A High Proliferation Rate Is

Current Biology 21, 45–52, January 11, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.11.049

ReportRequired

for Cell Reprogramming and Maintenanceof Human Embryonic Stem Cell Identity

Sergio Ruiz,1 Athanasia D. Panopoulos,1 Aıda Herrerıas,4

Karl-Dimiter Bissig,2 Margaret Lutz,3 W. Travis Berggren,3

Inder M. Verma,2 and Juan Carlos Izpisua Belmonte1,4,*1Gene Expression Laboratory2Laboratory of Genetics3Stem Cell CoreThe Salk Institute for Biological Studies, 10010 North TorreyPines Road, La Jolla, CA 92037, USA4Center of Regenerative Medicine in Barcelona,Doctor Aiguader 88, 08003 Barcelona, Spain

Summary

Human embryonic stem (hES) cells show an atypical cell-

cycle regulation characterized by a high proliferation rateand a short G1 phase [1,2]. In fact, a shortened G1 phase

might protect ES cells from external signals inducing differ-entiation, as shown for certain stem cells [3]. It has been sug-

gested that self-renewal and pluripotency are intimatelylinked to cell-cycle regulation in ES cells [4–6], although little

is known about the overall importance of the cell-cyclemachinery in maintaining ES cell identity. An appealing

model to address whether the acquisition of stem cell prop-erties is linked to cell-cycle regulation emerged with the

ability to generate induced pluripotent stem (iPS) cells byexpression of defined transcription factors [7–11]. Here, we

show that the characteristic cell-cycle signature of hES cells

is acquired as an early event in cell reprogramming. Wedemonstrate that induction of cell proliferation increases

reprogramming efficiency, whereas cell-cycle arrest inhibitssuccessful reprogramming. Furthermore, we show that

cell-cycle arrest is sufficient to drive hES cells toward irre-versible differentiation. Our results establish a link that

intertwines the mechanisms of cell-cycle control with themechanisms underlying the acquisition and maintenance

of ES cell identity.

Results

Acquisition of a High Proliferation Rate Is an Early Eventin Cell Reprogramming

Human induced pluripotent stem (hiPS) cells are molecularlyand functionally similar to human embryonic stem (hES) cellsin terms of pluripotent gene expression and the ability tosupport differentiation into the three embryonic germ layers[7–11]. In agreement, we have observed that the characteristiccell-cycle structure of hES cells, comprised of a large numberof cells in S phase and an abbreviated G1 phase [1, 2], is alsoacquired in established hiPS cell lines (see Figure S1 andSupplemental Results available online). Thus, this led us toinvestigate whether the acquisition of this profile is an earlystep in the process of reprogramming. To this end, we usedkeratinocytes, a cell source with high reprogramming effi-ciency, where nascent iPS cell colonies arise between

*Correspondence: belmonte@salk.edu

10 and 14 days during the reprogramming process [12].Therefore, we analyzed the cell-cycle profile of emerging iPScell colonies as identified by the stem cell surface markersTra-1-81 and Tra-1-60, 12 days after the expression of OCT4,SOX2, KLF4, and cMYC (OSKC) [13] (Figure 1A). The Tra-1-60+

