6
Different telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells Elisa Varela a , Ralph P. Schneider a , Sagrario Ortega b , and Maria A. Blasco a,1 a Telomeres and Telomerase Group, Molecular Oncology Program, and b Transgenics Unit, Biotechnology Program, Spanish National Cancer Research Centre, Madrid E-28029, Spain Edited by Inder M. Verma, The Salk Institute, La Jolla, CA, and approved July 15, 2011 (received for review April 6, 2011) Murine embryonic stem (ES) cells have unusually long telomeres, much longer than those in embryonic tissues. Here we address whether hyper-long telomeres are a natural property of pluripotent stem cells, such as those present at the blastocyst inner cell mass (ICM), or whether it is a characteristic acquired by the in vitro expansion of ES cells. We nd that ICM cells undergo telomere elongation during the in vitro derivation of ES-cell lines. In vivo analysis shows that the hyper-long telomeres of morula-injected ES cells remain hyper-long at the blastocyst stage and longer than telomeres of the blastocyst ICM. Telomere lengthening during derivation of ES-cell lines is concomitant with a decrease in heterochromatic marks at telomeres. We also found increased levels of the telomere repeat binding factor 1 (TRF1) telomere-capping protein in cultured ICM cells before telomere elongation occurs, coinciding with expression of pluripotency markers. These results suggest that high TRF1 levels are present in pluripotent cells, most likely to ensure procient capping of the newly synthesized telomeres. These results highlight a previously unnoticed difference between ICM cells at the blastocyst and ES cells, and suggest that abnormally long telomeres in ES cells are likely to result from continuous telomere lengthening of proliferating ICM cells locked at an epigenetic state associated to pluripotency. Nanog | Sox2 | Oct4 | embryo M ouse embryonic stem (ES) cells are pluripotent, proliferate indenitely, and bear very long telomeres (13). ES cells emerge from preimplantation blastocyst-stage embryos (4), but how this process takes place is largely unknown. In previous studies, we observed that telomeres of mouse ES cells were much longer than those of mouse embryonic broblasts (MEFs) of the same genetic background (5), which are typically obtained at embryonic day 13.5 (E13.5). This observation raised the issue of whether blastocyst inner cell mass (ICM) cells, which are the natural equivalents of ES cells, also have hyper-long telomeres. If this is the case, then telomeres must shorten during fetal development, despite high telomerase activity (68). An alternative explanation emerges, however, that hyper-long telomeres in ES cell are aberrant and may result from the in vitro establishment and expansion of ES cells. ES-like pluripotent stem cells can be generated from differ- entiated cells (i.e., MEFs) by using dened factors, giving rise the so-called induced pluripotent stem (iPS) cells, which are consid- ered functional equivalents of ES cells (916). We recently showed that iPS telomeres increase in length during and after nuclear reprogramming until reaching ES cell hyper-long telo- meres. This elongation process occurs concomitantly to lower density of trimethylated histones H3K9 and H4K20 at the telo- meric chromatin (5). Furthermore, hyper-long telomeres were not observed in iPS cells derived from rst-generation telomerase- decient MEFs, indicating that they do not originate from a selective reprogramming of a subset of parental cells with very long telomeres; instead, they result from an active telomere elongation by telomerase during and after nuclear reprogram- ming (5). Notably, early passage iPS cells had shorter telomeres than those of ES cells from the same genetic background and only acquired ES cell-like hyper-long telomeres after several passages in vitro (5). These ndings suggest that hyper-long telomeres in iPS cells are the consequence of in vitro expansion of these cells, lending support to the possibility that a similar scenario may be true also for established mouse ES cell lines. Results To directly address these possibilities, we rst analyzed telomere length at different stages of mouse embryonic and fetal de- velopment, including morula, blastocyst, E7.5, E10.5, and E13.5 (Materials and Methods). Embryo sections were hybridized with a telomeric probe and telomere length was measured at a single- cell level by using the telomapping technique (6) (Materials and Methods). We observed that average telomere length signicantly increased from morula to the blastocyst stage (Fig. 1A) and that, although average telomere length was shorter at E7.5 compared with the blastocyst stage, it was maintained constant from E7.5 until E13.5, in agreement with the presence of high telomerase activity throughout embryo development (8, 1721). Strikingly, ES cells processed in parallel showed much longer telomeres than those of blastocyst cells (Fig. 1A). To discard that differences in telomere length are caused by changes in probe accessibility, chromatin status associated to developmental stage, or ploidy, we performed quantitative-FISH (Q-FISH) with a centromeric major satellite probe and found no signicant differences in centromeric uorescence (Materials and Methods and Fig. S1). In this regard, centromeres and telomeres have been reported to share the same heterochromatic marks (22). We next performed a separate analysis of telomere length in trophectoderm (TE) cells versus ICM cells within the same blastocysts by using telomapping. Blastocysts cells were grouped into three categories according to their average telomere uorescence intensity and a color was as- sociated to each group (Fig. 1B, Top). Most of the cells with the longest telomeres (red color) localize to the ICM, and only a few to the trophectoderm (Fig. 1B), and the mean telomere length for the ICM was signicantly higher compared with the TE (Fig. 1B, Bottom). Notably, telomeres of ICM cells were shorter than those of established ES-cell lines, suggesting that ES-cell telomeres un- dergo a signicant lengthening during ES-cell in vitro expansion, in analogy to that previously reported for iPS cells (5). To test this nding, we analyzed in-parallel telomere length in blastocysts and two independent ES-cell lines at both early and late passages by telomapping. Mean telomere length of the ICM was signicantly higher than that of the MEFs and trophectoderm cells and of a similar length to early passage ES cells (passage 5) (89 and 83 Kb, respectively) (Fig. 1C). Telomere length further increased from passage 5 (83 kb) to passage 12 (around 125 kb) (Fig. 1C). In addition, the increased recombination rates of ES cells compared with MEFs (23) could account for the increased heterogeneity in telomere length found in increasing passages of ES cells. By per- forming Q-FISH with a centromeric major satellite probe, we ruled out that these differences in telomere length were because of Author contributions: E.V. and M.A.B. designed research; E.V., R.P.S., and S.O. performed research; E.V. and M.A.B. analyzed data; and E.V. and M.A.B. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1105414108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105414108 PNAS | September 13, 2011 | vol. 108 | no. 37 | 1520715212 CELL BIOLOGY Downloaded by guest on November 13, 2020

