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RESEARCH ARTICLE

Telomerase-mediated telomere elongation from humanblastocysts to embryonic stem cells

Sicong Zeng1,2,*, Lvjun Liu1,2,*, Yi Sun1,2,3, Pingyuan Xie1,2, Liang Hu1,2,3, Ding Yuan1,4, Dehua Chen3,Qi Ouyang1,2,3,4, Ge Lin1,2,3,4,{ and Guangxiu Lu1,2,3,4,{

ABSTRACT

High telomerase activity is a characteristic of human embryonic

stem cells (hESCs), however, the regulation and maintenance of

correct telomere length in hESCs is unclear. In this study we

investigated telomere elongation in hESCs in vitro and found that

telomeres lengthened from their derivation in blastocysts through

early expansion, but stabilized at later passages. We report that the

core unit of telomerase, hTERT, was highly expressed in hESCs in

blastocysts and throughout long-term culture; furthermore, this was

regulated in a Wnt–b-catenin-signaling-dependent manner. Our

observations that the alternative lengthening of telomeres (ALT)

pathway was suppressed in hESCs and that hTERT knockdown

partially inhibited telomere elongation, demonstrated that high

telomerase activity was required for telomere elongation. We

observed that chromatin modification through trimethylation of

H3K9 and H4K20 at telomeric regions decreased during early

culture. This was concurrent with telomere elongation, suggesting

that epigenetic regulation of telomeric chromatin may influence

telomerase function. By measuring telomere length in 96 hESC

lines, we were able to establish that telomere length remained

relatively stable at 12.0261.01 kb during later passages (15–95). In

contrast, telomere length varied in hESCs with genomic instability

and hESC-derived teratomas. In summary, we propose that correct,

stable telomere length may serve as a potential biomarker for

genetically stable hESCs.

KEY WORDS: Telomere, Telomerase, Embryonic stem cells, Wnt–b-

catenin signaling

INTRODUCTIONMammalian telomeres are composed of tandem repeats of

(TTAGGG)n DNA sequences and telomere-associated proteins,

which protect chromosome ends and maintain chromosomal

stability (Blackburn, 2001; Palm and de Lange, 2008). The

telomeres of somatic cells progressively shorten with each cell

division due to the end-replication problem (Blasco, 2005). When

telomeres reach a critically short length, cells exhibit genomic

instability and undergo cell cycle arrest, replicative senescence or

apoptosis (d’Adda di Fagagna et al., 2003). Telomere

maintenance is principally associated with telomerase activity,

which protects telomere integrity through the addition of single

stranded TTAGGG repeats onto the 39-ends of chromosomes.

Telomerase activity is generally silenced or found at very low

levels in somatic cells, and telomerase expression is insufficient

to maintain telomere length in most adult stem cells, which limits

cell division (Redaelli et al., 2012). In contrast, most tumor cells,

germline cells and embryonic stem cells (ESC) exhibit high

levels of telomerase expression. An important complementary

maintenance mechanism is the alternative lengthening of

telomere (ALT) pathway, in which telomeres are elongated by

homologous recombination at a later stage of replication. The

ALT pathway has been found to be activated in some tumors

(Henson et al., 2002) and during early embryo development (Liu

et al., 2007).

Human ESCs (hESCs) are derived from the inner cell mass of

human blastocysts. They are capable of indefinite division and are

considered to be a candidate cell source for regenerative

medicine. High telomerase activity is an important

characteristic of ESCs. Our current knowledge of telomere

maintenance in ESCs is largely derived from studies in mouse

ESCs (mESCs): telomere elongation has been observed during in

vitro derivation of mESCs (Varela et al., 2011); loss of

telomerase activity in mESCs resulted in progressive telomere

loss, genomic instability, aneuploidy and telomere fusion,

eventually leading to reduced growth rates (Niida et al., 1998);

telomerase overexpression enhanced self-renewal, improved

resistance to apoptosis and increased proliferation in mESCs

(Armstrong et al., 2005). Tetraploid embryo complementation

experiments have shown that telomerase-deficient mESCs with

short telomeres lose their ability to generate complete ESC-pups

(Huang et al., 2011). In contrast, induced pluripotent stem cells

(iPSC), which resemble ESCs in many ways, with long telomeres,

generate chimeras at a higher efficiency than those with shorter

telomeres (Huang et al., 2011).

In combination, these reports indicate that telomerase plays a

key role in ESC proliferation. Furthermore, correct telomere

function is required to maintain the pluripotency of ESCs and a

correlation exits between telomere length and self-renewal.

