Molecular Mechanisms Controlling the Cell Cycle

Molecular Mechanisms Controlling the Cell Cyclein Embryonic Stem Cells

Essam M. Abdelalim

Published online: 18 August 2013# Springer Science+Business Media New York 2013

Abstract Embryonic stem (ES) cells are originated from theinner cell mass of a blastocyst stage embryo. They can prolif-erate indefinitely, maintain an undifferentiated state (self-re-newal), and differentiate into any cell type (pluripotency). EScells have an unusual cell cycle structure, consists mainly of Sphase cells, a short G1 phase and absence of G1/S checkpoint.Cell division and cell cycle progression are controlled bymechanisms ensuring the accurate transmission of geneticinformation from generation to generation. Therefore, controlof cell cycle is a complicated process, involving several sig-naling pathways. Although great progress has been made onthe molecular mechanisms involved in the regulation of EScell cycle, many regulatory mechanisms remain unknown.This review summarizes the current knowledge about themolecular mechanisms regulating the cell cycle of ES cellsand describes the relationship existing between cell cycleprogression and the self-renewal.

Keywords ES cells . Self-renewal . G1 phase . Pathway .

Pluripotency

Introduction

Embryonic stem (ES) cells are originated from the inner cellmass of themammalian blastocyst. They were first establishedfrommice in 1981 [1], and from human in 1998 [2]. Like their

counterparts in the inner cell mass, ES cells can differentiateinto all derivatives of three primary germ layers as well asgerm cells. The potential of ES cells to differentiate into anycell type of the body raised the hope for treatment of incurablediseases.

The ability of ES cells to maintain the self-renewal andpluripotency is associated with their ability to remain in aproliferative condition, which requires a unique transcription-al profile. ES cells divide rapidly with a short generation timeof approximately 8–10 h in murine ES cells [3, 4], and 8–16 hin human ES cells [5]. ES cells undergo successive symmet-rical cell divisions to generate new progeny structurally andfunctionally equivalent to the mother cells [3, 6]. ES cell cycleprogression is controlled by mechanisms that ensure the rapidreplication and accurate transmission of genetic material todaughter cells. The cell cycle is divided into four distinctphases; S phase is a period of chromosome replication, andM phase is the period of chromosome transmission. G1 andG2 act as gap phases, which temporally separate S from Mphase [6] (Fig. 1). It has been found that pluripotent cells in theepiblast have a cell cycle profile lacking fully formed G1 andG2 gap phases in which a high proportion of time (approxi-mately 60 %) is devoted to S phase. A similar cell cyclestructure has been reported for ES cells [4, 7]. ES cells havean unusual cell cycle structure, consists mainly of S phasecells and a truncated G1 phase [4, 8]. In human ES cells, 65 %of the time, the cells reside in S phase and 15% of the times inG1 phase [5] (Fig. 1). Also, the induced pluripotent stem cells(iPSCs) close to these trends, suggesting that rapid cell divi-sion and shortening of the cell cycle are essential forpluripotency [9–11]. When ES cells are differentiated, thecells accumulate in G1 phase and show a cell cycle lengthenedto more than 16 h, as seen in somatic cells [6] (Fig. 1). Thevery short G1 phase of pluripotent cells increases the rate ofcell division since the length of S phase is similar to other celltypes [6, 12]. Also, it is possible that a short G1 phase may berequired for maintaining ES cell pluripotency [13].

The G1 phase is a gap period between cytokinesis andDNA synthesis. During the G1 phase, a cell monitors the

E. M. Abdelalim (*)Department of Cytology and Histology, Faculty of VeterinaryMedicine, Suez Canal University, Ismailia 41522, Egypte-mail: [email protected]

E. M. AbdelalimStem Cell and Regenerative Medicine Research Centre,Qatar Biomedical Research Institute, Qatar Foundation, Doha, Qatar

E. M. AbdelalimMolecular Neuroscience Research Center, Shiga University ofMedical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan

Stem Cell Rev and Rep (2013) 9:764–773DOI 10.1007/s12015-013-9469-9

environments for the presence of growth factors and nutrients,as well as evaluates the integrity of its genome. These func-tions are achieved through a check point at the G1/S transition[14]. At the early G1 phase, the growth factors activate theexpression of the D-cyclins. In somatic cells, G1 to S phasetransition requires overcoming the G1-to-S restriction check-point, where the cell cycle becomes independent of externalgrowth factors and is detected by the inactivation of thephosphorylated retinoblastoma (pRB) protein followed bythe release of E2F factors [15]. Unlike somatic cells, whichdepend on mitogen signaling to proceed through the G1/Stransition, ES cells proliferate in a mitogen-independent man-ner, which leads to a short G1 phase [4, 7].

At this point, there is a considerable body of literaturedocumenting the factors involved in the regulation of ES cellcycle. In this review, I discuss the unique features of the EScell cycle, and how these unique features are linked to the self-renewal and stemness of ES cells. I also focus on recentadvances related to the molecular mechanisms regulating thecell cycle progression in ES cells

Role of Cyclins and Cyclin-Dependent Kinases (Cdks)in ES Cell Cycle Regulation

Cyclins are unstable proteins, synthesized and degraded atspecific times during the cell cycle. Cyclin protein levels areregulated by transcriptional mechanisms and at the level ofprotein stability [6]. Cyclin-dependent kinases (Cdks) are agroup of protein kinases, activated by the formation of acomplex with cyclins, and phosphorylate a number of cellcycle proteins essential for S phase progression. They promotecell cycle entry, control the G1-S transition, and play anessential role in the rapid cell cycle progression of ES cells

[4, 16]. Cdks are activated by their binding to cyclins whichare expressed at different levels during the cell cycle [9](Fig. 2). In ES cells, the levels of Cdks remain constant, butexpression of cyclins changes within cell cycle. Fluctuationsin levels of cyclins allow activating different Cdks, forcingtransitions between specific phases of the cell cycle [9].

During normal cell cycle progression, the early events ofG1 phase are enhanced by the activity of Cdk4 and Cdk6 incomplex with cyclin D [17]. Cyclin D is expressed in G1phase in response to extracellular signals and associates withCdk4 or Cdk6 (Cdk4/6), and activate the phosphorylation ofretinoblastoma protein (pRB). Cyclin E associates with Cdk2and enhance phosphorylation of RB protein [18]. During Sphase cyclin A binds to Cdk2. At the beginning of G2 phasecyclin A binds to Cdk1 and drive the progression through G2phase. During G2 phase cyclin B is expressed and binds toCdk1. The activity of complex cyclin B/Cdk1 leads to nuclearenvelope breakdown and onset of prophase in mitotic divi-sion. Kinase activity of cyclin/Cdk complexes is negativelyregulated by cyclin–dependent inhibitors (CKI). CKIs are twogroups (Cip/Kip and INK4) based on their structures and Cdktargets. Cip/Kip family members bind to cyclin D–, E and A–dependent kinases and inhibit their activity. INK4 familymembers bind only to Cdk4/6 and block binding domain forD cyclins or disrupt the association between Cdk4/6 andcyclin D. All CKIs contain motif that enable them to bindboth cyclin and Cdk subunits [19] (Fig. 2).

Fig. 2 Control of cell cycle by cyclins and cyclin-dependent kinases(Cdks). Complexed with Cdk4 or Cdk6 regulate early G1 phase; theCyclin E-Cdk2 complex is required to intiate S phase; Cyclin A togetherwith Cdk1 or Cdk2 is then responsible for the continuation of S phase andfor entry into mitosis; and the B-type cyclins complexed with Cdk1enhance entry into mitosis. Cip/Kip family members bind to cyclin D–,E and A–dependent kinases and inhibit their activity. INK4 familymembers bind only to Cdk4/6 and disrupt the association betweenCdk4/6 and cyclin D