Different telomere-length dynamics at the inner cell mass ... · ESP7 Mean length ± SE (kb):81,93 ±1,06 Number of cells/ES:1129/2 Telomeres < 25kb: 2,7 % Telomeres > 60kb:

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Page 1: Different telomere-length dynamics at the inner cell mass ... · ESP7 Mean length ± SE (kb):81,93 ±1,06 Number of cells/ES:1129/2 Telomeres < 25kb: 2,7 % Telomeres > 60kb:

Different telomere-length dynamics at the inner cellmass versus established embryonic stem (ES) cellsElisa Varelaa, Ralph P. Schneidera, Sagrario Ortegab, and Maria A. Blascoa,1

aTelomeres and Telomerase Group, Molecular Oncology Program, and bTransgenics Unit, Biotechnology Program, Spanish National Cancer Research Centre,Madrid E-28029, Spain

Edited by Inder M. Verma, The Salk Institute, La Jolla, CA, and approved July 15, 2011 (received for review April 6, 2011)

Murine embryonic stem (ES) cells have unusually long telomeres,much longer than those in embryonic tissues. Here we addresswhether hyper-long telomeres are a natural property of pluripotentstem cells, such as those present at the blastocyst inner cell mass(ICM), or whether it is a characteristic acquired by the in vitroexpansion of ES cells. We find that ICM cells undergo telomereelongation during the in vitro derivation of ES-cell lines. In vivoanalysis shows that the hyper-long telomeres of morula-injectedES cells remain hyper-long at the blastocyst stage and longer thantelomeres of the blastocyst ICM. Telomere lengthening duringderivation of ES-cell lines is concomitant with a decrease inheterochromatic marks at telomeres.We also found increased levelsof the telomere repeat binding factor 1 (TRF1) telomere-cappingprotein in cultured ICM cells before telomere elongation occurs,coinciding with expression of pluripotency markers. These resultssuggest that high TRF1 levels are present in pluripotent cells, mostlikely to ensure proficient capping of the newly synthesizedtelomeres. These results highlight a previously unnoticed differencebetween ICM cells at the blastocyst and ES cells, and suggest thatabnormally long telomeres in ES cells are likely to result fromcontinuous telomere lengthening of proliferating ICM cells locked atan epigenetic state associated to pluripotency.

