 mM NaCl, 5% dry milk), the membranes were probed with anti-cyclin E (Sigma C4976), anti-cyclin A (Santa Cruz sc-751), or anti-pRB monoclonal antibodies (ref. no. 554136; BD Biosciences). Blots were incubated with horse-radish peroxidase¨Ccoupled anti-mouse, or anti-rabbit IgG, and developed using enzymatic chemiluminescence reagents (Amersham, Buckinghamshire, U.K., http://www.amersham.com).  _( q  u' Y8 l' w1 w8 \
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Population Doubling Time and Cell Cycle Duration of the ORMES-1 ESCs7 o  a$ A1 H. v: v7 n

ORMES-1 cells were grown on mitotically inactivated MEF feeder cells in the presence of 20% FBS  revealed that there is a statistical difference in the growth rate between the periods of day 0¨C3 and day 4¨C5 (p

Figure 1. Population doubling time and expression of Oct-4 and Ki67 markers in ORMES-1 cells. ORMES cells were plated on inactivated MEF at a density of 0.3 x 105 cells per cm2. (A): Cell numbers were counted using a trypan blue exclusion assay at each time point (two to three replicates) in two independent experiments. Means ¡À SEM are indicated on the graph. (B): Immunohistofluorescent detection of Oct-4 (revealed by Cy3) in undifferentiated ORMES-1 cells between day 1 and day 6 of culture. Curve represents the mean percentages of Oct-4 cells within colonies (n = 7¨C14) of ORMES-1 cells. (C): Immunohistofluorescent detection of Oct-4 (revealed by Cy3) in ORMES-1 cells at days 2, 4, and 6 of the culture. (D): Immunohistofluorescent detection of Oct-4 (revealed by Cy2) and Ki67 (revealed by phycoerythrine) in ORMES-1 cells at day 3 of the culture. Scale bar = 10 µM (C, D). Abbreviations: Cy, cyanin; MEF, mouse embryonic fibroblast; ORMES-1, Oregon Monkey ES.' I4 @+ g$ @4 y  ^- A" u; {
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To determine whether the variations in growth rates were paralleled by changes in the expression of pluripotency markers, colonies were immunolabeled to detect expression of the Oct-4 transcription factor (Fig. 1B, C). At day 1, 69% ¡À 22% (n = 14) of cells within colonies expressed Oct-4. This percentage peaked at 99% ¡À 1% (n = 10) at day 3, to decline to 80% ¡À 30% (n = 10) at day 6. Therefore, all subsequent experiments were carried out on ORMES cells between day 3 and 5, which corresponds to the optimal growth phase, when 99% of cells express the pluripotency marker Oct-4. Within the ORMES colonies, 99% ¡À 1% of Oct-4  cells were cycling cells, as shown by double immunolabeling against Ki67 and Oct-4 (Fig. 1D).
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Doubling time is a crude measure of proliferation rates and is contaminated with cell death and spontaneous differentiation. In order to accurately measure the mitotic cycle duration, we performed time-lapse videomicroscopy analysis on individual ORMES-1 cells. Cells were infected with SIV-eGFP, an SIV-based lentiviral vector expressing the eGFP, at a multiplicity of infection of 100 viruses per cell so as to transduce the eGFP gene into approximately 2%¨C3% of the cell population. Infected cells were propagated for 1 month and checked weekly for expression of eGFP by fluorescent microscopy. The fraction of eGFP-expressing cells did not vary with time, indicating identical rates of growth for infected and noninfected cells (data not shown).
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Cells were grown on inactivated MEF. Four days after replating, the proliferative behavior of individual eGFP  cells was recorded by time-lapse videomicroscopy during a 48-hour period. The time between two successive mitoses was calculated for 32 individual cells. As shown in Figure 2, the cell cycle duration of individual ORMES-1 ranges from 12¨C21 hours (median value, 15 hours).+ |' y; ]) w5 X9 G4 z7 m
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Figure 2. Cell cycle duration of individual Oregon Monkey ES (ORMES-1) cells measured by time-lapse videomicroscopy two passages after infection with lentiviral vector expressing enhanced Green Fluorescent Protein (eGFP). Histogram represents the duration of 32 individual cell divisions for a time period of 48 hours.
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ORMES-1 ESCs Have an Unusual Cell Cycle Distribution
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Next, we examined the distribution of ORMES-1 cells in cell cycle phases using single-color flow cytometry analysis after DNA staining with PI. Cells were harvested at day 4 of the culture, corresponding to the maximum growth rate (Fig. 1A). To rule out a putative bias due to the presence of growth-inactivated MEF, cultures were first incubated for 24 hours with 50 µM BrdU in order to label all proliferating cells. Subsequent analysis of DNA content stained with PI was restricted to the BrdU  cells (Fig. 3A, B). Under these conditions, the fractions of G1, S, and G2/M phase cells were 16% ¡À 3%, 56% ¡À 3% and 28% ¡À 5%, respectively,. ORMES-1 cells were induced to differentiate by withdrawal of MEF and culture on gelatine-coated dishes for 1 week. Under these conditions, of G1, S, and G2/M phase cells were the fractions 45% ¡À 2%, 29% ¡À 6%, and 26% ¡À 5%, respectively (Fig. 3C). Therefore, differentiation of ORMES-1 cells is accompanied by dramatic changes in the cell cycle distribution, characterized by a large increase in the proportion of cells in G1 phase and a decrease in the proportion of cells in S phase.

