Lengthened G1 Phase Indicates Differentiation Statusin Human Embryonic Stem Cells

Ashley Calder,1,2 Ivana Roth-Albin,1 Sonam Bhatia,1,2 Carlos Pilquil,1 Jong Hee Lee,1 Mick Bhatia,1

Marilyne Levadoux-Martin,1 Jamie McNicol,1 Jennifer Russell,1 Tony Collins,1 and Jonathan S. Draper1–3

The cell cycle in pluripotent stem cells is notable for the brevity of the G1 phase, permitting rapid proliferationand reducing the duration of differentiation signal sensitivity associated with the G1 phase. Changes in thelength of G1 phase are understood to accompany the differentiation of human embryonic stem cells (hESCs), butthe timing and extent of such changes are poorly defined. Understanding the early steps governing the differ-entiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discoveryapplications. Here we report the first use of real-time cell cycle reporters in hESCs. We coexpressed thechromatin-decorating H2B-GFP fusion protein and the fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fusion protein, a G1 phase-specific reporter, in hESCs to measure the cell cycle status in live cells. We foundthat FUCCI-G1 expression is weakly detected in undifferentiated hESCs, but rapidly increases upon differen-tiation. hESCs in the G1 phase display a reduction in undifferentiated colony-initiating cell function, under-scoring the relationship between G1 phase residence and differentiation. Importantly, we demonstrate inter- andintracolony variation in response to chemicals that induce differentiation, implying extensive cell–cell variationin the threshold necessary to alter the G1 phase length. Finally, gain of differentiation markers appears to becoincident with G1 phase lengthening, with distinct G1 phase profiles associated with different markers of earlyhESC differentiation. Our data demonstrate the tight coupling of cell cycle changes to hESC differentiation, andhighlight the cell cycle reporter system and assays we have implemented as a novel avenue for investigatingpluripotency and differentiation.

Introduction

The coupling of differentiation and cell cycle length isintrinsically linked in many species, with changes in cell

cycle phase profiles occurring in coordination with embryonicdevelopment [1–4]. Early embryos of fly [5,6], frog [7,8], zebrafish [9], mouse [10], rat [11], and human [12] all demonstrateshortened cell cycle lengths with truncated gap phases thatlengthen as development progresses. The unique cell cycleproperties of the early embryo can be captured in vitro duringthe derivation of permanent pluripotent embryonic stem cells(ESCs) from blastocyst-stage mammalian embryos. ESCs ofmouse [13], rat [14], and human [15], all maintaining theshortened cell cycle observed in the originating embryos. Inhuman ESCs, the total cell cycle length is understood to be*16-h long, primarily due to a truncated G1 phase length [15].Differentiation of pluripotent and multipotent cells leads tolengthened cell cycle by virtue of G1 phase extensions [16,17],with the corollary that pathways that regulate pluripotency of

human embryonic stem cells (hESCs) establish truncated cellcycle parameters [18,19].

The tight regulation of the G1 phase length during devel-opment appears to stem from the role G1 phase plays in cellfate decisions [20–23], with a short G1 phase hypothesized tohelp isolate cells from promiscuous differentiation signals.Modulation of cell cycle regulatory molecules that control G1checkpoints and passage through G1 to S phase alters theability of a cell to undergo apoptosis, enter or remain in qui-escence, or differentiate. The differentiation process appearsto be regulated primarily by negative regulators of prolifera-tion, such as p21 and p27. Levels of p21 or p27 increase duringthe differentiation of a broad range of cells, including oligo-dendrocytes and erythroid progenitors [24–26], intestinalepithelial cells [27], and keratinocytes [28]. These G1 phaseregulatory mechanisms also function in hESCs; levels of p21and p27 both increase on differentiation [29].

Despite these clear trends, the details of the kinetics of cellcycle changes, such as G1 phase extension onset during lineage-

1McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario,Canada.

2Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.3Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

STEM CELLS AND DEVELOPMENT

Volume 22, Number 2, 2013

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2012.0168

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specific differentiation of hESCs, remain unanswered. In-vestigating the cell cycle status in mammalian cells has beenlimited by traditional techniques, including BrdU or Ki67 la-beling, which necessitates the compromising of cell viability,preventing the prospective isolation of live cells in distinct cellcycle phases. Sakaue-Sawano and colleagues recently brokethis impass by developing a live-cell reporter system [fluores-cence ubiquitination cell cycle indicator (FUCCI)] that providesunparalled access to the cell cycle progress of viable cells [30].FUCCI-G1 is a G1-phase-indicating live fluorescent reportercreated by fusing the orange fluorophore mKO2 [31] to an N-terminus fragment of Cdt1 [30]. Truncated Cdt1 in this fusionprotein contains only the regulatory elements responsible for itsdegradation and lacks DNA-licensing capabilities. Tight, cellcycle-dependent degradation of Cdt1 results in orange fluo-rescence only during the G1 phase. We deployed the G1-phase-reporting element of the FUCCI system in human ESCs to in-vestigate historical observations describing the existence of atruncated G1 phase in ESCs. We linked the FUCCI-G1 reporterto H2B-GFP, which demarcates chromatin [32], to ensure thatour dual reporter would be compatible with automated mi-croscopy and software-based image quantification. The dualreporter system we have compiled represents a novel multi-plexing of 2 extant reporters in hESCs, producing a powerfulnew tool for investigating cell cycle changes.

Here we use these live-cell reporters of the cell cycle statusto describe the cell cycle kinetics of undifferentiated hESCs,and how differentiation imparts changes in the duration oftotal cell cycle and G1 phase length. We provide data illus-trating the relationship between lineage-specific differentia-tion and cell cycle changes, while also providing a functionalproof linking the G1 phase length with the differentiationstatus.

Methods

Cell culture

H1 and H9 hESC lines [33] were cultured on X-ray-inactivated MEFs on 1.0% gelatin-coated tissue culture plates(Falcon) as previously described [34]. Briefly, cells werepassaged by treatment with 1 mg/mL Collagenase IV (In-vitrogen) at 37�C followed by mechanical scraping. hESCswere maintained in a KO DMEM (Invitrogen), with 15%serum replacement (Invitrogen), 2 mM Gluta-Max (Invitro-gen), 100mM nonessential amino acids (Invitrogen), and8 ng/mL bFGF (Peprotech).

