Gap 1 Phase Length and Mouse Embryonic Stem Cell Self-Renewal

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

Victor C. Li, Andrea Ballabeni, and Marc W. Kirschner1

Department of Systems Biology, Harvard Medical School, Boston, MA 02115

Contributed by Marc W. Kirschner, April 24, 2012 (sent for review February 21, 2012) In somatic cells, the length of the G1 phase of the cell cycle is tightly ESCs, were viable and therefore presumably did not prematurely linked to differentiation, and its elongation can drive differentia- differentiate their stem cells (18, 19). In contrast, it was recently tion in many cases. Although it has been suggested that the situa- reported that olomoucine II, another CDK inhibitor, could in- tion is very similar in embryonic stem cells (ESCs), where a rapid cell duce mRNA up-regulation of differentiation markers in mouse cycle and a short G1 phase maintain the pluripotent state, evidence ESCs and that CDK2 siRNA could induce morphology changes has been contradictory. Here we show that, in murine ESCs, elon- characteristic of differentiation (20). In human ESCs exposure to gation of the cell cycle and elongation of G1 are compatible with roscovitine and other CDK inhibitors sometimes led to a loss of their pluripotent state. Multiple methods that lengthen the cell pluripotency markers but other times did not (21–24). We have cycle and that target cyclin-dependent kinase, retinoblastoma pro- now reexamined this question by using several methods to affect tein, and E2F activity all fail to induce differentiation on their own cell-cycle progression through the G1 phase. These results sup- or even to facilitate differentiation. The resistance of murine ESCs port the idea that the undifferentiated state of mouse ESCs can to differentiation induced by lengthening G1 and/or the cell cycle persist even with a highly elongated G1 phase and therefore that could allow for separate control of these events and provide new elongation of G1 does not in itself promote differentiation. opportunities for investigation and application. Results stemness | proliferation | decoupling Expression of p21 and p27 Lengthens G1 but Does Not Induce Differentiation. To promote G1 elongation, we introduced the n many somatic cell culture models, progression through the cell CDK inhibitors p21 and p27 into ESCs. The p21 and p27 pro- Icycle is essential to maintain the undifferentiated state (1–4). If teins belong to a family of proteins that are normally induced the cell cycle is lengthened or arrested, for example, in G1 by during differentiation and after DNA damage. Overexpression overexpression of G1 cyclin-dependent kinase (CDK) inhibitors of p21 or p27 is sufficient in many somatic cell culture models to (e.g., p21, p27, p16), differentiation will generally result. Several induce both lengthening and differentiation of the G1 cell cycle explanations have been proposed for this linkage of differentiation through the inhibition of CDK1 and CDK2 activity (1). and proliferation. One of them is that G1 may be a period that is When we cotransfected p21 or p27 into J1 ESCs with a plasmid more susceptible to differentiation signals from the external en- expressing EGFP to label the cells, we found that both proteins vironment (5). Continuous passage through G1 could, under these increased the percentage of G1 cells and decreased the percentage conditions, limit the total amount of differentiation signal received of cells in S phase, as determined by DNA content (+10.7% G1 over time. The observation that an inhibitor of TGF-β signaling, and approximately −10.3% S phase) (Fig. 1A). This change in the SnoN, is a substrate of the anaphase-promoting complex/cyclo- DNA content is similar to the gradual change in the cell-cycle some (APC/C) in G1 indicates that different cell-cycle states may structure as cells undergo differentiation, where p21 and p27 differ in their responsiveness to exogenous signals (6). In a similar levels similarly rise (16). To see how much of an effect the CDK way, passage through the cell cycle may also limit the opportunity inhibitors have on proliferation rate, we examined living cells in for epigenetic changes to take place. Such changes associated with real time after tagging p27 with mCherry to construct a fusion differentiation may include a remodeling of chromatin and acti- protein and then expressing it in ESCs. We found that cells vation or repression of lineage-specific gene expression. That G1 expressing p27 had much longer cell-cycle times compared with length may be limiting epigenetic changes is supported by the ob- cells that contained background levels of mCherry fluorescence − servation that many transcription factors are removed from con- (Fig. 1B; p27+ cells, mean 27.3 h; p27 cells, mean 12.4 h). Thus, densed chromatin during mitosis (7). The next G1 would then be expression of p27 generated cell-cycle times in ESCs that are as a critical period to initiate new transcriptional programs, which long or longer than most cultured somatic cells (compare, for may not be feasible if the cell rapidly proceeds through this stage example, the 27.3-h generation time of ESCs expressing p27 to into S phase. Finally, it has been suggested that rapid progression 25.3 h for mouse embryonic fibroblasts and 22.1 h for NIH 3T3 through G1 limits accumulation of the activity of the G1/S check- cells) (16). point protein retinoblastoma (Rb) or Rb family members, which We tested whether p21 and p27 overexpression could induce normally promote differentiation (8), because Rb is both phos- differentiation under standard self-renewing conditions [with phorylated and inactivated simultaneously by CDKs. In this view, leukemia inhibitory factor (LIF)] by assaying Oct4, Nanog, and when the cell cycle is lengthened or arrested in G1, more of the Rb stage-specific embryonic antigen 1 (SSEA-1) levels. We wanted to protein will be dephosphorylated and activated, allowing activity do this at the single-cell level so that we could choose cells with of differentiation-inducing transcription factors. high levels of CDK inhibitor expression. To do so, we used Embryonic stem cells (ESCs) have a very short G1 phase and high proliferation rate. Like somatic cells, they may use this