Figure 3. Cell cycle distribution of ORMES-1 cells as measured by flow cytometry. ORMES-1 cells were grown in the presence of 50 µM BrdU for 24 hours to label all proliferating cells. Cells were then processed for detection of BrdU incorporation and analysis of DNA content. (A): Dot plot representation of DNA/BrdU biparametric analysis for ORMES-1 and inactivated MEF. DNA was stained with PI (x-axis), and BrdU (y-axis) was revealed with FITC-conjugated anti-BrdU. (B): Histogram representation of the cell cycle distribution of ORMES-1 cells gated as the BrdU  cell population (gate R1). (C): Histogram representation of the cell cycle distribution of ORMES-1 after differentiation induced by withdrawal of MEF and culture on gelatine-coated dishes for 1 week. (B, C): Histograms show one representative experiment. Values are means ¡À SEM calculated from three independent replicates. Frequencies of cells in each phase of the cell cycle were calculated using MODFIT-LT software (Verity Software House, Topsham, ME, http://www.vsh.com). Abbreviations: BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate; MEF, mouse embryonic fibroblast; ORMES-1, Oregon Monkey ES; PI, propidium iodide; R1, region 1.4 y9 Z0 C* r7 I7 q) ^6 l8 Z: O

Duration of Individual Phases of the Cell Cycle of ORMES-1 and ORMES-6 Cells7 e" f& l. l# Z( F
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We investigated the duration of the individual phases of the cell cycle in undifferentiated, self-renewing ORMES-1 and ORMES-6 cells by combining the BrdU cumulative labeling technique . TG1 G2/M (length of G1 G2/M phases) is determined by projecting the extrapolated 100% LI value onto the x-axis. Projection on the negative limb of the x-axis determines TS. So as to monitor the cell cycle of undifferentiated cells and to exclude spontaneously differentiating cells from the calculation, the LI was computed within the Oct-4 cell population (LI was calculated as the proportion of Oct4 cells that were labeled with BrdU). Three independent BrdU cumulative labeling experiments were performed on ORMES-1 cells (Fig. 4A). Cumulative BrdU labeling returned TC values ranging from 16 (blue curve) to 20 hours (green curve). TS and TG1 G2/M values were comprised between 8 (blue curve) and 10 hours (green curve). One experiment of cumulative BrdU labeling in ORMES-6 cells (black curve) returned TC, TS, and TG1 G2/M values of 17, 7, and 10 hours, respectively.

Figure 4. Duration of individual phases of the ORMES-1 and ORMES-6 cell cycles by means of cumulative BrdU incorporation and PLM techniques. (A): Determination of TC (length of total cell cycle), TS (length of S phase), and TG1 G2/M (length of G1 G2 M phases) by cumulative BrdU incorporation. ORMES-1 and ORMES-6 cells were plated at a density of 0.3 x 105 cells per cm2. Three days after plating, cells were refed with fresh medium containing 50 µM BrdU, further cultured for 1¨C10 hours, then processed at regular time intervals for dual detection of Oct-4 expression and BrdU incorporation. The four curves correspond to the percentages of BrdU /Oct4 cells (LIs) calculated in four independent experiments  come from sister cultures). Abbreviations: BrdU, bromodeoxyuridine; LI, labeling index; ORMES, Oregon Monkey ES; PLM, percentage of labeled mitosis.