and Tra-1-81+ populations of cells displayed a cell-cycleprofile similar to that observed in hES cells (H9 cell line), incontrast to the Tra-1-602 and Tra-1-812 populations (Fig-ure 1A). Next, we also analyzed the level of proliferation, bymeasuring the percentage of bromodeoxyuridine (BrdU) incor-poration in emerging iPS cell colonies that express the stemcell marker Nanog. We determined that the percentage ofBrdU+ cells in hES and the nascent Nanog+ iPS cell colonieswas similar and was in contrast to the reduced levels ofBrdU+ cells observed in the somatic cell of origin (Figure 1B).To further evaluate these findings, we developed a cellularassay to identify early hiPS cell colonies based on the expres-sion of a green fluorescent protein (GFP) gene driven by theendogenous OCT4 promoter. It has been shown that reactiva-tion of endogenous pluripotent gene expression is a necessaryand early event for successful reprogramming [14]. Therefore,we differentiated a knockin OCT4GFP humanH1 ES cell line [15]into a fibroblast-like population of cells (dFib-OCT4GFP; Fig-ure S2). Transduction of dFib-OCT4GFP cells with OSKC reac-tivated the expression of the endogenous OCT4 locus, whichcorrelated with the appearance of GFP (Figure S2B). More-over, we determined that emerging GFP+ hiPS cell coloniesare still not transcriptionally identical to hES cells, in contrastto established GFP+ hiPS cell lines, thus suggesting that thispopulation represents iPS cells that are not yet fully mature(Figure S2C). In experiments to assess proliferation, weobserved a comparable percentage of BrdU+ cells in hES cellsand early GFP+ hiPS cells that was significantly higher than thepercentage observed in the parental population (Figure 1C).Moreover, we were able to detect highly proliferative GFP+

colonies as early as 10 days after transduction with OSKC (Fig-ure 1D). Finally, we analyzed the percentage of cells positivefor phosphohistone H3, a well-defined mitotic marker, tofurther assess the acquisition of proliferative abilities inemerging hiPS cell colonies. We observed that GFP+ coloniesderived fromdFib-OCT4GFP cells, aswell as Tra-1-60+ coloniesderived from keratinocytes, showed a similar percentage ofphosphohistone H3+ cells compared to hES cells, which wassignificantly higher than the percentages found in the startingcell populations (Figures 1E and 1F). Collectively, these datastrongly suggest that the characteristic proliferative abilitiesof hES cells are acquired as an early event in the generationof hiPS cells.

Induction of Cell Proliferation Promotes Formation

of hiPS Cells, Whereas Cell-Cycle Arrest InhibitsCell Reprogramming

It has been reported that the expression of OSKC triggersa senescence response and induces a G1 cell-cycle arrest[16]. Cell-cycle progression through G1 to S phase requiresthe stepwise phosphorylation and functional inactivation ofretinoblastoma (pRb), which regulates cell-cycle progressionthrough the binding to and inhibition of E2F transcription

Figure 1. The Characteristic Cell-Cycle Signature of Human Embryonic

Stem Cells Is Acquired Early in Reprogramming

(A) Cell-cycle distribution analyzed by 5-ethynyl-20-deoxyuridine (EdU)

incorporation of the Tra-1-60- and Tra-1-81-positive and -negative popula-

tions of cells obtained 12 days after the first infection (OCT4, SOX2, KLF4,

and cMYC; OSKC) of keratinocytes (Ke). H9 cells were included as a positive

control for staining. Two independent experiments were performed, and

5000 cells are represented in each dot plot.

(B) Percentage of Nanog+/BrdU+ cells in H9 and nascent human induced

pluripotent stem (hiPS) cells compared to thepercentageofBrdU+ cells in ker-

atinocytes (Ke). Aminimumof tenNanog+colonieswereevaluated in two inde-

pendent experiments. Cells were incubated with 10 mM BrdU for 30 min.

Results in (B), (C), (E), and (F) are shown as average6 standard deviation (SD).