Generation of reporter constructs and stablehESC reporter lines

The FUCCI reporter system was obtained from MBL Inter-national. The H2BGFP-F2A-mKO2Cd1-IRES-Puro (H2GFOIP)fragment was generated by synthesis of the F2A-mKO2-Cdt1fragment (GeneArt; Life Technologies) and subsequent inser-tion of a H2B-GFP fragment between upstream KpnI and ApaIsites. pCAG H2GFOIP was created by ligating the H2GFOIPinsert [AvrII (blunt)/BglII digested] into a pCAG expressionvector [Age (blunt)/BglII digested], and validated by sequenc-ing (MOBIX; McMaster University).

About 20mg of linearized CAG-H2GFOIP expressionplasmids were electroporated into H1 [33] and H9 (WiCell)hESC parental lines. PiggyBac H1 and H9 hESC lines were

created with 20mg of pB-CAG-H2GFOIP plus 5 mg of trans-posase pCYL43 (Sanger Institute, UK). The pB-CAG vectorwas generously provided by Dr. Andras Nagy. Electro-poration conditions were based on those previously de-scribed [35]. Cells were selected with puromycin (1mg/mL),and individual colonies manually picked, and expandedonce established.

Differentiation

Small-molecule inducers of differentiation were diluted ina complete hESC medium (defined above). Compounds andfinal concentrations used were 1.0% dimethyl sulfoxide(DMSO) (Sigma-Aldrich); 3 mM HMBA (Sigma-Aldrich);40 mM RRD-251 (Sigma-Aldrich); and 40mM LY294002 (Cal-biochem).

Endoderm differentiation protocols were based on thosepreviously described [36]. hESCs were grown to near con-fluence, and then washed 1· with phosphate buffered saline(PBS), and placed in a basal endoderm differentiation me-dium RPMI (Sigma-Aldrich) with 2 mM Gluta-Max supple-mented each day as follows: day 1: 100 ng/mL activin A(Peprotech) and 25 ng/mL Wnt3a (Peprotech); day 2:100 ng/mL activin A and 0.2% fetal bovine serum (FBS); day3: 100 ng/mL activin A and 2% FBS.

Neural differentiation was based on a previously de-scribed protocol [37]. Monolayer hESCs were cultured to*60% confluency, and then the hESC medium without bFGFsupplemented with 5 mM SB431542 (Tocris) and 5 mM dor-somorphin (Torcris) was added to cells. The medium waschanged every 2 days. Cells were then grown in DMEM-F12(Invitrogen) with 1· N2 supplement (Invitrogen) and 20 ng/mL bFGF for 6 days. The medium was changed every 2 days.

Fluorescence-activated cell sorting

hESCs were dissociated to single cells with TrypLE (In-vitrogen), stained with the viability dye 7-aminoactinomycinD (7-AAD; Immunotech) to exclude dead cells. For the A2B5sort, co-stained with A2B5 antibody (1:100; APC conjugated;Miltenyi Biotec; #130-093-58), diluted in a staining buffer (1%FBS in PBS with 1 mM EDTA) for 30 min on ice, and thenwashed 2 times with staining buffer, and stained with 7-AAD. Populations were fractionated using Aria II (BDBiosciences).

Indirect immunofluorescence

CAG H2GFOIP hESCs were passaged and differentiatedas described above. Cells were washed once with 1· PBSwith Mg + 2/Ca + 2, fixed at room temperature for 8 min with4% paraformaldehyde (PFA) in 1· PBS, and washed 3 timeswith 1· PBS. Fixed cells were permeabilized with ice-cold100% methanol for 2 min at room temperature and washed 3more times with PBS. Before staining, cells were blocked for15 min at room temperature with 1% BSA (Sigma-Aldrich) in1· PBS and washed once with 1· PBS. Antibodies werediluted in a blocking solution. Primary antibodies were in-cubated at 4�C overnight, washed 3 times with 1· PBS, andsecondary antibodies incubated for 1 hr at room temperature.Stained cells were stored in 1· PBS with Hoechst 33342nuclear stain. Primary antibodies used were as follows:OCT4 (1:200; BD #611203), NANOG (1:400; Cell Signalling

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#4903), SOX2 (1:300; BD #561469), p21 (1:400; Cell Signalling#2947), p27 (1:400; Cell Signalling #3686), Ki-67 (1:100; SantaCruz #sc-23900), Cyclin B1 (1:50; Cell Signalling #4138),LAMIN A/C (1:100; Santa Cruz #sc-6215), EOMES (1:50;Abcam #AB23345), GATA4 (1:300; Santa Cruz #sc-9053),TCF2 (1:250; BD #612504), SOX1 (1:150; R&D #AF3369), andSKP2 (1:100; Santa Cruz #sc-7164). Secondary antibodiesused were as follows: goat anti-mouse AF-647 (1:500; Invitro-gen #A-21238), donkey anti-rabbit AF-647 (1:500; Invitrogen#A-31571), or rabbit anti-goat AF-647 (1:500; Invitrogen #A-21446). Nuclei were co-stained with Hoechst 33342.

Imaging, analysis, and cell tracking

All imaging of fixed cells was performed on a CellomicsArray Scan HCS Reader (Thermo Scientific) or a OperettaHigh Content Screening System (Perkin Elmer). For live-cellimaging, H2GFOIP cells were passaged and plated as de-scribed above. Two days after passage, the plates wereplaced in a BioStation CT-integrated cell culture observationsystem (Nikon) for observation. Colonies were visually se-lected for time-lapse observation; phase, GFP, and mKO2images were acquired every 3–6 h for dose–curve experi-ments and every 15 min for high-resolution cell-tracking ex-periments for 3 days.

Image analysis of immunofluorescence and reporter fluo-rescence was performed using Accapella high-content andanalysis software (Perkin Elmer). Cell nuclei were identifiedby Hoechst 33342 staining, and the fluorescence intensity ofthe same nuclei in the FITC, Cy3, and Cy5 channels wasmeasured. Custom MatLab (Mathworks) scripts were thenused to quantify the fluorescent intensity of each nuclei in allchannels and output scatter plots and statistics.

Cells were tracked with the MTrackJ plugin for ImageJ(rsbweb.nih.gov/ij/). Cell measurements for H2BGFP andmKO2-Cdt1 intensities from each track were plotted withPrism 5 (GraphPad). Lineage trees were generated usingFigTree v1.3.1 software (http://tree.bio.ed.ac.uk/software/figtree/).

Reverse transcriptase quantitative polymerize chainreaction and transgene copy number assay

Approximately 500,000 cells per population for each ex-periment were collected. mRNA was extracted (RNAeasykit; Qiagen), and synthesized to cDNA (iScript kit; Bio-Rad).Reverse transcriptase quantitative polymerize chain reaction(RT-qPCR)s were run with SYBR Green (Bio-Rad) on aCFX96 Touch� Real-Time PCR Detection System (Bio-Rad).Bio-Rad CFX Manager software was used for analysis.