So as to estimate the duration of G2/M (TG2/M), the PLM method was implemented on sister cultures of the four independent experiments. After a 1-hour exposure to BrdU, the time interval required for 100% labeling of mitotic figures corresponds to the passage through G2 and M (Fig. 4B). So as to restrict the analysis to undifferentiated, self-renewing ORMES cells, the percentage of BrdU-labeled mitotic figures was calculated within the Oct-4  population. In ORMES-1 cells, this analytical procedure returned maximum values for G2/M of 9 (blue curve) to 10 hours (green curve). When T values are substracted from TG1 G2/M values of the same experiment, this indicates that TG1 is reduced, ranging from a few minutes (blue and green curves) to 1 hour (red curve). In ORMES-6 cells, the PLM method indicates that the maximum value of TG2/M is 7 hours, returning a TG1 of 3 hours (Fig. 4B). These results show that the relative duration of G1 phase with respect to TC is comprised between 10% and 20% in ORMES, as shown in the fluorescence-activated cell sorting (FACS) profile (Fig. 3B).1 h& J0 N! W( t, M2 T

DNA Damage Checkpoints in ORMES-1 Cells% Y7 ~4 n1 ^% a0 x! M
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To determine whether ORMES-1 cells have intact cell cycle response to DNA damage, we analyzed the effects of ¨Cirradiation on the cell cycle. In somatic cells, DNA damage results in rapid decrease in the S-phase population, due to p53- and Cdc25A-dependent mechanisms that prevent G1 cells with DNA damage from entering S phase . As shown in Figure 5, the G1-phase fraction in ESCs harvested 3¨C18 hours after exposure to 6 Grays of ionizing radiation decreased from 22% ¡À 0.1% to 7% ¡À 0.5%. In contrast, the G2-phase fraction increased from 36% ¡À 1% to 70% ¡À 2%. These results indicate that ORMES-1 ESCs do not have a functional DNA damage¨Cresponse pathway for growth arrest in G1. By contrast, they exhibit growth arrest in the G2 phase. Note that the fraction of cells in the G1 phase in the nonirradiated cell population (22%) was larger than the fraction of G1 cells found in the ESC population analyzed in Figure 3B (16%). The presence of feeder cells in the irradiation experiment largely accounts for this difference as the vast majority of feeder cells were in G1 (data not shown).
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Figure 5. DNA damage checkpoints in ORMES-1 cells. ORMES-1 cells were plated at a density of 0.3 x 105 cells per cm2 on MEF. Three days after plating, cells were irradiated (6 Gy) and further cultured for 3¨C18 hours before being processed for analysis of DNA content by flow cytometry. (A): Cell cycle distributions of irradiated ORMES-1 cells as measured 0¨C24 hours after irradiation. (B): Percentages of ORMES-1 cells in G1, S, and G2/M phases of the cell cycle as a function of time after irradiation (means ¡À SEM were calculated from three independent experiments). Percentages were calculated using MODFIT-LT software (Verity Software House, Topsham, ME, http://www.vsh.com). Abbreviations: MEF, mouse embryonic fibroblast; ORMES-1, Oregon Monkey ES 1; PI, propidium iodide.
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Elevated Levels of Cyclins A and E Throughout the Cell Cycle in ORMES-1 Cells