(C) Quantification of the percentage of BrdU+ cells in early emerging hiPS

colonies obtained 14 days after the first infection of dFib-Oct4GFP cells

Current Biology Vol 21 No 146

factors [17]. This phosphorylation is mediated by cyclin-dependent kinases (CDKs), which require for their activationthe binding of cyclins (Cyc) [18]. Therefore, formation of theCycD-CDK4/6 and CycE/A-CDK2 complexes allows progres-sion of cells into S phase. Conversely, cell-cycle arrest ismediated through the expression of CIP/KIP (p21, p57, andp27) and INK4 (p15, p16, p18, and p14) family members ofCDK inhibitors (CKIs) [17–20]. We tested whether overcomingthe OSKC-induced cell-cycle arrest in order to establish thehES cell-cycle structure in hiPS cells was an important eventduring the acquisition of pluripotency. Thus, we investigatedwhether modulation in the expression of proteins involved inthe control of the G1-to-S phase transition, and hence theproliferation status, would affect the efficiency of cell reprog-ramming. To this end, we designed a panel of lentiviruses en-coding short hairpin RNAs against pRb, CDK2/4, CycE1, andCycD1/D2 (Figure S3A) and retroviruses encoding differentcell-cycle proteins (p15, p16, p21, CycD1/D2, CDK1/2/4, andCycE2) and coinfected them with OSKC in keratinocytes.Downregulation of pRb induced an average 3-fold increasein the efficiency of reprogramming (Figure 2A). Interestingly,we observed that no colonies arose from keratinocytes whenCycE1 (a CDK2 activator) or CycD2 (a CDK4 activator) wasdownregulated (Figure 2A), whereas no changes in reprogram-ming efficiency were detected following CDK2, CDK4, orCycD1 downregulation (Figure 2A). We also found that ectopicexpression of the cell-cycle arrest mediators p15, p16, or p21resulted in almost no hiPS cell colony formation (Figure 2B).Moreover, we observed that expression of CycD1, CycD2,and CycE2, but not CDK1, CDK2, or CDK4, increased reprog-ramming efficiency up to more than 2-fold compared tocontrols (Figure 2C). Similar results were obtained with eitherIMR90 or BJ fibroblasts (data not shown).Next, we hypothesized that the expression of a cyclin

together with its CDK catalytic partner, rather than its expres-sion alone, might have a more profound effect on reprogram-ming efficiency. Indeed, we observed a dramatic increase inreprogramming efficiency, up to 10-fold, by coexpressingCycD1/CDK4 in BJ fibroblasts, a cell type that typicallyreprograms less efficiently than keratinocytes (Figure 2D).Importantly, we observed that changes in cell proliferationcorrelated perfectly with changes in reprogramming efficiency(Figures 2E–2G). Moreover, we further used cumulative BrdUlabeling, which provides information about the rate of S phaseentry and the length of the cell cycle [21], to determine thateither overexpression of p16, p15, or p21 or downregulationof CycD2 or CycE1 resulted in a permanent cell-cycle arrestin the population of cells subjected to reprogramming.However, overexpression of CycD1, CycE2, or CDK4/CycD1or downregulation of pRb resulted in a constant increasedrate of cells entering into S phase over the time of the analysis

with OSKC. H9 cells as well as the original dFib-Oct4GFP population of cells

were included for comparison. A minimum of ten GFP+ colonies were

evaluated in two independent experiments. Cells were incubated with

10 mM BrdU for 30 min.

(D) BrdU immunofluorescence in hiPS cells obtained 10 days after the first

infection of dFib-Oct4GFP cells with OSKC. A representative example is

shown.

(E and F) Quantification of the percentage of phosphohistone H3+ cells in

hiPS colonies obtained 12 or 14 days after the first infection of keratinocytes

(Ke, E) or dFib-Oct4GFP cells (F), respectively, with OSKC. H9 cells as well as

the original population of cells were included for comparison. A minimum of

ten GFP+ (E) or Tra-1-60+ (F) colonies were evaluated in two independent

experiments.

Figure 2. Reprogramming Efficiency Is Influenced by Modulations in the Expression of Proteins Involved in the G1-to-S Phase Transition

(A–D) Human cultures of keratinocytes (A–C) or BJ fibroblasts (D) were infected with retroviruses encoding the four reprogramming factors (OCT4,

SOX2, KLF4, and cMYC), and either lentiviruses encoding short hairpin RNAs (shRNAs) against the indicated proteins (A) or retroviruses encoding the indi-

cated proteins (B–D). Relative reprogramming efficiencies normalized to the efficiency observed in pMX-GFP- or pLVTHM-infected keratinocytes respec-

tively are shown with the fold changes indicated. Uninfected cells were used as a negative control for all experiments. Error bars represent the SD.