Nanog | Sox2 | Oct4 | embryo

Mouse embryonic stem (ES) cells are pluripotent, proliferateindefinitely, and bear very long telomeres (1–3). ES cells

emerge frompreimplantation blastocyst-stage embryos (4), but howthis process takes place is largely unknown. In previous studies, weobserved that telomeres of mouse ES cells were much longer thanthose of mouse embryonic fibroblasts (MEFs) of the same geneticbackground (5), which are typically obtained at embryonic day 13.5(E13.5). This observation raised the issue of whether blastocystinner cell mass (ICM) cells, which are the natural equivalents ofES cells, also have hyper-long telomeres. If this is the case, thentelomeres must shorten during fetal development, despite hightelomerase activity (6–8). An alternative explanation emerges,however, that hyper-long telomeres in ES cell are aberrant andmayresult from the in vitro establishment and expansion of ES cells.ES-like pluripotent stem cells can be generated from differ-

entiated cells (i.e., MEFs) by using defined factors, giving rise theso-called induced pluripotent stem (iPS) cells, which are consid-ered functional equivalents of ES cells (9–16). We recentlyshowed that iPS telomeres increase in length during and afternuclear reprogramming until reaching ES cell hyper-long telo-meres. This elongation process occurs concomitantly to lowerdensity of trimethylated histones H3K9 and H4K20 at the telo-meric chromatin (5). Furthermore, hyper-long telomeres were notobserved in iPS cells derived from first-generation telomerase-deficient MEFs, indicating that they do not originate froma selective reprogramming of a subset of parental cells with verylong telomeres; instead, they result from an active telomereelongation by telomerase during and after nuclear reprogram-ming (5). Notably, early passage iPS cells had shorter telomeresthan those of ES cells from the same genetic background and onlyacquired ES cell-like hyper-long telomeres after several passagesin vitro (5). These findings suggest that hyper-long telomeres iniPS cells are the consequence of in vitro expansion of these cells,

lending support to the possibility that a similar scenario may betrue also for established mouse ES cell lines.

ResultsTo directly address these possibilities, we first analyzed telomerelength at different stages of mouse embryonic and fetal de-velopment, including morula, blastocyst, E7.5, E10.5, and E13.5(Materials and Methods). Embryo sections were hybridized witha telomeric probe and telomere length was measured at a single-cell level by using the telomapping technique (6) (Materials andMethods). We observed that average telomere length significantlyincreased from morula to the blastocyst stage (Fig. 1A) and that,although average telomere length was shorter at E7.5 comparedwith the blastocyst stage, it was maintained constant from E7.5until E13.5, in agreement with the presence of high telomeraseactivity throughout embryo development (8, 17–21). Strikingly, EScells processed in parallel showed much longer telomeres thanthose of blastocyst cells (Fig. 1A). To discard that differences intelomere length are caused by changes in probe accessibility,chromatin status associated to developmental stage, or ploidy, weperformed quantitative-FISH (Q-FISH) with a centromeric majorsatellite probe and found no significant differences in centromericfluorescence (Materials and Methods and Fig. S1). In this regard,centromeres and telomeres have been reported to share the sameheterochromatic marks (22). We next performed a separateanalysis of telomere length in trophectoderm (TE) cells versusICM cells within the same blastocysts by using telomapping.Blastocysts cells were grouped into three categories according totheir average telomere fluorescence intensity and a color was as-sociated to each group (Fig. 1B, Top). Most of the cells with thelongest telomeres (red color) localize to the ICM, and only a few tothe trophectoderm (Fig. 1B), and themean telomere length for theICM was significantly higher compared with the TE (Fig. 1B,Bottom). Notably, telomeres of ICM cells were shorter than thoseof established ES-cell lines, suggesting that ES-cell telomeres un-dergo a significant lengthening during ES-cell in vitro expansion, inanalogy to that previously reported for iPS cells (5). To test thisfinding, we analyzed in-parallel telomere length in blastocysts andtwo independent ES-cell lines at both early and late passages bytelomapping. Mean telomere length of the ICM was significantlyhigher than that of the MEFs and trophectoderm cells and ofa similar length to early passage ES cells (passage 5) (89 and 83Kb,respectively) (Fig. 1C). Telomere length further increased frompassage 5 (83 kb) to passage 12 (around 125 kb) (Fig. 1C). Inaddition, the increased recombination rates of ES cells comparedwith MEFs (23) could account for the increased heterogeneity intelomere length found in increasing passages of ES cells. By per-formingQ-FISH with a centromeric major satellite probe, we ruledout that these differences in telomere length were because of

Author contributions: E.V. and M.A.B. designed research; E.V., R.P.S., and S.O. performedresearch; E.V. and M.A.B. analyzed data; and E.V. and M.A.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105414108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1105414108 PNAS | September 13, 2011 | vol. 108 | no. 37 | 15207–15212