Fig. 1 Schematic illustrations showing the cell cycle of somatic cells (a)and undifferentiated ES cells (b). Note the difference between somaticand ES cells in the length of the cell cycle phases. Cell cycle is dividedinto four distinct phases: G1, S, G2 and M phase. Cells duplicate theirDNA during S phase and divide it equally between the two daughter cellsduring M phase. G1 and G2 act as gap phases, which temporally separateS from M phase

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In mruine ES cells, all cyclins and Cdks are expressedthroughout the cell cycle [4, 6, 8, 12], with the exception ofCdk1-cyclin B that is selectively activated before mitosis [4],and Cdk4-cyclin D1 complexes, which demonstrate littleactivity in murine ES cells [8, 9, 20]. The high levels of Cdkactivity in murine ES cells are due to the absence or very weakexpression of CKIs that are linked to the relatively high levelsof the cyclins throughout the cell cycle [9, 21]. Cdk2 isconsidered the main Cdk in ES cells [4, 12]. It has been shownthat the rapid progression of cell cycle in murine ES cells isdue to an unusually high Cdk2 activity in undifferentiated EScells, which is not under the control of cell cycle [4]. This highlevel of Cdk2 activity is a result of continuous expression ofboth cyclin E and A throughout the cell cycle of murine EScells [4]. Consistent with these findings, it has been found thatinhibition of Cdk2 activity with Cdk2 inhibitor, OlomoucineII, suppresses murine ES cells proliferation by accumulatingthe cells at G1 phase and preventing G1-S transition withoutaffecting on ES cell pluripotency [22]. After differentiation,the check point pathway, at the G1/S phase transition, isestablished. As a result, Cdk4/Cdk6-Cyclin D and Cdk2-Cyclin E activity becomes cell cycle-regulated and responsiveto external signals, leading to elongated G1 phase [6]. Also, indifferentiated cells, Cdk activity is inhibited during G1 be-cause of degradation of cyclins and existence of inhibitorproteins, like p21 [23].

In human ES cells, cyclin D1, D2, and D3 have beenshown to be increased in G1 and cyclin D2, A, E, and B1 tobe increased at G2, at the transcription level [5, 24]. ThemRNA levels of cyclins A2, B1, and B2 have the highestexpression levels compared to the other cyclins [5]. Sincecyclins can be regulated post-transcriptionally, the proteinlevels of cyclins show slightly different expression pattern inhuman ES cells: the cyclin B1 protein is increased around G2/M, similar to murine ES cells [6, 24, 25]. In contrast to murineES cells, where cyclin E levels remain stable throughout thecell cycle, protein levels of cyclin E increase around the G1/Stransition and cyclin A protein levels are increased in late G1/S through G2/M in human ES cells [24, 25].

In human ES cells, it has been found that knockdown ofCdk2 activity delays the G1-S transition and leads to G1 arrest[24, 26]. Also, exposure of human ES cells to DNA damagedelays G1 phase though the downregulation of Cdk2 activitywhich is mediated by inhibition of Cdc25A [27]. In contrast tomurine ES cells, Cdk2 activity is also required for cell fatedecisions in human ES cells [24, 28].

Role of Retinoblastoma in ES Cell Cycle Regulation

Retinoblastoma (RB) is considered as a negative regulator ofthe cell cycle progression and a positive regulator of cellulardifferentiation. Unphosphorylated RB binds proteins such as

E2F family members and co-repressors, which allow chroma-tin remodeling acitivities [29]. G1 to S progression is regulatedby RB family, which includes the pRB, p107 and p130 pro-teins. The hypophosphorylated state of RB family inhibits theexpression of genes required for S-phase entry through directlybinding to the E2F family of transcriptional factors, resulting intranscriptional suppression of these genes. During normal cellcycle progression, phosphorylation of pRB by cyclin/Cdkcomplexes can suppress the ability of RB to bind to E2F andallow cell cycle progression through G1 phase [30, 31].