property to inhibit differentiation and preserve their pluripotent Author contributions: V.C.L. and M.W.K. designed research; V.C.L. performed research; state (5, 9–16). Although plausible, direct evidence for this idea V.C.L. contributed new reagents/analytic tools; V.C.L., A.B., and M.W.K. analyzed data; has been limited and equivocal. Early experiments treating D3 and V.C.L. and M.W.K. wrote the paper. mouse ESCs with the CDK inhibitors roscovitine and Ro09-3033 The authors declare no conﬂict of interest. found no effect on the pluripotency markers Oct4 and Rex-1 (17). 1To whom correspondence should be addressed. E-mail: [email protected]. Furthermore, it was later shown that knockout mice of CDK2, the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. main CDK thought to be responsible for G1/S progression in 1073/pnas.1206740109/-/DCSupplemental.

12550–12555 | PNAS | July 31, 2012 | vol. 109 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1206740109 Downloaded by guest on September 26, 2021 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.

reporter GFP lines for Oct4 and Nanog (called Oct4-GiP and noticed no significant increase (within approximately twofold) in Nanog-GFP, respectively) along with the J1 line for measuring any of these transcripts (Fig. 1D). SSEA-1 levels by immunostaining. By introducing mCherry-tagged p21 and p27 into these lines, we could specifically examine Knockdown of CDK2 or Cyclin A with siRNA Reduces the Proliferation cells expressing high levels of the CDK inhibitors by flow cytom- Rate but Does Not Induce Differentiation. Although some previous etry. At 4 d after p21 and p27 addition, we found that cells that studies reported that a reduction of CDK2 protein is capable continued to express high levels of CDK inhibitor contained of inducing differentiation in human ESCs and producing similar levels of Oct4, Nanog, and SSEA-1 as control cells morphological changes similar to differentiation in mouse ESCs expressing mCherry (Fig. 1C and Table S1). Furthermore, there (21–24), these results are inconsistent with the fact that CDK2 was no significant decrease in pluripotency marker expression knockout mice are viable unless the knockout mice compensated between cells expressing high levels of CDK inhibitors versus by other functional changes. To reexamine the effect of reducing background levels, indicating that progressive elongation in G1 CDK2 under more acute conditions, we used siRNA to knock did not lead to differentiation. Similarly, we observed no drop in down CDK2 levels in our ESC lines. We were able to achieve a Oct4, Nanog, or SSEA-1 even at 10 d after p21/p27 addition knockdown efficiency of ∼50% in the mean fluorescence of the (Table S1). As an additional control, we were able to reproduce population (Fig. S1). CDK2 knockdown slowed down the growth the induction of differentiation by p27 in the somatic neuroblas- rate of ESCs by ∼14% but did not lead to any changes in cell-cycle CELL BIOLOGY toma differentiation model N1E-115, as previously reported (1). A fi In addition to observing no indication of a drop in pluripotency structure (Fig. 2 ) compared with a nonspeci c control siRNA. markers, we also observed no significant increases in genes that Thus, each phase of the cell cycle was lengthened, including G1. are used to characterize differentiated lineages and therefore With regard to morphology, we did not see the enlargement and might suggest differentiation. To compare the populations of cells flattening of the cells associated with differentiation, even at 4 d expressing CDK inhibitor, we FACS-sorted the cells that were after the knockdown (Fig. S2). There was also no change in the positive at 48 h posttransfection and assayed Fgf5 and Msx1 levels of Oct4, Nanog, or SSEA-1 at 4 d after knockdown (Fig. 2B). (ectoderm), Brachyury (mesoderm), GATA4 and GATA6 (en- These results support the conclusions about the dispensability of doderm), and Cdx2 (trophectoderm) by quantitative PCR. We CDK2 that were suggested by the mouse knockout studies.