Although telomerase activation and restoration of telomere

length are essential for cellular reprogramming (Marion et al.,

2011), telomere maintenance and its biological significance have

not been fully investigated in hESCs. In this study, we monitored

telomere length and maintenance in 96 hESC lines in vitro, from

their derivation in blastocysts throughout early and prolonged

culture. Our data revealed that hESCs preserved telomerase

activity and acquired elongated telomeres during early culture

and these stabilized during later culture. Further investigation

1Institute of Reproductive and Stem Cell Engineering, Central South University,Changsha 410001, China. 2Key Laboratory of Stem Cells and ReproductiveEngineering, Ministry of Health, Changsha 410008, China. 3Reproductive &Genetic Hospital of CITIC-Xiangya, Changsha 410001, China. 4NationalEngineering and Research Center of Human Stem Cell, Changsha 410001, China.

*These authors contributed equally to this work.

{Authors for correspondence: (linggf@hotmail.com; lugxdirector@aliyun.com)

Received 20 March 2013; Accepted 19 November 2013

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 752–762 doi:10.1242/jcs.131433

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indicated that molecular genotyping in aberrant hESCs andteratomas might also be involved in telomere lengthening. These

finding suggested that a correct, stable telomere length could be abiomarker for genomically stable hESCs.

RESULTSTelomere elongation in hESCs occurs during the early expansionin vitro

Four hESC lines, chHES-8, chHES-20, chHES-22 and chHES-69,

were prepared from blastocysts and set to an initial telomerelength, as described in Materials and Methods. Cells at interphasewere analyzed by telomere quantitative fluorescence in situ

hybridization (Q-FISH) during early (#5) and later (.15)passages. Fig. 1A gives the mean fluorescence intensities asarbitrary units (a.u.) relative to LY-S cells. The results revealed

that hESCs from later passages had the longest telomeres, andthat hESCs from early passages had longer telomeres than those

in blastocysts. To verify these observations, telomere length wasmeasured in seven hESC lines at different passages by telomeric

terminal restriction fragment (TRF) assays (Fig. 1B). The resultsconfirmed that early-passaged hESCs (,15) had shortertelomeres than late-passaged hESCs (P,0.05). Furthermore, theelongated telomeres in late-passaged hESCs (15–95) were

maintained at a relatively stable length (P.0.05; supplementarymaterial Table S1; Fig. S1). These results demonstrated thattelomeres in hESCs progressively elongate during early

expansion in vitro, eventually reaching a relatively stable lengthonce the cells have established.

Telomerase regulates telomere lengthening in hESCsTelomerase lengthens telomeres by adding telomeric repeats ontochromosome ends. Telomerase reverse transcriptase (hTERT) is

the limiting factor for telomerase activity. We therefore comparedhTERT expression levels in the inner cell mass of six blastocysts

Fig. 1. Telomeres in hESCs lengthen during early stages of expansion. (A) Q-FISH analysis of telomere length in blastocyst-stage cells (at day 6 afterfertilization) and hESCs. The red spot represents the telomeres. (Left) Telomere length is given in arbitrary units of fluorescence (a.u.); n5number of single cells.(B) TRF assay to determine the telomere length in hESCs at different passages was performed using a biotin-labeled telomeric DNA probe (left). Meantelomere lengths were calculated in seven cell lines (hESC-8, 26, 20, 22, 69, 137 and 177) at different passages (right). P-values were determined using theindependent samples t-test.

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with those in four hESC lines at passage P2. The results showed aconsistently high level of hTERT expression in both groups

(Fig. 2A,B) suggesting that hTERT may play a key role in theregulation of telomere length in hESCs. To determine whetherhTERT expression continued to influence telomere elongation inhESCs during early culture, we measured telomere length in

early-passaged cells after silencing hTERT expression by

lentiviral shRNA interference. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and telomeric

repeat amplification protocol (TRAP) assay showed that bothhTERT expression and telomerase activity decreased by ,70%(supplementary material Fig. S2A,B). In order to investigate themechanism by which telomerase activity influenced telomere

amplification, we measured telomere length in hESCs at different

Fig. 2. Telomere lengthening in hESCs during early passages is telomerase dependent. (A) Real-time qRT-PCR analysis of TERT expression in theinner cell mass and hESCs shows that hESCs initially retain expression levels of pluripotency factors and TERT. The inner cell mass from six unusedblastocysts and cells from four hESC lines at passage P2 were examined. (B) TERT expression levels in hESCs from initial to late passages. The P-valuewas determined using the independent samples t-test. (C) Metaphase cells from the indicated passages were analyzed using the Q-FISH assay. Thehistograms show the frequency of different telomere lengths in TERT-silenced hESCs. The results are from two or more independent experiments; P,0.05.(D) Cell growth curves of cells infected with shRNA control or shTERT. The cells were counted every day; the data are from two independent experiments.