A cell type that is cycling rapidly, including pluripotentstem cells have low level of active RB to permit for a rapid cellcycle progression through G1/S. Pluripotent stem cells lack atypical E2F/pRB mediated transition in late G1, which nor-mally support commitment to S phase entry at the restrictionpoint [6, 20, 32]. Several reports indicate that the RB pathwayis present in ES cells. It has been found that RB and its twofamily members, p107 and p130, are expressed in murine andhuman ES cells [31, 33]. In ES cells, RB and its familymembers are mainly in their hyperphosphorylated state be-cause ES cells contain high level of cyclin/Cdk activity, whichprevent RB family to bind with E2F transcription factors, andin turn allows an R-point-independent short transition throughG1 phase [6, 34]. However, suppression of Cdk activity,which may lead to hypophosphorylation of RB family, hasbeen found to arrest ES cell cycle progression [26, 27]. RBknockout murine ES cells showed no self-renewal defects.Also, murine ES cells which are simultaneously triple knock-out for RB gene family (RB , p107 and p130) are viable andproliferate normally [30, 31, 35], indicating that RB family arenot necessary for normal growth of ES cells. However, aprevious study demonstrated that RB inhibition leads to in-creased genomic instability in murine ES cells [36]. Takentogether, these observations suggest that RB family membersare not required for normal ES cells growth, but they may berequired for genomic stability.

Role of p53 in ES Cell Cycle Regulation

The tumor suppressor p53 is essential to maintain the genomeintegrity [37]. It has been shown that p53 can lead to cell cyclearrest in response to DNA damage and induce proapoptoticsignaling. Also, p53 can block G1 phase by activation of p21[38, 39].

Inmurine ES cells, p53 is highly expressed in the cytoplasmunder basal conditions [40, 41]. There are discrepancies be-tween results from different research groups regarding the roleof p53 pathway in cell cycle regulation in ES cells. Thepossible explanations of these discrepancies are the use ofdifferent ES cell lines, and the use of different experimentalprotocols. Although earlier studies had shown that the p53-mediated response is inactive in ES cells due to the

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cytoplasmic sequestration of p53 [40], the recent studies re-ported that p53 is activated and translocated to the nucleus afterDNA damage, and induce differentiation of ES cells by direct-ly suppressing Nanog expression in ES cells [42–44]. It hasbeen reported that after UV irradiation, the ability of murine EScells to form colonies is more strongly decreased in p53-positive murine ES cells than in those which lack the p53 [45].

However, unlike murine ES cells, human p53 is localizedin the nucleus of human ES cells at a low concentration, andthe exposure of human ES cells to DNA damage induces p53-dependent cell cycle arrest rather than differentiation [33].Interestingly, a recent study showed that p53 may be func-tional in unstressed murine ES cells, where chemical inhibi-tion of p53 leads to suppression of ES cell proliferation byaccumulating the cells in the G1 phase of the cell cycle [44].Taken together, these findings suggest that p53 has dualfunctions in ES cells. On the one hand, it induces apoptosisand ES cell differentiation after exposure to DNA damage,and on the other hand, it promotes ES cell proliferation byregulating the ES cell cycle under standard culture conditions.

Several reports have studied the function of p53-p21 path-way in stem cells [40, 43, 46–48]. The arrest of cell cycle inG1 phase is mediated by Cdk inhibitor p21, a downstreamgene target of p53 [21]. In human ES cells, in response todifferentiation signals, p53 is transiently activated and binds tothe p53-responsive elements (p53REs) of downstream genetarget p21 to enhance the accumulation of cells in G1 phaseleading to elongate of G1 and prolong the cell cycle [49].Exposure of human ES cells to DNA damage causes a cellcycle arrest at G1 phase accompanied by accumulation andphosphorylation of p53. Moreover, the DNA damage in-creases the expression of p21 mRNA without any change inthe p21 protein level suggesting that p21 is not a regulator ofG1/S progression in human ES cells [27, 50]. However,overexpression of p21 in human ES cells results in an increasein the percentage of cells in G1 phase [51]. A recent studyshowed that the p21 protein is not detectable in undifferenti-ated human ES cells under basal conditions and after DNAdamage because the translation of p21 mRNA is prevented bythe miRNA pathway, particularly miR-302 s [50].