Li et al. PNAS | July 31, 2012 | vol. 109 | no. 31 | 12551 Downloaded by guest on September 26, 2021 (Fig. 2A). Again there was no detectable change in pluripotency marker levels at 4 d after the knockdown (Fig. 2B).

Effects of Mutant CDK2, E2F1, and Rb Overexpression. Any given method for elongating G1 could affect pluripotency by indirect means, so we attempted some alternative approaches to affect CDK, Rb, or E2F activity. We overexpressed dominant-negative CDK2 (dnCDK2) and dominant-negative E2F1 (dnE2F1) as well as both WT and constitutively active forms of Rb. The dnCDK2 consists of the human CDK2 with an aspartyl residue replacing an asparaginyl residue in the ATP binding site (D145N). This mutant presumably acts by competitively inhibiting the binding of the WT CDK2 to its cyclin binding partners. It was previously shown to induce G1 arrest in human U2OS osteosarcoma cells (27). To generate the dnE2F1 mutant, we used the DNA binding domain of mouse E2F1 (amino acids 90–186). This region is homologous to the 95–191 region of human E2F1, which was previously shown to block S-phase entry in 3T3 cells (28). The dnE2F1 mutant presumably acts to block binding of WT E2F1 to its DNA targets. Finally, we constructed a WT (WTLP) and a mutant (7LP) version of the human Rb large pocket (i.e., the region of Rb that binds E2Fs basic helix–loop–helix factors such as MyoD, amino acids 37–928). The putative dominant-negative mutant contains inac- tivating mutations in seven phosphorylation sites, rendering it uninhibitable by CDK phosphorylation and, hence, constitutively active. Introduction of the 7LP mutant into Rat-1 cells was pre- Fig. 2. Effects of CDK2 and cyclin A knockdown on the cell cycle and plu- viously shown to induce growth arrest in G1 (29). Each of these ripotency. (A) Effect of knockdown on proliferation rate and cell-cycle constructs was tagged with mCherry. structure by DNA content. (B) Flow cytometry analysis of Oct4 reporter, Nanog reporter, and SSEA-1 levels at 4 d after siRNA treatment. Overexpression of these constructs led to clear cell-cycle effects but not differentiation. The dnCDK2 led to a similar percentage of G1 cells but decreased S phase and increased G2/M percentage of − A To further examine whether loss of CDK2 activity could induce cells ( 9.4% S phase, +11% G2/M) (Fig. 3 ). The dnCDK2 also differentiation, we knocked down cyclin A, the principal partner of led to a 20% slower proliferation rate, which implies a longer time spent in G1 (Fig. 3B). The WT Rb construct had a small effect on CDK2 at the G1/S transition. This experiment is difficult to per- increasing S-phase percentage and decreasing G2/M percentage, form because complete removal of cyclin A is lethal (25). We and this effect was more pronounced in the mutant Rb construct therefore tested whether a partial knockdown of cyclin A might (WT: +3.5% S phase, −3.8% G2/M; mutant: +10.2% S phase, lead to some loss of self-renewal. Cyclin A siRNA, which achieved −9.2% G2/M). However, both Rb constructs had little or no effect a population mean knockdown of ∼75% (Fig. S1), did not gen- on proliferation rate (3% and 6% decrease for WT and mutant, erate any significant changes to ESC morphology (Fig. S2). The respectively). The dnE2F1 construct led to both an increase in G1 knockdown did, however, lead to an accumulation of cells in G2/ cells (+26.7% G1) and a 28% slowdown in proliferation rate. We M, as previously reported (26), and a 77% reduction in growth rate found no significant changes in the pluripotency markers Oct4,