Transgene copy number assay was performed on a ViiA�7 real-time PCR system (Life Technologies) using TaqMan�

assays (Life Technologies) for RNase P (Catalog #: 4403326),hTERT (Assay ID: Hs06055639_cn), and GFP (Assay ID:Mr00660654_cn) on genomic DNA extracted from wild-typehESCs and H2GFOIP clones. The GFP-transgene copynumber was relatively quantified using CopyCaller� soft-ware v2.0 (Life Technologies), where RNase P was used as aninternal control for each sample, and hTERT was used as thecalibrator with a known copy number of 2 in the humangenome. Efficiencies of hTERT and GFP assays were assessedusing cycle threshold values (Ct) for 1:5-fold dilutions of H1H2GFOIP clone 6 genomic DNA, ranging from 100 to 0.8 ng.

(Supplementary Fig. S1B; Supplementary Data are availableonline at www.liebertonline.com/scd). Cycling parametersfor the assays were 95�C for 10 min and (95�C for 15 s and60�C for 60 s) for 40 cycles.

Colony-initiating cell assay

H2GFOIP cells were grown in a standard hESC mediumuntil confluent, and sorted by fluorescence-activated cellsorting (FACS) into FUCCI-G1 + and FUCCI-G1 - popula-tions as described above. Isolated populations of cells wereplated in triplicate at densities of 2.5 · 104, 5.0 · 104, or 1 · 105

cells per well of an MEF-coated 6-well plate. A second rep-licate of 5.0 · 104 cells was grown with Rock inhibitor Y27632added to the medium for 24 h after plating to increase cel-lular viability [38]. Cells were grown for 12 days until es-tablished colonies appeared, and then fixed and stained foralkaline phosphatase activity with a VECTOR Red AlkalinePhosphatase Substrate Kit (Vector laboratories). Plates offixed and stained cells were scanned on a flatbed scanner(Canon), and image analysis and quantification performed inImageJ using custom scripts to identify colony units.

Cell motility assay

Eight-mM pore Boyden chamber inserts were precoatedwith Matrigel and then 1 · 105 H2GFOIP cells seeded in afeeder-conditioned hESC medium (FCM) supplemented with10mM Y27632. After 24 h, the FCM was substituted for freshFCM (control) or 10% FBS in the upper and lower chamber,the latter serving as a motility and differentiation inducer.After 48 h, the top of the porous membranes was cleared byusing a cotton-tipped applicator, and then the migrated cellswere fixed with 4% PFA. The inserts were then imaged usingthe Operetta High Content Screening System.

Statistical analysis

All graphs generated from the automated image analysisare derived from at least 2 cell lines and an n of between 3and 6. Each n involved the analysis of > 10,000 cells. Errorbars show standard error of the mean (SEM). Prism 5(GraphPad) was used for statistical analysis. Statistical sig-nificance: * = p < 0.01, ** = P < 0.001, ***p < 0.0001.

Results

Generation of hESC lines stably expressinga cell cycle reporter

To establish the kinetics of the G1 phase change duringhuman ESC differentiation, we employed the FUCCI re-porter system, which has previously been shown to be anaccurate gauge of cell cycle position and is compatible withthe development of live mice [30]. Pluripotent ESCs are no-ted for their truncated G1 phase, so we utilized the FUCCI-G1 reporter, a fusion of the orange fluorescent protein mKO2to a fragment of Cdt1 (herein referred to as FUCCI-G1) [30].The periodic nature of the G1 phase during the cell cycle,combined with and the potential for mosaic expression oftransgenes in ESCs [39], presented the possibility that theexpression pattern observed in hESC lines carrying theFUCCI-G1 reporter lines could be hard to interpret. To ad-dress this, we modified the FUCCI-G1 reporter to include a

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permanent nuclear marker that would confirm the ubiqui-tous expression of the FUCCI-G1 transgene in live cells.Fusion of the histone-binding protein H2B to GFP (H2B-GFP)effectively demarcates nuclei [32], without the need for DNAstaining, which can be cytotoxic and cytostatic [40]. Weused a picornaviral 2A sequence [41] to link H2B-GFP toFUCCI-G1, creating a bicistronic message that ensured si-multaneous, but separate, translation of the H2B-GFP andFUCCI-G1 proteins (H2GFOIP; Fig. 1A). We placed theH2GFOIP dual-reporter cassette under the control the pow-erful CAG ubiquitous promoter [42] and linked by an IRESsite to a puromycin selection cassette. We then generatedstably expressing clones of H1 and H9 hESCs containing theH2GFOIP reporter by electroporation, and picked > 3 clonesof each cell line for expansion, which had *1–18 copies ofthe H2GFOIP transgene inserted (Supplementary Fig. S1A).Despite the variability in number of transgenes inserted, allhESC clones containing the H2GFOIP reporter behavedsimilarly and displayed the same phenotype when observedunder a fluorescence-based microscope: FUCCI-G1 wasabsent or weakly expressed in colonies that were mor-phologically undifferentiated; however, numerous brightFUCCI-G1-positive nuclei were found to surround the un-differentiated hESC colonies or were found in clusters thatcorresponded to morphologically differentiated hESC colo-nies (Fig. 1B and Supplementary Fig. S2).

Validation of FUCCI-G1 reporter fidelityin human cells

To ensure that the FUCCI-G1 reporter displayed the sameG1 phase cell cycle restriction in human cells as observed inmouse, we co-stained H2GFOIP-expressing hESCs withmarkers of the cell cycle that indicate cell cycle position. Theexpression and distribution of Ki67 can be used as a guidefor identifying cell cycle position [15,43]. HESCs expressingFUCCI-G1 displayed a pattern of Ki67 distribution mostfrequently associated with the G1 phase (Supplementary Fig.S3A, B). SKP2 is an E3 ubiquitin ligase that is expressedthroughout the S and G2 phases of the cell cycle, but is de-graded during the G1 phase [44]. Co-staining of H2GFOIPhESCs with SKP2 and subsequent coexpression quantifica-tion demonstrated that FUCCI-G1 expression displays vir-tually no overlap with SKP2 expression in hESCs(Supplementary Fig. S3C, D). CYCLIN B1 is cytoplasmicfrom the S phase through late G2, then nuclear during mi-tosis, and degraded during G1 [45]. CYCLIN-B1 co-stainingof H2GFOIP hESCs revealed that FUCCI-G1 was expressedin cells that were absent for CYCLIN-B1 expression (Sup-plementary Fig. S3E). Together, these observations show thatFUCCI-G1 expression is restricted to the G1 phase of the cellcycle in human cells.