(E–G)PercentageofBrdU+cells inkeratinocytes (EandF)orBJfibroblasts (G)4daysafter infectionwithOSKCpluseither retrovirusesexpressing thenotedproteins

or lentiviruses expressing the indicated shRNAs. Note the different percentage of BrdU+ cells between the experimental controls, pMX- or pLVTHM-infected ker-

atinocytes (E and F, respectively), due to the different viral deliverymethod (retrovirus versus lentivirus) used. Two independent experiments were performed, and

the results arepresentedasaverages6SDof at least 300cells scored in threedifferent fields inbothexperiments.Cellswere incubatedwith 10mMBrdU for 60min.

(H)Upperpanel:numberofGFP+coloniesper105cellsseededfollowing infectionofdFib-Oct4GFPcellswith therespective retroviruses.Twoindependentexperiments

wereperformed,and the results arepresentedas theaverage6SDof twobiological replicatesperexperiment. Lowerpanels:morphological aspect andGFPexpres-

sion or Nanog staining of colonies obtained from the different conditions 8 days (lower left) or 16 days (lower right) after first infection. Scale bars represent 100 mm.

n indicates number of independent experiments. Sc indicates scrambled shRNA.

High Proliferation Rate and ES/iPS Identity47

Figure 3. Cell-Cycle Arrest in Human Embryonic Stem Cells Induces Differentiation

(A) Western blot analysis of GFP-, p15-, p16-, and p21-inducible H9 cell line protein extracts 24 hr after treatment with 200 ng/ml doxycycline.

(B) Cumulative BrdU labeling curves for the GFP-, p15-, p16-, and p21-inducible H9 cell lines. After 24 hr of culture with or without doxycycline, fresh BrdU

was added to the medium every 2 hr, and the BrdU labeling index was analyzed at different time points as indicated. The BrdU labeling index indicates the

proportion of DAPI-stained nuclei that were positive for BrdU incorporation. Each time point represents the average and standard deviation of triplicate

counts of at least 500 nuclei in different fields in two biological replicates. The upper dashed line represents the maximum labeling index, which was similar

independent of doxycycline treatment. The time necessary to reach the maximum labeling index corresponds to the total cell-cycle length minus the length

of the S phase (TG2+M+G1) [21].

Current Biology Vol 21 No 148

High Proliferation Rate and ES/iPS Identity49

compared to control cells (Figure S3B). In summary, ourresults suggest that alleviation of the cell-cycle arrest inducedby OSKC, by promoting S phase entry, increases the efficiencyof cell reprogramming, whereas a further induction of cell-cycle arrest has the opposite effect (Figures 2E–2G).

We next examinedwhether the observed increase in reprog-ramming efficiency following induced proliferation was due toan acceleration in the rate of reprogramming or to an increasein the number of cells amenable to being reprogrammed. Tothis end, we infected dFib-OCT4GFP cells with OSKC alone,OSKC+CycD1, or OSKC+CDK4/CycD1. The appearance ofthe first GFP+ colonies following OSKC expression weredetected as early as 8 days after infection (Figure 2H). Surpris-ingly, we did not observe differences in the rate of cell reprog-ramming upon increasing cell proliferation, as mediated byCycD1 or CycD1/CDK4, because all conditions showedGFP+ colonies by day 8 (Figure 2H). However, in agreementwith our previous findings, we observed many more colonieswhen CycD1 or CycD1/CDK4 was overexpressed (Figure 2H).Collectively, these data demonstrate that an increase in theproliferation rate induces a burst in the number of cellsamenable to being reprogrammed, rather than modifying thekinetics of the reprogramming process.