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.

12552 | www.pnas.org/cgi/doi/10.1073/pnas.1206740109 Li et al. Downloaded by guest on September 26, 2021 Nanog, and SSEA-1 after introducing these constructs into our ESC lines (Fig. 3C).

Aberrant Effects Caused by Small-Molecule CDK Inhibitors. Probably the most available and common approach to perturb cell-cycle progression is the use of small-molecule kinase inhibitors. Al- though potent kinase inhibitors are now widely available, each has a range of potential enzyme targets, and some have a very broad range. Nevertheless, they are potentially useful because they produce acute effects, and the dose–response range can easily be ex- plored. Several groups have previously used small molecules (e.g., roscovitine) targeted to CDKs but with a host of off-target effects on ESCs and found drastically different results on promoting dif- Fig. 5. Rb dephosphorylation generated by p21 and p27 overexpression. (A) ferentiation (17, 20). To explore the effects of these small molecule Schematic of Rb inactivation during the G1/S transition in somatic cells. In inhibitors further, we applied both roscovitine and CVT-313, mouse ESCs, CDK activity is high, and Rb appears to be largely in the a more specific CDK2 inhibitor, to ESCs. Titrations of both drugs hyperphosphorylated form. (B) Gel-shift analysis shows that the active, hypophosphorylated form of Rb accumulates from p21/p27 overexpression. showed that they affect the rate of cell proliferation at low mi- cromolar concentrations (Fig. 4A). Furthermore, both exhibited changes on cell-cycle structure (Fig. S3). CVT-313 induced a de- with the level normally observed for differentiated cells (−90% crease in S phase percentage and an increase in G2/M percentage Oct4, −98.5% Nanog, and −98.5% SSEA-1). Thus, in cases of (e.g., at 8 μM: −11.6% S phase, +14.6% G2/M). Roscovitine in- differentiation, the Oct4, Nanog, and SSEA-1 levels drop to al- duced fluctuations in S and G2/M percentages across the range of most undetectable levels while, in response to these two drugs, concentrations tested (for 5, 10, 15, and 20 μM: −8.3%, +3.4%, appreciable levels of Oct4, Nanog, and SSEA-1 remain. These +6.7%, and −6.5% S phase; +7.9%, −7.6%, −1.7%, and +10.6% results suggest to us that this drop may not be recording differ- G2/M). In both cases, the estimated G1 fraction did not change entiation but rather some other generally toxic effect of the drug. dramatically over the range of concentrations. On top of this general effect, a small fraction of roscovitine-trea- To examine their effect on pluripotency, we applied a chronic ted cells had further reduced levels of Oct4, Nanog, and SSEA-1, dose of drugs at concentrations that reduced the proliferation rate indicating that differentiation could be induced by roscovitine in to approximately a third of normal (Fig. 4A,4μM CVT-313 and some cells. Because this effect was not observed with CVT-313 10 μM roscovitine). At the end of 10 d of treatment, we observed cells, the effect appeared to be roscovitine-specific and not at- small decreases in Oct4, Nanog, and SSEA-1 levels for both drugs tributable simply to the reduction in proliferation rate, which was in the majority of the population (Fig. 4B). The decrease was similar for both drugs. Cell morphology correlated with the mo- minor (−33% Oct4, −60% Nanog, and −43% SSEA-1) compared lecular data. CVT-313-treated cells were unchanged compared with untreated ESCs (Fig. S4). The majority of cells also maintained an undifferentiated colony morphology in response to roscovitine. A distinct minority exhibited morphological signs of differentiation in both the Oct4 reporter line and the J1 line; they were larger, flatter, and generally resembled fibroblasts.