A minority of pluripotent hESCs are in the G1 phase

Previous attempts to quantify the distribution of hESCs inthe phases of the cell cycle have been performed on bulkcultures of cells, under the assumption that all cells within agiven culture are equivalent. More recently, we have dem-onstrated that heterogeneity due to spontaneous differenti-ation is a significant and confounding issue wheninvestigating the properties of pluripotent stem cells [46]. To

negate the caveats associated with the heterogeneity com-monly found in hESC cultures, we co-stained the H2GFOIPhESC lines with markers of pluripotency and then measuredthe coincidence of FUCCI-G1. Cultures of undifferentiatedH2GFOIP hESCs were co-stained with antibodies to thepluripotency markers OCT4, NANOG, and SOX2. The ex-pression of all 3 pluripotency markers, and to a lesser extentthe proliferation marker Ki67, was almost completely re-stricted to the colonies of morphologically undifferentiatedhESCs and displayed little overlap with bright FUCCI-G1expressing cells (Fig. 1C). A converse pattern was observedfor the high overlap of FUCCI-G1 expression with that of p21or p27, both CDK inhibitory proteins that are associated witha lengthened G1 phase and differentiation. To quantify therelationship between pluripotency and the lengthened G1phase at the cell level, we measured the overlap betweenantibody staining and the FUCCI-G1 expression by high-content automated imaging and analysis (see SupplementaryFig. S4 for summary of workflow). This analysis revealedthat a minority of cells expressing pluripotency markers alsoexpressed FUCCI-G1, in direct contrast to the majority ofspontaneously differentiated cells expressing p21 or p27 (Fig.1D). In addition, the expression of p21 was also most closelyassociated with the hESCs displaying brighter expression ofFUCCI-G1 (Supplementary Fig. S5). The overlap of OCT4 withp21 in undifferentiated cultures was < 2% (data not shown).

We next performed a colony-level quantification of FUC-CI-G1 and pluripotency marker expression (see Supple-mentary Fig. S6 for summary of workflow). Images acquiredfrom the high-content automated imaging were stitched to-gether in silico to form seamless montages covering*0.28 cm2 of a well, permitting software-based identificationand quantification of colonies and their properties. This as-say demonstrated that the majority of colonies in undifferen-tiated cultures of hESCs display a high-average OCT4:FUCCI-G1 intensity ratio of 4.6:1 (Fig. 2A, B). Treatment of H2GFOIPcells with 1% DMSO for 48 h did not impact the OCT4:FUCCI-G1 intensity ratio in every colony to the same extent, but didreduced the average OCT4:FUCCI-G1 intensity ratio to 1.5:1(Fig. 2B–D). These data illustrate the strong relationship be-tween FUCCI-G1 expression and the loss of OCT4 at thecolony level, while also demonstrating hESC intercolonyvariation in response to differentiation stimuli.

These observations were consistent between the H1 andH9 hESC lines, and describe that a minority of pluripotenthESCs are in the G1 phase of the cell cycle, and that theexpression of FUCCI-G1 is most likely to be found in dif-ferentiating hESCs.

FUCCI-G1 expression in hESCs revealsfunctional differences

The high correlation between hESCs in the G1 phase anddifferentiation markers (Fig. 1) suggested that expression ofthe FUCCI-G1 reporter identifies hESCs with functionallydistinct characteristics. To test this hypothesis, we fraction-ated H2GFOIP hESC cultures by expression of the FUCCI-G1reporter into FUCCI-G1-negative ( - ) and FUCCI-G1-positive ( + ) populations using FACS and then performing acolony-initiating cell (CIC) assay, a functional measure ofself-renewal capacity [47]. FUCCI-G1 + and FUCCI-G1 -fractions were seeded at a range of densities on MEF-coated

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FIG. 1. A G1 phase reporter is expressed at low levels in pluripotent human embryonic stem cells (hESCs). (A) Schematic ofthe H2BGFP-F2A-mKO2Cd1-IRES-Puro (H2GFOIP) cell cycle reporter used in this study. (B) Representative micrographsof phase-contrast and fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fluorescence of an undifferentiated colonyof H2GFOIP hESCs. Note the expression of FUCCI-G1 around the colony periphery. (C) Representative micrographs de-tailing the co-localization of FUCCI-G1 reporter expression with indirect immunofluorescence antibody staining for OCT4,NANOG, SOX2, Ki67, p21, and p27. (D) Graph of the overlap of FUCCI-G1 expression with the antibodies used in (C) inundifferentiated H2GFOIP cultures. All scale bars = 100 mM. Color images available online at www.liebertpub.com/scd

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FIG. 2. Colony-based analysis of FUCCI-G1 expression. (A) Micrograph montages of representative control H2GFOIP hESCcolonies and cells treated with 1% dimethyl sulfoxide (DMSO) for 48 h. Scale bar= 200mM. (B) Graphs depicting the average fluo-rescent intensity OCT4 and FUCCI-G1 expression in individual colonies of control and 1% DMSO-treated H2GFOIP hESC cultures. (C)Micrographs with hESC colonies outlined and numbered that were chosen for cell-level analysis in (D). (D) Histograms showing OCT4and FUCCI-G1 expression profiles for the hESC cells comprising control colony 9 and 48 hr 1% DMSO treated colony 4.

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6-well plates and then recultured for 12 days in undifferen-tiated hESC growth conditions. We then used alkalinephosphatase activity to assay the number of pluripotentcolonies [48]. The FUCCI-G1 - fraction consistently gave riseto significantly more alkaline phosphatase-positive coloniesthan the FUCCI-G1 + cells across all seeding densities(Fig. 3A, B). Treatment with Y27632, a Rho kinase inhibitor,has been demonstrated to improve the cloning efficiency ofhESCs [39]. Post-FACS treatment of both FUCCI-G1 popu-lations with Y27632 elevated the cloning efficiency in bothfractions by more than 10-fold, although the FUCCI-G1 -cells continued to give rise to significantly more pluripotentcolonies than the FUCCI-G1 + fraction.

We next assessed the cell cycle properties of H2GFOIPhESCs in a cell motility assay. Undifferentiated hESCs dis-play strong epithelial bonds mediated by E-Cadherin, whichare lost during differentiation to more motile mesenchymal-like cells [49]. H2GFOIP cells grown in 10% FBS for 48 hdisplayed a sixfold increase in motility in an 8-mM-poretranswell chamber assay, when compared to cells in controlcells treated with FCM (Fig. 3C). Regardless of treatment,approximately half of all cells that demonstrated motility bytransiting through the 8-mM transwell pores expressedFUCCI-G1 (Fig. 3C, D).