We investigated cyclin E and cyclin A expression by immunoblot analysis. Undifferentiated ORMES-1 cells expressed high levels of cyclin A and cyclin E, and these levels decreased dramatically after differentiation induced by low-density culture in feeder-free conditions for 5 days (Fig. 6A). To determine whether cyclin E and cyclin A levels fluctuate during cell cycle progression as observed in somatic cells or, alternatively, whether their expression is not cell cycle¨Cdependent like in mouse ESCs , cyclin E and cyclin A were analyzed by in situ immunofluorescence in the ORMES-1 cell population; 97% ¡À 2% and 78% ¡À 4% of Oct4 ORMES-1 cells expressed cyclin E and cyclin A, respectively (Fig. 6B, C). By contrast, only a small fraction of differentiated Oct4¨CORMES cells expressed cyclin E (6% ¡À 5%) and cyclin A (22% ¡À 5%) (Fig. 6B, D). Together, these data show that cyclin E is expressed throughout the cell cycle in ORMES-1 cells, indicating that its expression is not cell cycle¨Cdependent. By contrast, not all Oct4 ORMES-1 cells expressed cyclin A, suggesting that cyclin A expression is downregulated in a subfraction of undifferentiated ORMES-1 cells.8 O2 V( g6 K' L4 `5 B
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Figure 6. Expression of cyclin E, cyclin A, and pRB in ORMES-1 cells. (A): Western blot analysis of the steady-state levels of cyclin E and cyclin A in undifferentiated ORMES-1 cells . p105 and p110 indicate molecular weights of hypophosphorylated and hyperphosphorylated pRB, respectively. Scale bar = 10 µM (C, D). Abbreviations: Cy, cyanin; ESC, embryonic stem cell; ORMES, Oregon Monkey ES; pRB, retinoblastoma protein.) ]- r  b; L2 P( {  t0 \* q. d
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Predominence of Hyperphosphorylated pRB in ORMES-1 Cells+ {. c$ C2 R8 F( ~8 X4 c' @/ J7 ~7 s
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In parallel, phosphorylation of the retinoblastoma gene product (pRB) was investigated by immunoblot analysis. Mostly hyperphosphorylated pRB¡ªspecific to the S, G2, and M phases of the cell cycle¡ªwas detected in undifferentiated ORMES-1 cells, whereas hypophosphorylated, G1-specific, pRB was predominant in differentiated cells (Fig. 6E). The predominance of hyperphosphorylated pRB in ORMES-1 cells reflects the cell cycle distribution of undifferentiated cells in which S and G2/M phases represent approximately 85% of the total cell cycle.

Dependency on Serum Stimulation and MEK Signaling for G1/S Transition in ORMES-1 Cells

We next addressed the question of whether rhesus monkey ESCs were dependent on persistent serum stimulation for progression in the cell cycle. After serum deprivation for 24 hours¡ªa duration sufficient for every cell of the population to pass at least once through any serum checkpoint¡ªno alteration was detected in the cell cycle distribution of ORMES-1 cells (2 test, p > .5), as shown by flow cytometry analysis (Fig. 7A). Pairwise comparisons between control and serum-deprived sister cultures did not reveal significant variations in the percentages of cells in G1 (p = .5), in S (p = .92), and in G2/M (p = .89) (Student¡¯s t-test, 3 experiments) (Fig. 7B).
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Figure 7. Dependency on serum stimulation and MEK signaling of ORMES-1 cells. ORMES-1 cells were plated at a density of 0.3 x 105 cells per cm2. (A¨CD): Two days after plating, cells were refed with fresh medium lacking serum, or containing the MEK inhibitors PD98059 (PD98) or U0126, or vehicle (DMSO) alone, and further cultured for 24 hours. Cells were then processed for DNA analysis by flow cytometry. Percentages of cells in each phase of the cell cycle were calculated using MODFIT-LT software (Verity Software House, Topsham, ME, http://www.vsh.com). (A): Cell cycle distribution of ORMES-1 cells cultured in the presence ( FBS) or absence (¨CFBS) of serum (one representative experiment). Distributions are compared by means of X2 test. (B): Histograms showing the percentages of ORMES-1 cells in G1, S, and G2/M phases (¡ÀFBS). Means ¡À SEM were calculated from three independent experiments and compared by means of Student¡¯s t-test. (C): Cell cycle distribution of ORMES-1 cells cultured in the presence of 25 µM PD98059, or 10 µM U0126, or vehicle alone (one representative experiment). Distributions are compared by means of X2 test. (D): Histograms showing the percentages of ORMES-1 cells in G1, S, and G2/M phases (¡ÀMEK inhibitors). Means ¡À SEM were calculated from three independent experiments and compared by means of Student¡¯s t-test. (E): ORMES-1 cells were propagated in the presence of PD98059 (dotted line), or in vehicle alone (plain line), for 7 days. Cell numbers were counted using a trypan blue exclusion assay at the indicated time points. Means ¡À SEM were calculated from two replicates. Abbreviations: DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; MEK, mitogen-activated protein kinase kinase; ns, non significant; ORMES, Oregon Monkey ES; PI, propidium iodide.9 s8 a2 N5 N4 \! ]8 y: d3 q9 F