G1-Lengthened ESCs Contain Hypophosphorylated, Active Rb. One way the short cell cycle of ESCs is thought to protect against differentiation is by constitutively phosphorylating the Rb protein (9). The Rb protein is a substrate of CDKs in G1 and has 16 Ser/Thr-Pro sites that potentially can be phosphorylated (27). In somatic cells, Rb is normally dephosphorylated exiting mitosis and progressively becomes rephosphorylated during the G1/S transition (Fig. 5A). We examined the effect of G1 lengthening on Rb phosphorylation by mobility shift on a Western blot. In J1 cells, Rb ran as the hyperphosphorylated species (Fig. 5B), which is the only detectable form of Rb so far in mouse ESCs (30). Addition of p21 or p27 led to the appearance of a second faster-running form, which was the hypophosphorylated form (here detected with anti–pan-Rb- phospho form pMG3-245 antibody). That this form was in fact the hypophosphorylated form of Rb was conﬁrmed by λ phosphatase Fig. 4. Pharmacological CDK inhibitors can induce multiple effects at sub- treatment, which showed a same size gel shift. Previous studies lethal concentrations. (ALeft) Proliferative dose–response curves of two have shown that the hypophosphorylated form in cycling cells is

small-molecule inhibitors: CVT-313, a CDK2 inhibitor, and roscovitine, a the active form that binds and suppresses E2F function (31). Thus, CELL BIOLOGY broader CDK inhibitor. (Right) Percentage of cells in G1, as determined by addition of p21 or p27 led to the expected accumulation of the FACS cell-cycle profile. (B) Flow cytometry analysis of pluripotency marker active, hypophosphorylated form of Rb that can bind E2F, but it levels after 10 d of treatment with sublethal concentrations of inhibitors. did not lead to differentiation. The CDK inhibitors induced a minor decrease in fluorescence marker levels (demarcated by a shift in arrow position over the major peak). Reductions in G1 Extension Does Not Potentiate Differentiation by LIF Withdrawal. marker levels of magnitude suggestive of differentiation could be found fi only in a fraction of cells in roscovitine-treated samples. (C) Morphologies of Although a lengthening of G1 might not be suf cient on its own ESCs treated with sublethal concentrations of inhibitors for an extended to undermine pluripotency and promote differentiation, it might period (10 d). CVT-313 did not induce noticeable changes, but roscovitine did potentiate differentiation driven by LIF withdrawal. To test this in some cells. possibility, we manipulated the cell cycle in G1 by introducing p21