These data unequivocally demonstrate that residence inthe G1 phase in hESCs is most closely associated with afunctionally differentiated phenotype, and that identificationof hESCs in the G1 phase can be used to enrich cells withdistinct functional properties.

Differentiated hESCs display diverseG1 phase profiles

FUCCI-G1 expression is low in cells expressing plur-ipotency markers; however, little is understood about the cellcycle status of hESCs that have committed to differentiate. Toexamine the relationship between germ layer differentiationand cell cycle phase distribution, we co-stained H2GFOIPhESCs that had either undergone spontaneous differentiationor been subjected to protocols that induced endoderm orneural differentiation. LAMIN A/C is an intermediate fila-ment protein that has been shown to be upregulated in thespontaneously differentiated cells surrounding undifferenti-ated hESC colonies [50], and can be associated with a mes-enchymal cell fate [51]. In undifferentiated cultures ofH2GFOIP hESCs, we found that LAMIN A/C expression wasrestricted to the cells outside of the undifferentiated hESCcolonies (Fig. 4A). Approximately one-third of LAMIN A/C-positive cells also expressed FUCCI-G1 (Fig. 4B). Induction ofendoderm differentiation [36] by H2GFOIP hESCs generatedcells that stained positive for the endoderm markers EOMES,GATA4, FOXA2, and TCF2 (Fig. 4A). Two-thirds of cells ex-pressing the early endoderm markers EOMES, GATA4, andFOXA2 also expressed FUCCI-G1 (Fig. 4B), whereas almostall TCF2- (HNF1B) positive cells also expressed FUCCI-G1.These data suggest that EOMES, GATA4, and FOXA2, allmark endoderm cells with similar proliferative profiles, but agreater proportion of TCF2-expressing cells is in G1, and ap-parently represent a population of cells with a slower

FIG. 3. Analysis of hESCs by FUCCI-G1 expression. (A) Representative 6-well plate from a colony-initiating cell (CIC) assayperformed on fluorescence-activated cell sorting (FACS) isolated FUCCI-G1-negative ( - ) and positive ( + ) hESCs. Graycolonies denote alkaline phosphatase activity. (B) Graph depicting results from CIC assay performed at 25k, 50k & 100 k(1k = 1,000) of FUCCI-G1 + (F + ) and FUCCI-G1 - (F - ). The 50 k dilution was also seeded in the presence of the Rho kinaseinhibitor Y27632 (Y27). n = 3. (C) Increase in hESC motility in a transwell assay when treated with 10% fetal bovine serum(FBS) in contrast to control cells in a feeder-conditioned hESC medium (FCM) (left), and the percentage of motile cellsexpressing FUCCI-G1 (right). (D) Micrographs of H2GFOIP cells that transited through the 8-mM transwell pores in 10% FBS.Scale bar = 50mM. Statistical significance: * = p < 0.01, ** = p < 0.001, ***p < 0.0001.

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proliferation rate. In contrast to the cell cycle phase profile ofendoderm cells, neural differentiation of hESCs inducedSOX1- and SOX2-positive cells, which both displayed a lowfrequency of cells in the G1 phase (Fig. 4B and SupplementaryFig. S7), an observation consistent with the rapidly prolifer-ating phenotype previously described for neural stem cells. Q-

RT-PCR on FACS-isolated populations of FUCCI-G1 + andFUCCI-G1 - from endoderm-differentiated hESCs demon-strated that markers associated with endoderm differentia-tion were enriched within the FUCCI-G1 + cells relative to theFUCCI-G1 - population (Supplementary Fig. S8). Similarly,pluripotency markers were depleted in the FUCCI-G1 +

FIG. 4. Overlap of FUCCI-G1 with lineage-associatedmarkers. (A) Representativemicrographs of indirect im-munofluorescence for LAMINA/C in undifferentiatedH2GFOIP hESCs and EOMES,GATA4, FOXA2, and TCF2 incells after 3 days of endodermdifferentiation. Scale bars = 100mM. (B) Graph depictingoverlap of FUCCI-G1 withdifferentiation markers.

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population. These observations, supported by our endoderm-associated antibody-based imaging analysis (Fig. 4A), dem-onstrate the preponderance for cells expressing endodermmarkers to be in the G1 phase of the cell cycle.

Time-lapse imaging reveals kinetics of G1 phaseentry during hESC differentiation

The inclusion of the H2B-GFP nuclear marker in theH2GFOIP dual-reporter system not only provided an indi-cator of the ubiquity of H2GFOIP transgene expression in thehESCs but also enabled live quantification of the total cellnumber within a colony by automated image analysis soft-ware. We next used live-cell imaging to record a time-lapseseries of the H2GFOIP hESCs in undifferentiated growthconditions or in response to conditions supplemented withdifferentiation agents (Supplementary Movie S1). Auto-mated software-based image analysis was then used to countall of the nuclei using H2B-GFP fluorescence and then cal-culated the fraction of cells expressing the FUCCI-G1 fusionprotein over the course of 3 days of treatment with differ-entiation agents (Fig. 5A). Four differentiation agents weretested: (1) two polar molecules, HMBA and DMSO, whichare known to initiate the differentiation of hESCs and mouseESCs; (2) the PI3K inhibitor LY294002; and (3) the RB/RAF-1interaction inhibitor RRD-251, which suppresses the hyper-phosphorylation of RB by RAF-1 in the G1 phase, preventingsubsequent entry into the S phase. Treatment of H2GFOIPhESCs with 1% DMSO leads to approximately a quarter ofthe hESCs entering the G1 phase, with the time-to-peakfraction of cells in the G1 phase occurring after around 60 hof treatment (Fig. 5B, C). In contrast, application of HMBA,RRD-251, and LY294002 all peaked at around 36 h of treat-ment (Fig. 5D and Supplementary Fig. S9), with half of thecells in RRD-251-treated cultures displaying high FUCCI-G1expression. Regardless of the time required to reach peakFUCCI-G1 expression, the shape of the FUCCI-G1 responsecurve follows a typical bell-shaped distribution, with a pla-teauless peak quickly tapering to lower FUCCI-G1-positivecell numbers by the end of the treatment course. Interest-ingly, the fraction of cells positive for the FUCCI-G1 reporterincreased in a dose-dependent manner for all differentiationagents tested, indicating that the individual cells withinhESC cultures possess a range of thresholds when respond-ing to differentiation stimuli. A second striking feature ap-parent from the time-lapse study of the H2GFOIP hESCs wasthe pattern of FUCCI-G1 expression in response to differ-entiation agents: initial upregulation of the FUCCI-G1 re-porter occurred in the center of the colony and radiatedoutward (Figs. 5B and 6). This rosette-like pattern of FUCCI-G1 expression is consistent with the restriction ofOCT4-positive cells to the edges of the colonies after 3 daystreatment with differentiation agents like HMBA (Supple-mentary Fig. S10A). Our data demonstrate that changes tothe G1 phase of the cells cycle, communicated by FUCCI-G1expression, is an early indicator of differentiation, and isdetectable in as little as 12–16 h after treatment of hESCcultures with differentiation agents. A 36-h treatment ofH2GFOIP hESCs with HMBA caused a marked change inboth NANOG, a sensitive marker of pluripotency [52], andFUCCI-G1 expression, but was insufficient to initiate a loss ofthe more widely expressed pluripotency marker OCT4 de-