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passages. Although telomere lengthening still occurred in hTERT-deficient cells, it was 50% slower than in wild-type hESCs

(Fig. 2C; supplementary material Fig. S2C). To exclude thepossibility that the growth rate influenced telomere lengthening,we compared growth curves between wide-type and hTERT-silenced hESCs, and found no significant difference between the

two cell types (Fig. 2D). These data suggested that telomeraseactivity is necessary for telomere elongation in hESCs duringearly expansion.

It has previously been suggested than telomere lengtheningoccurs in telomerase-negative cancers and during embryonicdevelopment through the ALT pathway, which is based on

chromosome recombination (Zeng et al., 2009). We thereforeinvestigated the ALT pathway in hESCs by examining the levels of

ALT-associated promyelocytic leukemia (PML) nuclear bodies(APB) and telomere sister chromatid exchange (T-SCE), which arethe hallmarks of ALT pathway activation (Muntoni and Reddel,2005). The data showed that APBs occurred in ,5% of U20S cells

(an ALT cell line) but were absent in hESCs at all passage steps(Fig. 3A). In addition, a low rate of T-SCE was observed in hESCsfrom both initial and late passages compared to U20S cells

(Fig. 3B). These results indicated that the primary mechanism fortelomere elongation and maintenance in hESCs is telomerasedependent, and that the ALT pathway has lesser influence.

Fig. 3. The ALT pathway is suppressed in hESCs. (A) ALT-associated promyelocytic leukemia nuclear bodies (APB) are absent in hESCs. Cells werestained with DAPI (blue) and PML (red) antibody combined with telomeric DNA-FISH (green). In the merged images yellow foci show APBs in ,5% ofU20S cells but none in hESCs. (B) Low levels of telomere sister chromatid exchange (T-SCE) occur in hESCs. BrdU was incorporated into cells from theindicated passages during a single round of DNA replication (,20 hours) and T-SCE analysis was performed using a telomeric C-strand probe (green). Theinset images show non-telomere exchange (two signals; chromosome A) and telomere exchange (three signals; chromosome B). The results were quantified;values are means 6 s.d. from three independent experiments. Statistical analysis was performed using the independent samples t-test.

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To further understand the mechanisms that regulate telomeraseactivity in hESCs, we focused on the Wnt–b-catenin signaling

pathway, which has previously been reported to play a major rolein regulating TERT expression and telomere length in mESCs(Hoffmeyer et al., 2012). Real-time qRT-PCR (Fig. 2A) andchromatin immunoprecipitation (ChIP) assays (Fig. 4A) were

employed to analyze b-catenin mRNA expression in hESCs fromblastocysts and throughout long-term culture. The resultsidentified the hTERT promoter transcription start sequence in b-

catenin pull-downs from hESCs, suggesting that b-catenin bindsdirectly to the hTERT promoter region. Therefore we investigatedwhether Wnt–b-catenin participated in the regulation of

telomerase activity in hESCs undergoing Wnt-3a treatment. Wefound that hTERT expression was upregulated and telomeraseactivity was increased in hESCs after 6 hours of Wnt-3a

treatment (Fig. 4B,C, respectively). In contrast, knockdown ofb-catenin led to decreased hTERT expression and decreasedtelomerase activity in hESCs. Taken together, these resultsindicated that the Wnt–b-catenin pathway was involved in

maintaining telomerase activity in hESCs. It has also beenreported that b-catenin may regulate telomerase activity inmESCs through the transcription factor KLF4. b-catenin and

KLF4 co-immunoprecipitated from hESCs (supplementarymaterial Fig. S3A), therefore we explored the interactionbetween b-catenin and KLF4 in transcriptional regulation of

hTERT. We cloned the core region of the hTERT promoter, whichharbors KLF4 binding sites (2211, +40), into PGL3 luciferasereporters and transfected these into hESCs. The data showed that

overexpression of either KLF4 or b-catenin led to increasedpromoter activity in hESCs, whereas b-catenin knockdown led toinhibition of promoter activity; furthermore, this could not berescued by KLF4 overexpression (supplementary material Fig.

S3B). These data supported the proposed role of b-catenin in theregulation of hTERT expression in hESCs.

Telomere elongation is associated with decreased histonemethylationOur results had revealed that telomere length in hESCs was

relatively stable at later passages. Therefore we speculated thatother factors were involved in regulating telomere length in

addition to telomerase activity. Previous reports have demonstratedthat specific chromatin modifications at telomere sites are involved

in the regulation of telomere length. TRF1, a telomeric DNAbinding protein, is a component of shelterin subunits associatedwith mammalian telomeres. It binds specifically to double-strandedtelomeric DNA and has been reported to enhance telomere

shortening in cancer cells by fastening to chromatin in telomeres,thereby reducing the affinity of telomerase to these regions(Seimiya et al., 2005). However, our data showed that TRF1

expression increased in hESCs at later passages (Fig. 5A). Thissuggested that different chromatin structures may exist in early andlate-passaged hESCs, in agreement with a previous report (Blasco,