It can be deduced from the above described data that thereis a difference in cell cycle regulation between human ES cellsand somatic cells. It has been reported that in human ES cells,the p53-p21 pathway may be involved in the regulation ofentry into differentiation instead of maintaining intact DNA[46], suggesting that p53-p21 pathway is non-functional inundifferentiated ES cells.

Role of microRNAs in ES Cell Cycle Regulation

The microRNAs (miRNAs) are a class of small non-codingRNAs (18–24 nucleotides in length). They down-regulate

gene expression by attaching themselves to messenger RNAs(mRNAs) and preventing their translation into proteins [52,53]. Specifically, miRNAs regulate cell proliferation byinteracting with cyclin/Cdk complexes.

Wang et al. named the miRNAs which are expressed inundifferentiated ES cells and they play roles in cell cycleregulation, ES cell-specific cell cycle-regulating miRNAs(ESCC miRNAs). ESCC miRNAs include; miR-291a-3p,miR-291b-3p, miR-294, miR295, and miR-302 [51]. ThesemiRNAs can rescue the defect in the cell cycle and enhancethe transition of the cells from G1 to S phase [51] by sup-pressing p21, RB, and Lats2 which are inhibitors along theCdk2-Cyclin E pathway [18] (Fig. 3).

An important role for miRNAs in the regulation of ES cellcycle has been inferred from the ES cell miRNA knockoutmodels through deletion of either Dicer or Dgcr8 [54–56],which are required for maturation of miRNAs [56]. It has beenfound that Dgcr8-null murine ES cells are viable and prolif-erate slowly due to the accumulation of the cells in the G1phase of the cell cycle, indicating a defect in the G1-S transi-tion [56]. Also, Dicer-deficient murine ES cells showed areduced proliferation rate and an altered cell cycle profile[54, 55]. Interestingly, it has been reported that 14 differentmiRNAs, which are highly expressed in ES cells, can rescuethe cell cycle defects in the Dgcr8-null cells [56]. Thesefindings indicate that miRNAs play an essential role in en-hancing the G1-S transition in ES cells.

It has been reported that Let-7 family members cansuppress self-renewal in the Dgcr8- null murine ES cellsbut not wild-type ES cells [57]. Furthermore, overexpressionof ESCC miRNAs into the Dgcr8- null murine ES cells,prevent loss of self-renewal induced by the let-7 miRNAs[57], indicating that the let-7 and ESCC miRNAs haveopposing effects on ES cell self-renewal (Fig. 3). Takentogether, these findings suggest that miRNAs which arenormally expressed in ES cells prevent let-7 from silencingES cell self-renewal.

In human ES cells, it has been reported that miRNAs arealso essential for the G2/M transition. Suppression of Dicer orDrosha results in accumulation of ES cells in the G2 phase[58]. Interestingly, miR-195 overexpression rescues this de-fect by inhibiting Wee1 levels. Wee1 is a kinase that is aninhibitor for the Cdk1-Cyclin B complex, which is essentialfor the G2/M transition [59] (Fig. 3). However, in murine EScells, lack of Dicer or Dgcr8 has no effect on G2 phase andmiR-195 does not rescue proliferation defects of Dgcr8-nullES cells, suggesting a difference between human ES cells andmurine ES cells. These data suggest that miR-195 may affecton targets that normally suppress the progression of the cellcycle at the G2/M transition in human ES cells [58].Furthermore, the overexpression of miR-372 significantlydownregulates p21 levels in Dicer-deficient human ES cells[58] (Fig. 3).


Also, miR-92b has been found to be required for G1-Stransition of the human ES cell cycle by its suppressing effecton the Cdk inhibitor 57 , a G1/S checkpoint gene [60]. Anoth-er study demonstrated that inhibition of miR-302 familymembers in human ES cells can suppress the cell cycle pro-gression and cause a G1 arrest [61]. The effect of miR-302 oncell cycle is mediated by the regulation of cyclin D1 and Cdk4post-transcriptionally [61], suggesting the important role ofthe miR-302 family in G1/S transition of ES cell cycle.