Li et al. PNAS | July 31, 2012 | vol. 109 | no. 31 | 12553 Downloaded by guest on September 26, 2021 is hard to assess objectively and quantitatively, and lineage-specific genes can often be expressed promiscuously in ESCs without af- fecting self-renewal (32). Given these criteria, several previously contradictory studies would not be in conflict with our conclusions (20, 24). Furthermore, any particular method used to elongate G1 and shorten the cell cycle may individually harbor potential arti- facts, which may be a reason why some previous studies have reached contradictory conclusions. We addressed this issue by using a total of 10 different methods involving the perturbation of G1 CDK activity, Rb, and E2F. Perhaps the most natural method for lengthening G1 was the overexpression of p21 and p27 because these genes are thought to be highly specific for their targets. Ex- pression of these genes induced a cell-cycle length beyond typical somatic cells and produced a cell-cycle structure that was elongated in G1. Some of the other methods generated effects that were more complicated and not just limited to lengthening the G1 phase. Given the potential off-target effects of small-molecule CDK inhibitors and their strong toxicity at slightly higher doses, it may be that their effects on G1 are uninterpretable. Lengthening G1 by p21 and p27 overexpression did not accel- erate differentiation induced by LIF withdrawal (as measured by Fig. 6. Effects of modulating G1 length on the kinetics of Nanog reporter Nanog loss) (Fig. 6). However, shortening G1 by overexpressing loss during LIF withdrawal. Nanog-GFP reporter ESCs were first transfected some G1 cyclins did produce a delay in differentiation. These for 24 h, then LIF was removed to begin kinetics measurement. Values in- results suggest that, although the natural lengthening of G1 during dicated are the means of GFP fluorescence in the Nanog-GFP line for the ESC differentiation may be important for the differentiation construct-expressing population after background correction. The mCherry process to proceed on schedule, it is not itself capable of driving curve is reproduced in light blue in each subsequent graph for reference. the process as it does in many somatic cell models. Provided that the short G1 is not involved in maintaining the ESC state, an interesting question is whether a basal rate of pas- and p27 into our Nanog reporter line and monitored the effect of sage through the cell cycle is required for the pluripotent state. removing LIF from the media. If lengthening the cell cycle pro- This scenario would suggest an alternative model in which there is moted differentiation, we would expect a more rapid loss of plu- a threshold of G1 length above which pluripotency cannot be ripotency markers on LIF withdrawal. In the opposite condition, maintained. Such a model may be possible, but it would need to be shortening the G1 period by expression of proteins that promote reconciled with the fact that, upon exit from the pluripotent state, the G1/S transition would be expected to delay or block differen- G1 lengthens gradually and not in a discrete step-wise fashion. At tiation induced by LIF withdrawal. There was no indication that the moment, we believe our data cannot exclude the possibility of lengthening G1 by overexpression of p21 or p27 facilitated differ- the threshold model because we may not have hit this threshold. entiation under G1 elongation (Fig. 6). In p21- and p27-expressing With CDK inhibitor overexpression, we have observed cell-cycle cells, LIF withdrawal was followed by identical kinetics of Nanog lengths elongated beyond that of typical somatic cells (mean cell- loss. In an attempt to shorten G1, we expressed cyclin A, cyclin E, cycle time of p27-expressing cells of 27.3 h vs. 22.1 h for NIH 3T3 and cyclin D individually (and tagged with mCherry). The in- cells and 25.3 h for mouse embryonic fibroblasts) (Fig. 1B). In the troduction of the new cyclin constructs all led to a decrease in the future, it will be interesting to try to induce even longer cell cycles. percentage of G1 cells by DNA content (Fig. S5), consistent with Why is the length of the cell cycle tightly tied to differentiation of their role as S-phase–promoting cyclins (G1: −10.4% cyclin A, somatic cells but not to ESCs? What is the nature of the block to −5.6% cyclin E, −6.9% cyclin D). Unlike p21 and p27, however, differentiation in both? At present, the mechanisms of both are some of the cyclins led to a short temporary delay in the kinetics of unknown, but one possibility raised by these experiments is that Nanog loss. The extent and duration of the delay were unique to the features unique to ESCs dictate their strong resistance to differ- different cyclins, with cyclin E having almost no loss of Nanog entiation under circumstances where G1 length or cell-cycle length during the first 2 d, cyclin D having a comparatively milder effect is perturbed. One such mechanism is the dependence on LIF sig- (∼25% loss of signal by day 2), and cyclin A having little if any naling or the requirement of blocking MAPK and FGF signaling to effect. Also, the effect of cyclin E appeared immediately (difference maintain pluripotent status. In those specific cases, the unique with mCherry control was detectable by day 1), whereas the effect combination of external signals could override the normal cell- of cyclin D only appeared later (detectable at day 2). Thus, there cycle trigger for differentiation. Another possibility is that the was no facilitating effect of lengthening G1, but shortening G1 by normal triggers for differentiation exerted by slowing of the cell overexpressing specific cyclins did slow down the rate of differen- cycle in G1 only take effect later during embryogenesis rather than tiation as measured by Nanog loss. in the ESC stage. Indeed, most of the documented somatic cell culture models in which G1 phase length has an effect have been Discussion models of late-stage, terminal differentiation (1). Much less is We have reexamined the notion that the short G1 of mouse ESCs known about the role of the cell cycle and Rb in differentiation in actively maintains their stem cell state. Our results support the these early embryonic stages. Surprisingly, our results from G1 conclusions of some previous reports (17–19) and dispute those of lengthening and Rb overexpression indicate that both may have others (20–24). The conflict may partially reflect differing criteria little to do with the regulation of differentiation in the ESC context. for assessing pluripotency. The criteria we used is a drop in pluri- Several authors have previously speculated on the relationship potency factors such as Oct4, Nanog, and SSEA-1. The experiments of ESCs to adult stem cells and cancer on the basis of cycling and were performed in single cells, where the potential heterogeneity the G1 phase. Specifically, it has been postulated previously that of the experimental treatment can be recognized. By themselves, blocking G1 phase-induced differentiation could be a common assessments of cell morphology or the expression of lineage-spe- strategy underlying self-renewal in ESCs and adult stem cells, ei- cific transcription factors may be misleading because morphology ther by constitutively cycling or entering quiescent G1, respectively