2007b). It is now accepted that the main histone modificationsoccur at the H3K9me3 and H4K20me3 sites in telomeric regions,and regulate telomere lengthening (Blasco, 2007a). To observe

changes in chromatin structure during telomere elongation, weperformed double immunofluorescence staining combined withconfocal scanning to measure the extent of colocalization ofH3K9me3 and H4K20me3 with telomeres. In agreement a previous

report, we found that colocalization was ,13% in humanfibroblasts (Varela et al., 2011). In addition, the colocalizationsignal for H3K9me3 and H4K20me3 with TRF1 was 27.9% and

16.7%, respectively, in early-passaged hESCs, reducing to 13.8%and 7.6%, respectively, in late-passaged hESCs (Fig. 5B). A ChIPassay not only showed that binding of these markers to telomeric

DNA decreased in late-passaged hESCs (Fig. 5C) but also that thebinding level to telomeres was stable in these hESCs. Theseobservations indicated that switching between heterochromatin

states might be involved in telomere lengthening in hESCs.

Telomere elongation is not associated with three-germ-layerdifferentiationRecovery of pluripotency and restoration of telomere length aretwin facets of somatic cell reprogramming (West and Vaziri,2010). Therefore, to explore the biological significance of telomere

lengthening, both early-passaged and late-passaged hESCs wereanalyzed using in vitro and in vivo differentiation assays. The datashowed that there were no significant differences in expression

levels of pluripotency markers, including AKP, OCT-4 SSEA-3,SSEA-4, TRA-1-60 and TRA-1-81 between the two passage

Fig. 4. The Wn–b-catenin pathway participates in TERT regulation in hESCs. (A) ChIP analysis of the hTERT promoter region following b-catenin pull-down. (B) Real-time qRT-PCR analysis of TERT expression in Wnt-3a-treated cells at different time points, and in b-catenin knockdown cells. (C) Quantitativeanalysis of telomerase activity in cells undergoing the indicated treatments. Data are means 6 s.d. from three independent experiments.

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groups (Fig. 6A). The hESCs from all the passages expressedthe triploblastic markers NGN3, HNF4 (endoderm markers),Tbx4 (mesoderm marker) and KRT17 (ectoderm marker) informed embryoid bodies, and developed b-tubulin-, AFP- and

SMA-positive cells after 3 weeks in vitro differentiation(Fig. 6B,C). Early- and late-passaged hESCs expressed similarproliferation markers (Fig. 6D) and displayed similar levels ofthree-germ-layer differentiation potential following in vivo

Fig. 5. Differences in chromatin structures between initial and late-passaged hESCs. (A) Western blot analysis of OCT4 and TRF1 levels in hESCs.(B) Heterochromatic markers are reduced in late-passaged hESCs. Fluorescence images show that H3K9me3 (green) or H4K20me3 (green) colocalize withTRF1 (red) in hESCs at the indicated passages. Images were captured by confocal microscopy. The bar graph gives the mean 6 s.d. from three or moreindependent experiments; statistical analysis was performed using the independent samples t-test; n5number of cells. (C) Telomeric DNA-associatedH3K9me3 or H4K20me3 in hESCs at different passages. The ChIP assay was used to analyze hESCs at initial or later passages using telomeric probes. Dataare means 6 s.d. of three independent experiments.

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teratoma formation (Fig. 6E). These data suggested that hESCsmaintain a pluripotent state throughout in vitro culture, and that

telomere elongation during early-stage hESC culture may not bedirectly related to three-germ-layer differentiation.

Aberrant telomere length is associated with genomic instability andteratoma formation in hESCsAs telomere length had been found to be stable after early culture,we measured telomere length after passage P15 in the 96 hESC

lines, including the widely used H9 cell line, to determine the

normal range of telomere length (Fig. 7A). The mean telomerelength was 12.0261.01 kb and did not follow Gaussian

distribution, with telomere lengths of 9.8–14.4 kb between the2.5th and 97.5th percentiles (supplementary material Table S2).We noted that the chHES-3 line, which has an unstable genomicstate and acquires progressive chromosomal abnormalities during

in vitro culture (Yang et al., 2008; Yang et al., 2010), hadsignificantly shorter telomeres (P,0.05) up to passage P60(Fig. 7B). We also observed a distinct elongation of telomeres in

teratomas compared with the original hESCs (Fig. 7C). Teratoma

Fig. 6. hESCs exhibit pluripotency markers during culture. (A) Alkaline phosphatase (AKP) activity and OCT-4, SSEA-3, SSEA-4, TRA-1-60 andTRA-1-81 expression were detected by immunofluorescence staining in both initial and late-passaged hESCs. Nuclei were stained with DAPI (blue; insets).(B) Real-time qRT-PCR assay of triploblastic markers in embryonic bodies. (C) Immunofluorescence staining was performed to detect AFP, SMA andb-tubulin triploblastic markers in 21- to 42-day differentiated hESCs. (D) FACS analysis of Ki67 expression in cells. (E) Tissues of the three germ layers weredetected in hESC-derived teratomas by Hematoxylin and Eosin staining and microscopy.