spite a significant increase in the percentage of OCT4 + cellsexpressing FUCCI-G1 (Supplementary Fig. S10B, C). Thesedata show that changes in the expression FUCCI-G1 is atleast as sensitive as NANOG in discerning perturbations inpluripotency, and that both FUCCI-G1 and NANOG re-spond more quickly to differentiation signals than OCT4.

Individual hESCs display variable FUCCI-G1expression and total cell cycle times

We next used the H2GFOIP hESC lines to perform a de-tailed study of the human pluripotent cell cycle. Time-lapseimaging was used to capture images of the H2GFOIP re-porter lines every 15 min over a period of 3 days(Supplementary Movie S2). The H2B-GFP fluorescence pro-vided a pan-cell-cycle nuclear marker that facilitated thetracking of individual cells while also enabling the discrim-ination of cells in the M phase by the significant increase inperceived H2B-GFP intensity due to chromatin condensationduring mitosis (Fig. 7E). We tracked over 100 motherand daughter cells in control and RRD-251-treated culturesand recorded the fluorescent intensity of the H2B-GFP andFUCCI-G1 reporters for each cell throughout the time-lapsestudy. H2GFOIP hESCs in control, undifferentiated condi-tions displayed a mean total cell cycle length of 15.8 h(SEM – 0.3; Fig. 7C). Interestingly, we observed not onlyvariation in the cell cycle length between different cells in theculture (Supplementary Fig. S11A) but also in the total cellcycle length for the same cell between consecutive mitosis(Fig. 7A). The addition of 40mM RRD-251 to the cells leads toalmost a doubling of the mean cell cycle duration (28.6 hSEM – 0.3; Fig. 7B, C and Supplementary Fig. S11A) and asignificant variability between the response of individualcells. The average duration of FUCCI-G1 expression was0.6 h (SEM – 0.1; Fig. 7D) in undifferentiated conditions, alower value than the *3-h G1 phase time derived by others[15]. RRD-251 extended the average FUCCI-G1 expressionduration by a factor of 12 (Fig. 7D), and elicited a range ofcell cycle changes, with some cells expressing FUCCI-G1 forover 50 h (Fig. 7B and Supplementary Fig. S11B), likely dueto G1 phase arrest. Importantly, all of our results show thatan increase in FUCCI-G1 expression is only observed directlyafter mitosis events, strengthening the fidelity of FUCCI-G1reporter in hESCs. Together, these data provide novel insightinto the kinetics of hESC proliferation, revealing extensivecell–cell variability in the cell cycle duration of pluripotentcells and response to stimuli that perturb the normal cellcycle transit.

Increased FUCCI-G1 expression durationis concomitant with acquisition of differentiationmarkers

It is currently unclear if G1 extension in human plu-ripotent stem cells occurs before or after the onset of dif-ferentiation. We next used our H2GFOIP reporter system,in combination with the tracking of single cells from time-lapse imaging, to address this question. H2GFOIP hESCswere placed in endoderm differentiation conditions andimaged over the course of 3 days, immediately fixed, andthen endpoint-stained for the endoderm marker GATA4.This approach allowed GATA4-positive cells to be

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identified and then tracked back through the time-lapsemovie, and a lineage tree was generated. In contrast to theregularity of lineage trees from control hESC culturesmaintained in undifferentiated conditions that display noGATA4 expression (Fig. 8A, D), the lineage trees of cells

that underwent differentiation into GATA4-positive cellsdisplayed less complexity (Fig. 8B). By 24 h of treatment inendoderm differentiation conditions, the average total cellcycle duration underwent a small, but statistically signifi-cant, increase from 15.8 h (SEM – 0.3) to 17.5 h (SEM – 0.5)

FIG. 5. Time-lapse imaging of FUCCI-G1 expression in hESCs. (A) Schematic representation of calculation used to derivethe FUCCI-G1 + fraction. (B) Time-lapse stills of H2B-GFP and FUCCI-G1 fluorescence taken at experiment start (0 h) and at12-h intervals in control or 1% DMSO-treated cells. (C, D) Fraction of FUCCI-G1 + cells during 4 days of treatment with adose range of (C) DMSO or (D) HMBA. Color images available online at www.liebertpub.com/scd

288 CALDER ET AL.

FIG. 6. Colony distribution of FUCCI-G1 during differentiation. Measurements of H2B-GFP and FUCCI-G1 fluorescenceintensity diagonally across the time-lapse stills shown in the figure demonstrate that initiation of FUCCI-G1 expression incells treated with 1% DMSO starts in the center of the colony and radiates outward. Black dotted line indicates the extent of thecolony boundaries in H2G-GFP and FUCCI-G1 plots.

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(Fig. 8C). At 48 h after the start of the endoderm differ-entiation treatment, the average cell cycle time had un-dergone a robust increase to 25.6 h (SEM – 1.0). GATA4staining of surrogate endoderm-differentiated H2GFOIPhESC cultures at the same time points revealed a similarpattern: a small, but significant, increase in GATA4-posi-tive cells by 24 h, followed by a robust increase at 48 h toalmost 30% of cells (Fig. 8D). These data demonstrate apositive correlation between an increase in the cell cyclelength and acquisition of GATA4 expression at 48 h. In-terestingly, although the total cell cycle length increased at24 h, this was not accompanied by a noticeable increase inFUCCI-G1 expression (Fig. 8E). However, by 48 h, FUCCI-G1 expression had increased in cells that were demon-strably GATA4 positive. Our results indicate that the ex-tension of the duration of FUCCI-G1 expression isconcomitant with the expression of differentiation markers

like GATA4, suggesting that differentiation and G1-lengthening are simultaneous events in hESCs.