Cell-Cycle Arrest Is Sufficient to Drive hES Cells towardIrreversible Differentiation

We have shown that acquisition of self-renewal and pluripo-tency during cell reprogramming is associated with thesuccessful overcoming of the cell-cycle arrest mediated byOSKC and the establishment of the ES cell-cycle structure.Next, we investigated whether self-renewal and pluripotencyare dependent on the atypical mode of cell-cycle regulationin hES cells. We generated H9 cell lines harboring doxycycline(dox)-inducible expression constructs for the cell-cycle arrestinducers p15, p16, and p21 to determine whether overexpres-sion of these proteins in hES cells poises them for differentia-tion by promoting changes in the cell-cycle structure. Wedetected a significant increase in the percentage of hES cellsin G1 phase as early as 24 hr after induction of p15, p16, orp21 expression, with p21 showing the most dramatic effect(Figures 3A and 3B and data not shown). The accumulationof cells in G1 phasemight indicate that a subpopulation of cellsis arresting in G1, but it could also suggest that cells are under-going a lengthening of G1 phase. To distinguish between thesetwo possibilities, we subjected our inducible hES cell lines tocumulative BrdU labeling. Via this approach, we were ableto establish the time spent in the G2, M, and G1 phases(TG2+M+G1) (see Supplemental Experimental Procedures fordetails).Weobserved that induction of p21 resulted in aperma-nent cell-cycle arrest (Figure 3B), whereas induction of p15 orp16 resulted in an increased TG2+M+G1 ofw40% (15.52 hr [dox]versus 11.03 hr [no dox]) or w30% (12.9 hr [dox] versus9.94 hr [no dox]), respectively. Importantly, we did not detectsignificant changes in TG2+M+G1 when GFP was induced(TG2+M+G1 = 8.71 hr [dox] versus 9.27 hr [no dox]) (Figure 3B).Next, we assessed the effects of p15-, p16-, or p21-mediated

(C) Cell morphology of the same cell lines in the absence of doxycycline (day

embryonic stem (hES) cell-like colonies observed in the case of p15- and p16-

(D) Immunofluorescence analysis of BrdU and Oct4 in the p15-, p16-, and p21-in

nies are outlined with dashed lines. Scale bar represents 100 mm. Cells were in

(E) Western blot analysis of the indicated proteins in the GFP- and p21-expres

(F) Immunofluorescence analysis of differentiationmarkers for themesodermal

AFP, albumin, and FoxA2) germ layers in the p15-, p16-, and p21-inducible H9 c

represents 100 mm.

cell-cycle changes on hES self-renewal and pluripotency. Pro-longed induction of p21 resulted in massive cell differentiationinto nonproliferative cells following 10 days of doxycyclinetreatment (Figures 3C–3F). In contrast, the expression of p15or p16 resulted in an intermediate phenotype in which highlyproliferative hES cell-like colonies were detected but weresurrounded by differentiated nonproliferative cells (Figures3C and 3D). We then performed gene expression and proteinanalysis of pluripotency and differentiation markers in theinducible H9 cell lines maintained in normal growth mediumor following 10 days of continuous doxycycline treatment(Figures 3D–3F and data not shown). We detected OCT4expression after overexpression of p15 or p16 in the areasthat morphologically resembled hES cells, but not in theirsurrounding differentiated populations (Figure 3D). However,OCT4 expression was lost after continuous p21-induced cell-cycle arrest in hES cells (Figures 3D and 3E). Furthermore, inhES cells where p15, p16, or p21 was induced, we detectedelevated expression levels of several differentiation markers,which was not observed in the control GFP line or in thesame lines not treated with doxycycline (Figure 3F and datanot shown).Next, we investigated whether the cell-cycle-induced differ-

entiation in hES cells is reversible. We also evaluated theminimum duration of cell-cycle inhibitor expression requiredto trigger differentiation. To this end, we analyzed the mor-phology as well as the expression of differentiation markersin cells where p21 expression was induced by adding doxycy-cline to the medium for the indicated number of days followedby its withdrawal at each indicated time point and culturing fora total of 10 days (Figure 4A). Interestingly, we observed thatonly 1 day of p21 induction resulted in cultures where morpho-logically hES-like cells were surrounded by differentiated cells(Figures 4B and 4C). We also determined that 3 days of contin-uous p21 induction was enough to trigger massive differentia-tion, and that withdrawal of doxycycline was not sufficientto rescue the differentiation phenotype (Figures 4B–4D).Consequently, continuous induction of p21 expression formore than 3 days resulted in stronger expression of severaldifferentiation markers (Figure 4D). In contrast, we did notobserve any morphological changes and/or expression ofdifferentiation markers after inducing expression of GFP(data not shown). Taken together, these data suggest thatdifferentiation induced by cell-cycle arrest in hES cells is anirreversible process.