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formation leads to a failure of ESC-derived cell therapy, evenfollowing successful engraftment and functional improvement

(Bongso et al., 2008), therefore we proposed that the variation intelomere length reflected genomic instability in hESCs. To confirmthis hypothesis, we performed copy number variation (CNV)analyses in hESCs and teratoma cells derived from corresponding

hESCs by SNP 6.0 array using the Affymetrix Genotyping Console4.1.4. Variations in copy number .100 kb between early and late-passaged hESCs or between hESCs and corresponding teratoma

cells were identified (supplementary material Table S4) and theirfrequency was calculated. The data showed that the difference in thenumber of novel CNVs between early and late-passaged hESCs was

not significant (Fig. 8A). The CNV data were analyzed further forthe hESC lines that had been found to have variations in telomerelength: aberrant chHES-3 cells, which have short telomeres, had a

total of 44 different CNVs .100 kb after culture for several months,of which 13 CNVs were .1 Mb; in contrast, normal chHES-3 cells

had only 8 CNVs .100 kb, none of which were .1 Mb. Inaddition, the number of novel CNVs in teratomas was greater than incorresponding hESCs (Fig. 8B). These data suggested that there is acorrect range of telomere lengths in hESCs, implying that variations

may correlate with genomic instability and the potential for teratomaformation in hESCs.

DISCUSSIONIt has previously been established that hESCs derived from theinner cell mass of blastocysts have the capacity for three-germ-

layer differentiation and self-renewal through unlimitedsymmetric cell division in vitro (He et al., 2009). Telomerelength and telomerase activity have also been associated with

Fig. 7. Correct, stable telomere length may reflect genomic stability in hESCs. (A) Analysis of telomere lengths in 96 hESC lines: circles indicatetelomere length at passages .15; triangles represent the chHES-3 cell line at different passages; squares represent teratomas derived from the cell linesindicated by gray circles. Percentile and Gaussian methods were employed. (B) Analysis of telomere lengths in the chHES-3 line at different passages showsabnormally shortened telomeres (P,0.05). Statistical analysis was performed using the independent samples t-test. (C) Telomere length of hESCs andderived teratomas.

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unlimited proliferation in cells. We have shown that a high levelof telomerase activity was maintained in hESCs from initial

derivation to long-term culture; however, the rate of telomerelengthening during the early expansions (,15 passages)stabilized in later passages and telomere length remained

relatively constant (12.0261.01 kb) for at least 95 passages.Similar observations have been reported in relation to cellreprogramming: telomere elongation was accompanied by thecapacity for restoration of self-renewal in iPS cells; and telomere

length was restored in dyskeratosis-congenita-derived iPS cells,which harbor genomic defects in the telomerase RNA component(TERC) (Marion et al., 2009; Agarwal et al., 2010). These

findings indicated that telomere length may be a specific indicatorof the acquisition, restoration and maintenance of self-renewalcapacity in hESC and iPS cells during their establishment in vitro.

Our findings also indicated that telomere elongation in hESCsduring early culture was telomerase dependent, as reducingtelomerase activity in hESCs led to a decrease in the rate of

telomere lengthening and suppression of the ALT pathway.The expression of Wnt family members in feeder cells was

reported to enhance proliferation and improve survival in ESCs(Hasegawa et al., 2012). In addition, Wnt–b-catenin signaling has

been linked to TERT expression in both mESCs and humancarcinoma cells (Hoffmeyer et al., 2012). Therefore we investigatedthe mechanism by which Wnt–b-catenin signaling might regulate

hTERT expression in hESCs. Our results indicated that b-catenininduced hTERT expression by binding to the hTERT promoterregion, suggesting that Wnt–b-catenin signaling regulates telomere

lengthening in hESCs by promoting hTERT expression. We alsofound that KLF4, which is closely associated with pluripotency, wasinvolved in regulating telomerase expression in hESCs, further

supporting a potential relationship between the regulation oftelomere length and the ability for self-renewal.