Taken together, these findings suggest that miRNA path-way is essential for proper progression of the ES cell cycle.

Other Factors Involved in ES Cell Cycle Regulation

Recently, it has been reported that knockdown or deletion ofCIBZ, which is a BTB domain zinc finger transcriptionalfactor, delays the progression of ES cell cycle by inhibitionof G1 to S transition. Conversely, ectopic expression of CIBZpromotes ES cell proliferation by accelerating G1 to S transi-tion [62]. Furthermore, AMP-activated protein kinase(AMPK), a key regulator of energy metabolism, is involvedin the regulation of G1/S transition [63]. Treatment of murineES cells with 5-aminoimidazole-4-carboxyamide ribonucleo-side (AICAR), an AMPK activator, induces cell cycle arrestthrough inhibition of G1 to S transition, activates p53/p21signaling pathway, and suppresses Nanog expression [63].Also, the ATP-Chk1 kinase pathway has been found to beimportant for normal cell cycle progression in ES cells. Sup-pression of ATR-Chk1 leads to stimulation of a p38-p21

pathway which triggers an intra-S-phase checkpoint and de-lays entry into mitosis [64].

Brain natriuretic peptide (BNP) is expressed in murine [65,66] and human ES cells [67], and has been found to play anessential role in ES cell cycle regulation [65, 66]. Inmurine EScells, knockdown of BNP or its receptor; natriuretic peptidereceptor A (NPR-A) suppress ES cell cycle progression byaccumulating cells in G1 phase [65, 66, 68, 69]. In fullconsonance with these findings, knockdown of NPR-A in-duces ES cell cycle arrest by activating p21 expression andinhibiting cyclin D1 expression [68, 69]. Also, murine EScells express GABAARs, which consider as negative regula-tors of ES proliferation by suppressing the cell cycle progres-sion [70]. The effect of GABAAR on ES cell cycle is mediatedby phosphorylation of H2AX in cell cycle-dependent andDNA damage-independent manners [70]. Also, BNP has anegative effect on the expression level of GABAARs,suggesting that BNP signaling is important for maintainingthe appropriate level of GABAAR in ES cells to promote EScell cycle [65, 66].

The importance of intracellular calcium signaling duringES cell cycle progression is previously reported. It has beenreported that murine ES cells exhibit oscillations of Ca2+

which are coincide with the transition from the G1 phase tothe S and that is essential for cell cycle progression [71].Furthermore, the functional store-operated calcium entry(SOCE) is expressed in murine ES cells and that inhibitionof SOCE attenuates ES cell proliferation, and arrests cells atthe G1 phase [72]. On the other hand, activation of SOCEusing 17b-estradiol increases murine ES cells proliferation

Fig. 3 A schematic representation of the role of microRNAs in theregulation of ES cell cycle. In murine ES cells, ESCC (ES cell-specificcell cycle-regulating) and let-7 miRNAs have opposing effects on the G1-S transition. The ESCC miRNAs promote the transition of the cells fromG1 to S phase by suppressing p21, RB, and Lats2 which are inhibitorsalong the Cdk2-cyclin E pathway. However, let-7 miRNAs have an

inhibitory effect of the G1-S transition by suppressing Cdc25a andcyclinD/Cdk4/6, which are positive regulator of G1-S transition. Inhuman ES cells, miR-372 inhibits the Cdk inhibitor p21, which sup-presses the G1/S transition, whereas miR-195 suppresses Wee1 kinasewhich is an inhibitor of Cdk1-cyclin B complex. Cdk1-cyclin B complexis required for the G2/M transition


[72]. In human ES cells, a recent study reported that mito-chondrial protein CBARA1, a regulator of mitochondrialcalcium uptake, is expressed specifically in human ES cells,and its knockdown suppresses ES cell self-renewal bydownregulating Oct4 expression and accumulating cells atG1 phase [73].