12554 | www.pnas.org/cgi/doi/10.1073/pnas.1206740109 Li et al. Downloaded by guest on September 26, 2021 (9). Our results argue against the particular mechanism of blocking Materials and Methods differentiation by constitutive cycling but suggest that alternative ESCs were cultured under standard conditions with Knockout DMEM (Invi- mechanisms continue to block G1 phase-induced differentiation in trogen) and 15% (vol/vol) ESC-qualified FBS (Invitrogen) supplemented with ESCs. In this sense, ESCs may still share an underlying similarity glutamine (Invitrogen), penicillin/streptomycin (Invitrogen), β-mercaptoe- with adult stem cells in their ability to proliferate slowly in G1 in an thanol (Sigma), nonessential amino acids (Invitrogen), and LIF (Millipore). undifferentiated state. It has also been suggested that the short G1 Cells were grown feeder-free on gelatin. Oct4-GiP cells were kindly provided and fast cycling in ESCs may be a mechanism by which oncogenic by Austin Smith (University of Cambridge, Cambridge, United Kingdom). Nanog-GFP cells were purchased from Millipore. factors in ESCs, such as PI3K signaling and Myc, maintain pluri- fi Detailed methods for differentiation assays, time-lapse imaging, RNAi potency (5). We believe that our ndings do not contradict this transfections, construction of fusion constructs and cotransfection experi- idea. Instead, they imply that these oncogenic factors may have ments, flow cytometry and staining, quantitative RT-PCR, growth curve/drug cell-cycle–independent functions in maintaining pluripotency in response curve measurements, and Western blotting are available in SI addition to promoting fast cycling. Thus, the short G1 may be ir- Materials and Methods. relevant or it may facilitate, but it cannot be sufficient for maintaining the pluripotent state. Finally, our results suggest that, ACKNOWLEDGMENTS. V.C.L. thanks William Kaelin, Andrew Murray, Paola because it is possible to separate proliferation from pluripotency Arlotta, Nicholas Dyson, Jodene Moore, the Nikon Imaging Center at Harvard Medical School, and members of M.W.K.’s laboratory for their ad- in ESCs, additional opportunities for investigation and/or thera- vice, suggestions, and reagents. M.W.K. was supported by National Institute peutic application will be available. of General Medical Sciences Grant GM26875.

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