Histone methyltransferase was reported to control telomere lengthin a knockout mouse model (Garcıa-Cao et al., 2004); and a decrease

in the heterochromatic markers H3K9me3 and H4K20me3 in thetelomeres of mESCs and iPSCs has been observed during telomerelengthening (Marion et al., 2011; Varela et al., 2011). These reports

suggested that chromatin status may influence its affinity fortelomerase and thereby control telomere length. By investigatingthis relationship we found a reduction in these methylation markers

in the telomeres of hESCs during early passages; however, this was

seen to stabilize during later passages, which was similar to thepattern observed for telomere lengthening. Therefore our data

support the hypothesis that chromatin modification plays a role incontrolling and maintaining telomere length, and that a telomerase-mediated mechanism dependent on chromatin modification may

control telomere length in hESCs.Although it has been suggested that demethylation may

increase activation of the ALT pathway in mESCs (Zalzmanet al., 2010), the characteristic features of ALT activation were

absent in this study. Furthermore, we observed telomereelongation in a parthenogenetic cell line (hESC-69), whichlacks long telomeres. These findings support a previous study

showing that ALT signaling can be suppressed by high levels oftelomerase activity in hESCs (Ford et al., 2001).

Maintaining a correct telomere length is one of the key factors for

successful cell replication. Studies on the relationship betweentelomere length and tumorigenesis have reported that a variation intelomere length may affect tumor progression and could be a potential

indicator for patient survival (Zhou et al., 2012). Telomerase-deficientmESCs (Liu et al., 2000) exhibit telomere attrition, leading togenomic instability during prolonged proliferation. Although it isacknowledged that maintaining genetic stability affects indefinite

hESC division, there has been little investigation to determine thecorrect length of telomeres. We found that the majority of hESCs inthis study maintained a relatively stable telomere length during long-

term culture, but abnormal shortening or elongation of telomeres wasobserved in aberrant chHES-3 cells and in hESC-derived teratomas.Genetic instability is acknowledged to be a major obstacle in hESC

therapy against tumorigenesis in vivo (Bongso et al., 2008). However,there is little consensus on a standard method for measuring the earlyevents of genetic instability in hESCs. Our study has demonstrated

that a stable telomere state was obtained after several months ofculture, in accordance with the time required for hESCs to establish in

vitro. This further supported our suggestion that telomere length mayindicate the level of genetic stability in hESCs. We therefore propose

that a correct, stable telomere length could serve as a potential markerfor the characterization of hESCs.

MATERIALS AND METHODSSample preparationThe hESC lines were obtained from the established hESC bank at our

center (Lin et al., 2009). The blastocysts used in this study were collected

Fig. 8. Copy number variation (CNV) changes inhESCs in vitro. (A) Variations in copy numbersbetween early and late-passaged hESCs.(B) Variations in copy number between samples withdifferent telomere lengths.

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from patients receiving in vitro fertilization treatment. Written, informed

consent was obtained from all patients donating surplus embryos for

research purposes. The study was approved by the Reproductive & Stem

Cell Engineering of Central South University and the Reproductive &

Genetic Hospital of CITIC-Xiangya Ethics Committee.

The initial telomere length was set in the inner cell mass of five clinical

unused blastocysts from which the hESCs were derived. The hESC lines

were established by mechanically separating the inner cell mass of 6-day

blastocysts and plating them onto feeder cells for culture (passage zero,

P0). Cells were routinely passaged every 7 days by mechanical cutting.

Cell cultureThe hESCs were cultured in serum-free K-SR medium containing KnockOut

DMEM (Gibco, Carlsbad, CA, USA) supplemented with 15% serum

replacement (Gibco), 0.1 mM b-mercaptoethanol (Sigma, St. Louis, MO,

USA), 1% nonessential amino acids (Gibco), 2 mM L-glutamine (Gibco) and

4 ng/ml human recombinant basic fibroblast growth factor (bFGF; Gibco), at

37 C in a humidified atmosphere of 5% CO2. For Wnt-3a treatment, 100 ng/ml

Wnt-3a was added to feeder-free cultured hESCs in N2B27 medium (Gibco)

with 10 ng/ml bFGF and cell samples were collected at different time points. To

construct the cell growth curve, hESCs were seeded onto 12-well plates. Fresh

medium was added every 24 hours and the cells were counted at 1-day intervals

using a NucleoCounter YC-100 automated cell counter (ChemoMetec,

Norway). LY-S, LY-R and U2OS cells were cultured in DMEM (Gibco)

medium with 10% fetal bovine serum under standard conditions. For embryoid

body formation, hESCs clones were cut and suspended in ES medium (enriched

natural seawater medium) without bFGF and cultured for 14 days to obtain

embryoid body cells; the medium was changed every 24 hours. For in vitro

differentiation, embryoid bodies were attached to slides and cultured for at least

3 weeks in ES medium without bFGF. For teratoma formation, 16106 ESCs

were embedded in 30% Matrigel and injected into the rear leg of a mouse.

Tumors were collected after 6–8 weeks. All animal studies were authorized by

the Xiang-Ya Institute of Animal Use and Care Committee.