Treatment of ORMES-1 cells for 24 hours with 25 µM PD98059, or with 10 µM U0126, to specifically inhibit MEK activity  did not result in any significant alteration in the cell cycle distribution (PD09805-treated vs. control cells, 2 test, p > .5; U0126-treated vs. control cells, 2 test, p > .5) (Fig. 7C). Pairwise comparisons between control and PD98059-treated sister cultures did not reveal significant variations in the percentages of cells in G1 (p = .3) in S (p = .4), and in G2/M, (p = .6) (Student¡¯s t-test, three experiments). Pairwise comparisons between control and U0126-treated sister cultures did not reveal significant variations in the percentages of cells in G1 (p = .35), in S (p = .56), and in G2/M (p = .71) Student¡¯s t-test, three experiments) (Fig. 7D).4 Q7 ]# p7 m  f; N

To confirm that MEK inhibition did not result in long-term alteration of the growth rate, ORMES-1 cells were propagated in the presence of PD98059, or with vehicle alone, for 7 days. Daily counts of cell numbers did not reveal a difference in growth rates between PD-treated and control cells (Fig. 7E). Taken together, these data indicate that ORMES-1 cells do not require persistent mitogenic stimulation and functional MEK signaling to progress through the G1/S transition.5 r; }$ M& ~, N" V# y0 \. Z6 h# `8 \" k

DISCUSSION. s/ D! `8 t% u3 J( c
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These results indicate that there are important similarities between murine and primate ESC cell cycle characteristics. Like their murine counterpart, rhesus monkey ESCs show a reduced G1 phase, a predominance of hyperphosphorylated RB, an absence of DNA-damage checkpoint in G1, the absence of cell cycle¨Cdependent expression of cyclin E, and lack of dependence on persistent serum stimulation and active MEK signaling during cell cycle progression.
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Using the BrdU cumulative labeling 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, predominantly returns the cell cycle duration of the slowest cycling cells . 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¨C21 hours. The reason ORMES-1 cells are heterogeneous in their cell cycle duration is unclear. It is unlikely to result from mutations that would heritably influence the cell cycle duration, because 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 positions 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 ESCs to extracellular matrix-associated cytokines provided by feeder cells. This could result in randomly influencing the cell cycle duration of individual ESCs.

The most striking feature of the murine ESC cell cycle, apart from being unusually rapid, is a short G1 phase, which represents approximately 15% of the total cell cycle duration, or 2 hours. The duration of the G1 phase of mouse ESCs was calculated by measuring the delay between release from mitotic block and onset of .) a! E. ]+ ?  P: I- ~+ I
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Which mechanism, therefore, underlies the rapid transit through the G1 phase in monkey ESCs? In mouse ESCs, there is compelling evidence that the G1S transition is not dependent on a functional cyclin D:Cdk4/6 RB:E2F pathway . Thus, mouse and monkey ESCs are likely to differ on the basis of the cell cycle¨Cdependent regulation of cyclin A. The heterogenous cell cycle duration that characterizes rhesus ESCs could result from discontinuous expression of cyclin A in a subset of, or in all, Oct4  cells.
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Mouse ESCs fail to undergo cell cycle arrest in the G1 phase in response to DNA damage . Whether a similar mechanism operates in primate ESCs remains to be determined.
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We show that rhesus monkey ESCs, like their murine counterpart . It remains to be determined whether a similar mechanism operates in primate ESCs.( z8 l) a# a2 ]0 d2 X  {4 Z7 W5 ?

CONCLUSION
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Our results show that rhesus monkey ESCs share several fundamental properties of the cell cycle with mouse ESCs. We propose that these unique properties, which are distinct from those of other mammalian non-transformed cell types, are fundamental characteristics of ESCs. In view of the present results, together with several reports pointing to common mechanisms in cell cycle regulation of mouse ESCs and other pluripotent cell types such as the pluripotent cells of the mouse epiblast , we hypothesize that these unique cell cycle characteristics of ESCs may contribute to stemness.

DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS

We thank V¨¦ronique Cortay for assistance in the immunohistochemical techniques, Florence Wianny for expert help with the cell cultures, and Ken Knoblauch for assistance in the statistical analyses. We are indebted to Henry Kennedy for advice and constant support as well as for critical reading of the manuscript. This work was supported by R¨¦gion Rhône-Alpes (Emergence, no. 0101681601 "Th¨¦matique prioritaire cellules souches", no. 0301455301), Association Française contre la Myopathie (INSERM/AFM no. 4CS016F), INSERM AVENIR 2002 programm. A-C.F. is supported by an INSERM postdoctoral grant. A-C.F. and G.M. contributed equally to this work.8 M4 [' ~9 [; Z/ z- y
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