Discussion

The cell cycle is a finely regulated process that enablescellular growth, replication, and differentiation. In particular,the G1 phase appears to function in the mechanism thatgoverns the choice between proliferation and differentiationof stem cell populations [53]. The unusually short G1 phasethat is common to pluripotent stem cells from a range ofspecies, including mouse, monkey, and human [13,15,54],seems to be an obligatory step in attaining pluripotency.Despite the striking nature of this observation, extant toolsfor unlocking the significance of the abbreviated G1 phasekinetics in pluripotent stem cells have been lacking. Here weprovide a significant new insight into the G1 phase and total

FIG. 7. Single-cell tracking of FUCCI-G1 expression in hESCs. (A–B) Graphs of H2B-GFP (green) and FUCCI-G1 (orange)fluorescent intensity for individually tracked cells in (A) control and (B) 40-mM RRD-251-treated cells. Peaks in the H2B-GFPare due to H2B-GFP condensation during mitosis. Orange peaks describe cells in FUCCI-G1 expression. (C, D) Graphs ofaverage cell cycle duration (time between mitosis); (C) and FUCCI-G1 expression duration (D) in control and RRD-251-treated cells. (E) Panel of H2B-GFP and FUCCI-G1 images from an individual cell tracked for 50 h, showing the condensationof H2B-GFP during mitosis at 1 h, and 2 daughter cells at 2 h. Images taken from cell tracked in (C) bottom right graph. Colorimages available online at www.liebertpub.com/scd

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cell cycle kinetics of undifferentiated and differentiatinghuman pluripotent stem cells, by deploying cell cycle re-porters that function in live cells. Our new data solidify thelinkage between a shortened G1 phase and pluripotency inhESCs, while providing compelling new insights that de-scribe the transition steps and functional outcomes linkingdifferentiation with changes to the cell cycle.

We have used the H2GFOIP reporter, in conjunction withmarkers of pluripotency, to provide a characterization of G1phase kinetics in hESCs that is not obscured by the hetero-geneity common in hESC cultures. We find that a smallminority of OCT4-, NANOG-, or SOX2-positive cells are inthe G1 phase of the cell cycle, as reported by FUCCI-G1. Ourdata for the percentage of hESCs in the G1 phase are lowerthan previous estimates for bulk cultures of hESCs, whichare typically in the range of 20% of hESCs in the G1 phase[15,55]. There are at least 2 potential reasons for this dis-crepancy: 1. By gating only on cells expressing OCT4, NA-NOG, or SOX2 when evaluating FUCCI-G1 expression, wehave filtered out differentiated cells that have longer G1phases. The inclusion of differentiated cells in the data setwould artificially inflate the percentage of cells in the G1phase. 2. The FUCCI-G1 reporter is under-reporting theduration of the G1 phase, probably due to the rapidity of theundifferentiated hESC G1 phase not permitting sufficient

time for full maturation of the mKO2 fluorescent protein.Other estimates for the G1 phase duration in hESCs haveproduced a result of about 3 h [15]. A 3-h G1 phase repre-sents a tight window for the mKO2 fluorescent protein,which has a 1.2-h maturation half-time [56], to fully maturebefore CDT1-mediated degradation occurs in the S phase.Indeed, our cell-tracking data show a small lag between theend of mitosis and the peak FUCCI-G1 expression (Fig. 8B,C), which may contribute to the average FUCCI-G1 ex-pression duration of 0.6 h in undifferentiated hESCs (Fig. 8D,E). We have shown that FUCCI-G1 expression always fol-lows mitosis, as expected for an accurate reporter of the G1phase (Fig. 8B, C). When the time from the end of mitosis tothe subsequent loss of FUCCI-G1 expression is measured inundifferentiated hESCs, a figure of *3 h is derived. There-fore, measuring the duration of FUCCI-G1 expression mayunderestimate the actual G1 phase length by up to 2 h. Thisobservation is consistent with our introduction of the FUC-CI-G1 reporter into undifferentiated mouse ESCs, whichhave a total cell cycle length of 8–10 h and a G1 phase lengthof *1.5 h [57,58], where it was difficult to detect even low-level FUCCI-G1 expression, except in morphologically dif-ferentiated cells (data not shown).

Thus, it is likely that both heterogeneity and fluorescentprotein maturation time may have some impact upon the G1

FIG. 8. FUCCI-G1 expression during endoderm differentiation. Representative lineage tree derived from tracking singleH2GFOIP hESCs in control-undifferentiated conditions (A) or endoderm differentiation (B) conditions for 3 days. The numbersabove each branch denote the total cell cycle time for that branch. Red squares denote cell death, and blue circles denote GATA4-positive cells. (C) Total cell cycle length in control or endoderm differentiation conditions at 24 and 48 h. (D) Percentage ofGATA4-positive cells in control or endoderm differentiation conditions at 24 and 48 h. (E) Graphs of H2B-GFP (green) andFUCCI-G1 (orange) fluorescent intensity for the branches of the endoderm lineage tree (B) marked by the blue and red dotted lines.Statistical significance: * = p < 0.01, ** = p < 0.001, ***p < 0.0001. Color images available online at www.liebertpub.com/scd

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phase profile reported here. Despite these caveats, our ob-servations provide a state-of-the-art evaluation and new in-sights into the G1 phase kinetics of undifferentiated hESCs.

One striking finding that has arisen from our use of a cyclestatus reporter in live cells is the ability to use G1 phase toisolate functionally distinct cell types from undifferentiatedhESC cultures. We have previously shown that lineage-associated markers are capable of isolating hESCs withdiscrete lineage differentiation preferences from undifferen-tiated cultures [46], but to our knowledge, this is the firstproof of functional differences between hESCs residing indifferent stages of the cell cycle. Human ESCs resident in theG1 phase display a significant reduction in the ability toinitiate new undifferentiated hESC colonies. This demon-stration underscores the significance of the coupling of cellcycle changes to differentiation in pluripotent stem cells, andoffers a new mechanism for isolating functionally distinctpopulations during the differentiation of hESCs.