AntibodiesThe following antibodies and telomeric probes were used in this study. Primary

antibodies: anti-OCT 3/4, anti-SSEA-3, anti-PML, anti-TRF2, anti-b-tubulin,

anti-AFP, anti-SMA (Santa Cruz Biotechnologies, CA, USA); anti-TRF1, anti-

H4K20me3, anti-KLF4 (mouse), anti-b-catenin (Sigma, St. Louis, MO, USA);

anti-SSEA-4 (R&D Systems, Minneapolis, MN, USA); anti-KLF4 (rabbit;

Abcam, Cambridge, MA, USA); anti-TRA-1-60, anti-TRA-1-81 (Chemicon-

Millipore, Billerica, MA, USA); anti-H3K9me3 (Cell signaling, Danvers, MA,

USA). Secondary antibodies were obtained from Cell signaling (Danvers, MA,

USA). FITC/CY3-labeled Telo-G/C probe was obtained from Dako (Denmark)

and the biotin-labeled Telo-G probe from TaKaRa (Japan).

PlasmidsThe coding sequence for b-catenin cDNA was blunt-end cloned into the

backbone of EcoRI-and SpeI-digested pSin-EF2-SOX2-Pur vector

(Addgene, Cambridge, MA, USA) to generate the pSin-EF2-CTNNB1-

Pur vector. The TERT promoter was PCR amplified from hESC genomic

DNA and the PCR product was digested by KpnI and HindIII and cloned

into KpnI–HindIII sites of the pGL3-Basic reporter vector (Promega,

Madison, WI, USA) to create Luc-Tert-250 bp vector. All primers and

sequences are listed in supplementary material Table S3.

qRT-PCRRNA extraction of inner cell mass and initial hESCs clones was

performed using a RealTime Ready Cell Lysis Kit (Roche, Switzerland)

according to the manufacturer’s protocol. Real-time qRT-PCR was

performed as previously described (Agarwal et al., 2010). All primers

and sequences are listed in supplementary material Table S3.

Telomere sister chromatid exchange fluorescence in situhybridizationCells were examined in metaphase using Q-FISH by culturing established

hESCs for 3–4 days and stalling them at metaphase by incubation with

0.1 mg/l colcemid for 1.5 hours. T-SCE analysis was performed using

chromosome orientation-FISH (CO-FISH) by adding 30 mM BrdU to the

culture medium 18 hours before colcemid treatment, as described above.

The cells were then collected, resuspended in 0.56% KCl buffer for

10 minutes and fixed three times in fixation buffer (methanol:acetic acid,

3:1). Metaphase spreads were prepared by dropping fixed cells onto glass

microscope slides. Cells in interphase were fixed directly onto

microscope slides before examination. The Q-FISH procedure was

performed as previously described (Gonzalo et al., 2006; Zeng et al.,

2009). In order to avoid non-specific signals, the procedure was

optimized by increasing the temperature to 45 C during the first step of

probe washing. LY-S and LY-R cell lines were used to generate a

calibration curve for each experiment. Telomere length was determined

by fluorescence intensity and was integrated using TFl-TELO software.

Terminal restriction fragment assayTRF analysis is highly reproducible and recognized as the gold standard for

telomere length measurement (Allshire et al., 1989). Genomic DNA was

isolated following standard procedures and digested with the HinfI

endonuclease at 37 C overnight. DNA samples were separated by pulsed

field electrophoresis on a 1% agarose gel to obtain a linear separation of 6–

20 kb. Alkaline transfer of the DNA was performed and the membranes

were hybridized using a biotin-labeled telomere probe. A Chemiluminescent

Nucleic Acid Detection Module (Thermo Fisher Scientific, Rockford, IL,

USA) was used for signal detection. Data processing and telomere length

analyses were performed using Quantity One software.

Immunofluorescence microscopyCells were pre-extracted in ice-cold PBS containing 0.5% Triton X-100 for

1 minute at 4 C. The cells were fixed and blocked before being incubated with

the specified primary antibodies at 37 C for 60 minutes, then washed and

incubated with the appropriate secondary antibodies for 45 minutes at 37 C.

The following antibodies were used: anti-PML, anti-TRF2, anti-b-TUBLIN,

anti-AFP, anti-SMA and anti-rabbit IgG (Santa Cruz, CA, USA), anti-TRF1,

anti-H4K20me3, anti-b-catenin (Sigma, MO, USA), anti-H3K9me3, all the

secondary antibodies (Cell signaling, USA). For combined telomeric DNA–

FISH analysis, the cells were immunostained, treated with 0.5 mg/ml RNase A,

dehydrated and incubated in hybridization solution containing 0.3 mg/ml Tel-C

PNA telomeric probes (Applied Biosystems, Carlsbad, CA, USA). The slides

were washed, dehydrated and mounted using anti-fading DAPI medium

(Dako, Carpinteria, CA, USA). The cells were then visualized by fluorescence

(Zeiss, Germany) or confocal (Olympus, Japan) microscopy. For confocal

microscopy, the images were captured as a series of z-sections separated by

0.27 mm, taken at a depth of 30–40 mm. Image data were acquired and

analyzed using confocal software (Olympus).