Relationship Between Cell Cycle and Pluripotency in ESCells

The relationship between cell cycle regulation andpluripotency of ES cells is controversial. It is believed thatES cells start to differentiate during G1 phase. A link betweenthe G1 phase and commitment of ES cells into differentiationhas been described [13, 74, 75]. It has been suggested that thelonger the ES cells stay in G1 phase, the more likely they areto be subjected to differentiation signals. Thus, control of theG1 phase may be important to control the gateway to differ-entiation [13, 74, 75].

Previous studies have shown that the Nanog, Myc, Sox2,andOct4 transcription factors enhance ES cell pluripotency byregulation of cell cycle genes and miRNAs [76–78]. It hasbeen demonstrated that Nanog directly regulate the progres-sion of the cell cycle by directly binding to Cdk6 and Cdc25A,which are the regulators of the G1/S [79]. The overexpressionof Nanog in human ES cells leads to an increase in ES cellproliferation by increasing the number of cells entering Sphase and reduces the time needed for S-phase entry due toan increase in the expression levels of Cdk6 and Cdc25A [79].

Also, E2F has been found to work as a regulatory cofactorfor Oct4 at the promoters of Oct4 target genes and thatORC1L (Origin recognition complex subunit 1-like), a directE2F target which mediates cell cycle progression, belongs tothe core Oct4 regulatory network [80].

Furthermore, Oct4 has been found to regulate geneslinked to cell cycle progression [81]. Inhibition of Oct4 leadsto downregulation of genes involved in ES cell proliferation,and an upregulation in the cell cycle inhibitor p21and expression of p63, which has been linked to differenti-ation [82].

It has been shown that inactivation of Myc reduces cellproliferation and cell cycle remodeling [83]. Several reportshave identified direct Myc targets including cyclins and Cdks[84–86], suggesting the role of Myc in cell cycle regulation.Myc enhances self-renewal by regulating the cell cycle regu-latory network and maintains pluripotency by repression ofGATA6 [83]. In human ES cells, the expression of c-Myc isthe highest in the G1 phase of the cell cycle, at the RNA level.However, it has been found that c-Myc is upregulated in S andG2 phases at the protein level [24].

In murine ES cells, Myc plays an important role in regu-lating the cell cycle, since the deleltion of myc leads to a

significant reduction in the population of cells in S phase withincrease of the G1 and the G2/M cell populations as well as toincreased levels of cell death [87].

Cdk2 has been linked to the cell cycle regulation in EScells. It has been reported that knockdown of Cdk2 activity inES cells delays the G1-S transition, which is sufficient toinduce differentiation of ES cells, suggesting the presence ofregulatory relations between cell cycle and pluripotency [24,26, 28]. In human ES cells, chemical inhibition [24] or knock-down of Cdk2 activity [88] results in accumulation of cells inG1 phase, delay in G1-S transition, and induces ES cellsdifferentiation. This linked relationship is further supportedby the observation that pluripotency factors Oct4, Nanog, andSox2 control the expression of key cell cycle regulatoryproteins such as Cdk1, cyclin D1, Cdk6, CDC25A, andCDC7 [9, 28, 74, 79].

Furthermore, Cdk2-associated protein 1 (Cdk2ap1;p12DOC-1), a suppressor of G1-S transition by inhibition ofCdk2, play a role in differentiation of murine ES cells thoughregulation of the phopshorylation level of pRB [89]. In theabsence of leukemia inhibitory factor (LIF), Cdk2ap1-deficient murine ES cells showed resistance to differentiation,and altered phosphorylation of pRB [89]. These findingssuggest that Cdk2ap1-mediated differentiation of murine EScells is elicited through the regulation of regulation of cellcycle.