The H2GFOIP reporter system has also permitted us tobegin addressing important, but unanswered, questionsconcerning how lineage-specific differentiation is coupled tochanges in the human cell cycle. We quantified the expres-sion of FUCCI-G1 in conjunction with differentiation mark-ers, like LAMIN A/C, GATA4, or TCF2, and demonstratedfor the first time that differentiation states marked by theexpression of different proteins do not necessarily displayequivalent fractions of cells residing in the G1 phase of thecell cycle. Our data also reveal variation between markers ofthe endoderm germ layer, with a lower fraction of EOMES-,GATA4-, or FOXA2-expressing cells in the G1 phase thanTCF2-positive cells. In the endoderm lineage, EOMES,GATA4, and FOXA2 expression is initiated earlier duringmouse embryonic development than the expression of TCF2[59–62], affording the possibility that EOMES, GATA4, andFOXA2 may identify an earlier, more-proliferative, precursorpopulation. Similarly, the cell cycle reporter we have de-ployed in hESCs now offers a realistic opportunity to explorethe cell cycle properties of a range of developmentally rele-vant tissues, allowing cell types to be indexed by their pro-liferation profile.

These data confirm that we have developed a powerfulnew assay for measuring the cell cycle properties of distinctprecursors and tissues that could have utility in future celltherapy applications. We are currently using the H2GFOIPreporter to screen conditions that modulate the fraction oflineage-marked cells that reside in the G1 phase, with theintent of identifying protocols that invigorate the prolifera-tive potential of lineages like endoderm.

Using the data from time-lapse microscopy experiments,we have developed a new assay for measuring cell cyclekinetics in cultured cells. The total cell cycle time for hESCshas been estimated to take from 16 h through to 30 + h[15,63,64]. Here we derive a definitive cell cycle time forhESCs as averaging around 15.8 h by tracking individualcells through consecutive mitosis events. Additionally, wecan use this same assay to detail changes in the G1 phaselength, and discriminate between a prolonged and arrestedG1 phase. Given that FUCCI-G1 expression increases inhESCs treated with compounds intended for chemothera-peutic use, such as HMBA [65] and RRD-251 [66], the systemwe describe here may provide a novel mechanism for de-scribing the cytostatic nature of compounds with potential

anticancer drugs. The extension of G1 phase can also presageinduction of a differentiation program, making this assayalso of a potential value in the discovery of compounds thatcause hESC differentiation.

A second notable observation arising from our time-lapsedata is the variation in the transit time through the cell cyclebetween independent hESCs, and even the daughter cells ofpreviously tracked cells. Our results show that pluripotent stemcells do not represent a uniform phenotype, but instead arehighly dynamic entities displaying diverse responses to internalor external stimuli. Our time-lapse data demonstrate that (1)FUCCI-G1 expression responds in a dose-sensitive manner tochemical differentiation agents, like DMSO and HMBA; and (2)that the cells at the center of a colony respond first to thesestimuli. These observations have corresponding implications (1)that some cells that are resident within hESC cultures require ahigher signaling threshold before committing to increaseFUCCI-G1 expression; and (2) the cells with lower thresholdsreside in the central regions of colonies.

The signaling thresholds that control hESCs fate are un-derstudied, but some progress has been made in describingthose that operate to control the self-renewal machinery inmurine pluripotency. In mouse ESCs, the signaling pathwaysthat control the undifferentiated state are now understood tooperate in an analog manner, and not with binary on–offthresholds [67], and that the spatial organization of the cellswithin the colony is directly related to the signal respon-siveness of a cell [68]. Measurements of the proliferationactivity of hESC colonies, drawn from static time points,demonstrate that the most mitotically active cells are locatedtoward the colony center [69]. Our data show that cells at thecolony center are also most susceptible to differentiationagents. Rapid proliferation is directly coupled to pluri-potency [70], so the central region may be a reservoir of themost naıve pluripotent cells in the colony. Lineage priming, aprocess in which pluripotent cells begin to differentiate, isknown to involve the extension of the G1 phase duration anda concomitant increase in the total cell cycle length [71]. Incombination with these factors, our data imply that a ring oflineage-primed cells may encircle the central pool of naıvepluripotent cells at the colony center.

Finally, we have used our H2GFOIP system to investigatethe order in which hESC differentiation and G1 lengtheningoccurs. The short G1-phase length found during early de-velopment has been suggested to help maintain a stem cellphenotype by reducing the duration of mitogen sensitivityassociated with the G1 phase [20–23]. However, addressingthe linkage between G1 phase length and differentiation istechnically challenging. In mouse, this question has beentackled in neural stem cells, where gain-of-function experi-ments with cell cycle proteins that are associated with dif-ferentiation, such as cdk4/cyclinD1, have demonstrated thatG1 lengthening is necessary and sufficient to induce differ-entiation of neural progenitors [72]. Similarly, knockdown inhESCs of CDK2, a cell cycle protein that helps drive cell cycleprogression from G1 to S phase, leads to cell cycle arrest anddifferentiation [73]. These informative experiments provideinsight into the linkage between the cell cycle machinery anddifferentiation, but do not necessarily describe the cell cyclephase behavior during the fate decisions that are made bycells in response to differentiation cues found during devel-opment. Here we induced differentiation in our H2GFOIP

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hESCs with an activin A-based endoderm differentiationprotocol, performed live imaging, and then endpoint stainedthe cells with GATA4. These data, in conjunction withGATA4 staining at discrete stages during the differentiationtime course, show that the magnitude of cell cycle length-ening correlates with the extent of GATA4 acquisition. Im-portantly, the expression of the FUCCI-G1 reporter increasesat around 48 h, the time point at which a robust increase inthe percentage of GATA4-positive cells is detectable. Al-though our results are correlative, they imply that G1lengthening occurs at approximately the same time as thegain of endoderm differentiation markers. Single-cell track-ing of hESCs containing the H2GFOIP reporter and a livefluorescent reporter of early cell lineage fate decisions,such as PAX6, GATA4, or BRACHY, may provide a de-finitive connection between differentiation and cell cyclechanges.

Here we have described significant new insights into thehuman pluripotent cell cycle and how it changes during theprocess of differentiation. Our data and tools provide newavenues for investigating and mapping the very early stagesof hESC response to differentiation stimuli via changes in thecell cycle, and could impact the identification of efficaciousdifferentiation strategies to generate therapeutically usefulcell types.

Acknowledgments

Funding was kindly provided by CIHR, SCN, andMRI GL2 OCRIT grants to JSD. JSD is also supported bya Canada Research Chair in Human Stem Cell LineageCommitment.

Author Disclosure Statement

No competing interests are declared.

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Address correspondence to:Dr. Jonathan S. Draper

Stem Cell and Cancer Research Institute (SCC-RI)McMaster University

Hamilton, Ontario L8S 4K1Canada

E-mail: draperj@mcmaster.ca

Received for publication March 30, 2012Accepted after revision July 23, 2012

Prepublished on Liebert Instant Online July 24, 2012

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