Chromatin immunoprecipitation assay and telomere dot-blotsChIP assays were carried out as previously described (Zeng and Yang,

2009; Hoffmeyer et al., 2012). Briefly, 26106 hESCs were used for each

experimental condition. Cells were sonicated after cross-linking to obtain

chromatin fragments ,0.5–1 kb in length. After pre-incubation, protein-

A-G–Sepharose slurry and the specified antibodies were added to the

samples followed by rotation for 2 hours at 4 C. The pellets were washed

and the chromatin was eluted from the beads before being hybridized

with the Tel-G (TTAGGG)9 probe or analyzed by real-time qRT-PCR.

Lentiviral shRNA constructsThe shRNAs were designed according to the sequences given in supplementary

material Table S3, and cloned to the MISSIONH pLKO.1-puro lentiviral

construct (Sigma). The viral particles were packaged and collected according to

the manufacturer’s instructions, added to hESC culture medium with 8 ng/ml

polybrene and incubated overnight. The second infection was performed the

following day. After infection, hESCs were selected and cultured with 1 mg/ml

puromycin in ES medium and passaged as usual.

Western blot analysis and co-immunoprecipitation (co-IP)Western blot analyses and immunoprecipitation were carried out following

standard procedures. Briefly, cell extracts were prepared in lysis buffer

containing 50 mM Tris-HCl (pH 7.5), 120 mM NaCl and 1% NP-40 with

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protease inhibitors. Cellular protein (0.5 mg) was immunoprecipitated

with the specified antibodies and protein A/G beads. The proteins were

then separated and analyzed by western blotting.

Telomerase activityQuantitative measurement of telomerase activity was performed using the

TRAPeze Telomerase detection kit or TRAPeze RT Telomerase detection kit

(Millipore, Bedford, MA, USA). Briefly, the cells were lysed on ice for 30 minutes

in CHAPS buffer and then centrifuged at 12,000 g for 20 minutes at 4 C. The

supernatant was collected and the protein concentrations were determined by BCA

protein assay. The TRAP assay was carried out according to the manufacturer’s

instructions, using a total protein extract of 200 ng in each reaction. Telomerase

activity was determined by electrophoresis of the PCR products in a 12.5% non-

denaturing polyacrylamide gel with silver staining, or by fluorometric detection and

real-time quantification of the PCR products using an ABI Prism 7500.

Luciferase assayESCs were separated by adding 0.02% EDTA and then seeded on 24-well

Matrigel-coated plates in mTeSR medium (BD Biosciences, San Jose, CA,

USA). The cells were co-transfected with the plasmids after 48–72 hours

using X-tremeGENE HP (Roche). Luciferase activity was then quantified in a

Dual-Luciferase Reporter Assay System after 72 hours. The reporter activities

were normalized to the internal control in order to minimize experimental

variability due to differences in cell viability or transfection efficiency. All

experiments were performed in duplicate and repeated at least three times.

Statistical analysisThe independent sample t-tests between groups were used to evaluate the

statistical significance of mean values by using SPSS 18.0. The level of

significance was set to P,0.05.

AcknowledgementsWe thank the IVF team of the Reproductive & Genetic Hospital of CITIC-Xiangyafor their assistance and Dr. Tan Yueqiu and Yunhai Liu for proof reading.

Competing interestsThe authors declare no competing interests.

Author contributionsS.Z. devised and designed experiments, analyzed and interpreted data and wrotethe manuscript; L.L. collected, analyzed and interpreted data; Y.S. provided studymaterial, analyzed and interpreted data; L.H. analyzed and interpreted data; P.X.,D.Y., D.C. and Q.O. provided study material or patients; G. Lin. and G. Lu.,devised and designed the experiments, provided financial and administrativesupport and approved the manuscript.

FundingThis work was supported by the National Basic Research Program of China (973program) [grant numbers 2011CB964900 and 2012CB944901 to L.G., L.G.]; theNational Natural Science Foundation of China [grant number 81222007,31271596 and 81101510 to L.G., L.G., S.Y.]; the Ministry of Science andTechnology of China (863 program) [grant numbers 2011AA020113 and2006AA02A102 to L.G., L.G.]; the Natural Science Foundation of Hunan Province[grant number 14JJ2004 to S.Y.]; and the Natural Science Foundation of HunanProvince [grant number 10JJ5043 to Z.D.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.131433/-/DC1

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