Discussion

Our data establish an essential role for cell proliferation in bothcell reprogramming and the maintenance of self-renewal andpluripotency in hES cells. We showed that alteration in therate of hES cell proliferation leads to a compromise in themain-tenance of self-renewal and pluripotency. In fact, we observedthat expressionof p15orp16 in hEScells induces a lengtheningof the average time spent in the G2, M, and G1 phases thatcorrelates with an increased propensity to differentiate. The

0) or 2, 6, and 10 days after p15, p16, or p21 induction. The small human

expressing lines are outlined by dashed lines. Scale bar represents 100 mm.

ducible H9 cell lines after 10 days of induction with doxycycline. Oct4+ colo-

cubated with 10 mM BrdU for 30 min.

sing H9 cell lines with or without doxycycline induction for 10 days.

(SMA), trophectodermal (Cdx2), ectodermal (Tuj1), and endodermal (GATA4,

ell lines following 10 days of induction with 200 ng/ml doxycycline. Scale bar

Figure 4. Differentiation Mediated by p21 Expression in hES Cells Is an Irreversible Process

(A) Schematic representation of the experiment performed. Briefly, p21 expression was induced and maintained as indicated by adding doxycycline to the

medium for the indicated number of days, followed by its withdrawal at each indicated time point. All cells were analyzed following a total of 10 days in

culture.

(B) Cell morphology of the GFP- and p21-inducible cell lines maintained for 10 days in the indicated conditions. The small hES cell-like colonies observed

after a single day of p21 induction are outlined by dashed lines. Scale bars represent 100 mm.

(C) Immunofluorescence analysis of the differentiation markers for the mesodermal (SMA), trophectodermal (Cdx2), ectodermal (Tuj1), and endodermal

(AFP, albumin, and FoxA2) germ layers in the p21-inducible H9 cell line after 3 days of induction with 200 ng/ml doxycycline followed by 7 days of continued

culture after its withdrawal. Scale bars represent 100 mm.

(D) Real-time PCR analysis of the pluripotency marker OCT4 and the specific differentiation markers for mesodermal (Msx1), trophectodermal (Cdx2),

ectodermal (Pax6 and FGF5), and endodermal (GATA4, GATA6, AFP, albumin, and FoxA2) germ layers in the p21-inducible H9 cell line after the induction

conditions indicated. Data are shown as relative averages 6 SD of two biological replicates analyzed in triplicate.

Current Biology Vol 21 No 150

lengthening presumably affects the G1 phase, given the factthat both p15 and p16 are well-known cell-cycle inhibitors ofG1 CDKs [17–20]. Interestingly, it has been proposed that thelength of the G1 phase may influence the fate of stem cellsand is a determinant in stem cell differentiation [3,22–24].

We also demonstrated that induction of cell-cycle arrestis sufficient to commit hES cells toward differentiation.