Further evidence for a link between cell cycle regulationand pluripotency comes from the studies of telomerase activ-ity in ES cells [90, 91]. Forced expression of telomerasereverse transcriptase (TERT) in human ES cells can promoteproliferation by increasing the percentage of cells in S phaseand reducing the percentage of cells in G1 phase [91]. Also,TERT overexpression leads to an increase in protein levels ofcyclin D1, transcription of Cdc6, an E2F target gene, and anincrease in pRB phosphorylation [91]. Silencing of TERTleads to reduced ES cells proliferation by increasing the per-centage of cells in G1 phase, with a decrease in cyclin D1 andCdc6 [91]. In murine ES cells, inhibition of TERT byoverexpression of one of its repressors, Zap3 , is sufficient toinhibit ES cell proliferation through the defect in cell cycleprogression [90]. These findings suggest that telomerase isrequired for ES cell cycle progression.

Taken together, these findings indicate that the prolonga-tion of cell cycle progression affects on the pluripotency statusof ES cells and lengthening of G1 phase provides a windowfor differentiation signals.

The phosphoinsitide 3-kinase (PI3K) signaling pathwayhas been shown to be important to control cell-cycle regula-tion and maintain pluripotency in ES cells [92, 93]. Treatmentof ES cells with the PI3K inhibitor LY294002 inhibits theproliferation, induces cell cycle arrest at the G1 phase, andinduces ES cell differentiation by suppressing Nanog expres-sion [75, 92, 93].


Furthermore, it has been found that Klf5 promotes the G1/Stransition by enhancing Akt phosphorylation and suppressingp21 [94].

A recent study has showed that cyclin E is involved inmaintaining the pluripotent state of ES cells. Overexpressionof cyclin E promotes ES cell self-renewal, and increased theresistance of ES cells to transient LIF withdrawal. However,loss of cyclin E1 expression in ES cells elongates G1 phaseand induces differentiation [95]. These findings suggest a linkbetween the G1 phase and commitment of pluripotent ES cellsinto differentiation

On the other hand, a previous study demonstrated thatlengthening G1 phase by overexpression of p21 and p27 didnot enhance differentiation induced by withdrawal of LIF.Also, shortening G1 phase by overexpressing G1 cyclinscause a delay in ES cell differentiation [96]. These findingssuggest that lengthening of G1 phase alone is not able to forcethe process of differentiation.

Conclusions and Perspectives

The study of ES cells is strongly associated with hopes for thefuture of regenerative cellular therapies. Therefore a thoroughunderstanding of the basic molecular mechanisms involved inthe cell cycle progression will facilitate efforts to develop newmethods to propagate ES cells in vitro, a key step toward theaim of using cellular therapeutics to replace damaged tissues.Over recent years, our knowledge about the cell cycle of EScells has greatly expanded. Many ES cell cycle-related pro-teins have been identified and functionally characterized in EScells. The studies reviewed here discuss the unique features ofthe ES cell cycle, and how these unique features are crucial forself-renewal and pluripotency of ES cells. Furthermore, Iprovide insights into the molecular mechanisms that regulateprogression through the ES cell cycle.

A key question, from the standpoint of cell cycle regulationin ES cells, is whether all ES cell types share common char-acteristics. Previous studies demonstrated some differenceshave appeared between cell cycle regulation in human andmurine ES cells. A more comprehensive understanding of thecell cycle machinery of different ES cell types will be acquiredin order to exploit ES cell cycle regulatory genes for enhanc-ing ES cell expansion.

Considerable work still remains to be done to completelyunderstand the regulatory networks and the signaling pathwaysinvolved in controlling the cell cycle progression in ES cells.Recent reports showed that the cell cycle regulation is tightlylinked to maintenance of ES cell pluripotency. Perhaps futurestudies should focus on identifying the changes in cell cyclekinetics before and after ES cell differentiation, andunderstating the relationship between cell cycle progressionand cell fate decision. I expect the discovery of additional

regulatory mechanisms of the cell cycle which will facilitatethe control of ES cells in vitro and enhance the reprogrammingefficiency of iPSCs.

Conflict of Interest I declare no potential conflicts of interest

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Molecular Mechanisms Controlling the Cell Cycle

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