Surprisingly, we were able to obtain differentiated cells whenculturing the cell-cycle-arrested cells in mTeSR1, a chemicallydefined feeder-free hES cell medium [25]. We further observedthat the differentiation triggered by inducing cell-cycle arrest inhES cells is an irreversible process. In fact, 3 days of cell-cyclearrest was sufficient to commit hES cells toward differentia-tion. These results suggest that hES cells are specified to

High Proliferation Rate and ES/iPS Identity51

maintain a high proliferation rate in order to support their self-renewal. In fact, whereas the expression of p15 or p16 insomatic cells, such as keratinocytes, induced a completecell-cycle arrest, the expression of the same proteins in hEScells still enabled proliferation to occur despite causing alter-ations to the cell-cycle structure. Moreover, we observedthat the cell-cycle properties found in hES cells are acquiredearly in the process of cell reprogramming. Overcoming thecell-cycle arrest induced by OSKC expression might be anearly and necessary event for successful reprogramming[16]. In fact, it has been reported that silencing of the Ink4/Arf locus occurs early in cell reprogramming [26]. Moreover,OCT4 is able to inhibit the expression of the p21 promoter[6]. In agreement, as early as day 10 days after infection ofdFib-OCT4GFP cells, we detected highly proliferative smallcolonies in which the endogenous pluripotent network wasalready reactivated (Figure 1D).

Finally, we showed that stimulation of cell proliferationimproves somatic cell reprogramming, whereas the inductionof cell-cycle arrest impairs this process. Indeed, some of thefirst genetic events during cell reprogramming are the inactiva-tion of the p53/p21 pathway [27–29] and the Ink4/Arf locus [26].Our results establish a perfect correlation between thestimulation or the inhibition of cell proliferation and the effi-ciency of cell reprogramming. Interestingly, we observedthat expression of different CDKs, such as CDK4 or CDK2,neither modified the percentage of cells in S phase norimpacted the reprogramming efficiency. Conversely, theexpression of their corresponding activators, CycD1/D2 orCycE2 respectively, positively increased both processes.This can be explained by the fact that cyclins are the limitingfactor in inducing CDK activity and promoting entry into Sphase. Consequently, coexpression of CycD1/CDK4 induceda higher accumulation of cells in S phase and increasedreprogramming efficiency up to 10-fold. However, we did notobserve variation in the efficiency of reprogramming after thecoexpression of CycE/CDK2, which correlated with theabsence of a change in proliferation status in cells subjectedto reprogramming (data not shown). Nonetheless, the exactmechanisms bywhich cell division affects reprogramming effi-ciency are unknown. It is possible that active promotion oftransition through S phase might enable epigenetic resettingof the genome and/or promote proliferation to increase thenumber of cells available for stochastic reprogramming.

Overall, we have demonstrated that self-renewal and pluri-potency rely on the particular cell-cycle regulation observedin hES cells. Consequently, we show that a high proliferationrate is a requirement for the acquisition and maintenanceof pluripotency and self-renewal of hES and hiPS cells. Insummary, we provide strong evidence showing that cell-cycleregulatory pathways and the pluripotency network are intri-cately connected to safeguard ES cell identity.

Supplemental Information

Supplemental Information includes three figures, Supplemental Results,

and Supplemental Experimental Procedures and can be found with this

article online at doi:10.1016/j.cub.2010.11.049.

Acknowledgments

We especially thank Yasuhiko Kawakami, Jesus Paramio, Geoff Wahl, Chris

Walsh, May Schwarz, Susie Camus, and Sergio Menendez for critically

reading and improving the final version of the manuscript. We also express

gratitude to Krystal Sousley for support at the Salk Institute Stem Cell Core,

Kristen Brennand for assistance with reprogramming experiments, Merce

Marti for excellent work at the Center of RegenerativeMedicine in Barcelona

Histology and Bioimaging Department, and the rest of the Belmonte

laboratory. S.R. was partially supported by the Instituto de Salud Carlos III

(CGCV-1335/07-3). A.D.P. was partially supported by National Institutes of

Health training grant T32 CA009370.Work in this manuscript was supported

by grants from Fundacio CELLEX, the G. Harold and Leila Y. Mathers

Charitable Foundation, Sanofi-Aventis, and the Ministerio de Ciencia e

Innovacion.

Work in the laboratory of J.C.I.B. was supported by Sanofi-Aventis.

Received: August 12, 2010

Revised: October 21, 2010

Accepted: November 17, 2010

Published online: December 16, 2010

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