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changes in probe accessibility, chromatin status, or ploidy (Materialsand Methods and Fig. S2). Of relevance, this continuous incrementof telomere length over passages in pluripotent cells is not observedin established immortal human (Fig. S3) or mouse cell lines (24),which show stable telomeres over passages. To test whether telo-mere length was further increased after passage 12, we expandedthe cells until passage 31 and performed telomapping analysis. Wefound that telomeres continued to increase their length at these latepassages, although the difference between passage 24 and 31 wasnot statistically significant (Fig. S4). In conclusion, the reset oftelomere length during development happens at the blastocyststage, in accordance with a previous report showing telomereelongation at the transition from morula to blastocyst (25). Im-portantly, we first demonstrate here that the longest telomereswithin blastocysts localize to the ICM, suggesting that telomereelongation specifically occurs in this subset of pluripotent embry-

onic cells. In addition, ES cells undergo a further increase in telo-mere length compared with ICM cells of the blastocyst.The telomere length of the blastocyst ICM was comparable to

that of ES cells at passage 5, raising the possibility that telomerelength of established ES cells was inherited from the blastocystICM, and telomere lengthening restricted to in vitro expansion ofestablished ES cell lines. To test this hypothesis, we sought to an-alyze in-depth telomere dynamics at the earliest steps during es-tablishment of ES cells. The very first step is the in vitro ICM,obtained from 3.5-d blastocysts upon removal of the zona pellucida.After a few days, colonies of about 1,000 cells are formed (schemein Fig. 1D; images of ICMcolonies in Fig. S5; see alsoMaterials andMethods). In vitro ICM colonies are individually trypsinized andtransferred to 96-well plates, where ES cells start to emerge. Fur-ther expansion leads to the establishment of ES cells (Fig. 1D). Wemeasured telomere length by telomapping in the ICM and troph-

Freq

uenc

yFr

eque

ncy

Freq

uenc

yFr

eque

ncy

ESP10

Mean l ength ± SE (kb):129,6 ± 2,26Number of cells/ES:564/2Telomeres < 25kb:0,1%Telomeres > 60kb: 95,1%

ESP8

Mean l ength ± SE (kb):118,2 ±1,54Number of cells/ES:670/2Telomeres < 25kb: 0,89 %Telomeres > 60kb: 91,49%

ESP9

Mean l ength ± SE (kb):151,3 ±3,2Number of cells/ES:236/2Telomeres < 25kb: 0,42 %Telomeres > 60kb: 96,6%

Telomere length (kb)

Telomere length (kb)

Telomere length (kb)

01020304050

0 100 200 300

0

5

10

15

0 100 200 300

010203040

0 100 200 300

01020304050

0 100 200 300

0

10

20

30

0 100 200 300

1066 cellsn=2

72 cellsn=11

48 cellsn=11

859 cellsn=2

1129 cellsn=2

670 cellsn=2

236 cellsn=2

564 cellsn=1

954 cellsn=2 434 cells

n=2

732 cellsn=2

Telo

mer

e le

ngth

(kb)

0

40

80

120

160

TE ICM (Bl)

ESP5

ESP6

ESP7

ESP8

ESP9

ESP10

ESP11

ESP12

MEF

*

** ***

****

*p<0.0001

C

A B0 – 40

40 – 60

60 – 100

Blastocyst sections

ICM-like TE-like

ICM (Bl)= ICM from blastocyst TE= Trophectoderm B= blastocyst

ES= embryonic stem cellsMEF= mouse embryonic fibroblasts

Telo

mer

e le

ngth

(a.

u.)

0

20

40

60

80

B TE ICM (Bl) ESP9 MEF

* **

*p= 0.0001

n=9117 cells n=9

117 cells

n=9117 cells

n=2603 cells

n=2307 cells

MEF

Mean l ength ± SE (kb):39,6 ± 0,7Number of cells/MEF: 732/3Telomeres < 25kb: 32,5 %Telomeres > 60kb: 16,17 %

ICM (Bl)

Mean l ength ± SE (kb):89,3 ± 2,8Number of cells/blastocysts:28/11Telomeres < 25kb: 0 %Telomeres > 60kb: 100 %

Trophectoderm (TE)

Mean l ength ± SE (kb):39,65 ± 1,41Number of cells/blastocysts:72/11Telomeres < 25kb: 18%Telomeres > 60kb: 4,2 %

ESP5

Mean l ength ± SE (kb):83,22 ±1,07Number of cells/ES:1086/2Telomeres < 25kb: 2,48 %Telomeres > 60kb: 72,9 %

ESP7

Mean l ength ± SE (kb):81,93 ±1,06Number of cells/ES:1129/2Telomeres < 25kb: 2,7 %Telomeres > 60kb: 72,5 %

055

110165220

0 100 200 300

048

1216

0 100 200 300

0

2

4

6

0 100 200 300

ICM(C)= ICM culturedICM(Bl)= ICM from blastocysts T(Bl)= Trophectoderm from blastocyst

96 well = ICM colonies transferred to 96-well plate

Telomere length (kb)Telomere length (kb)

ICM (blastocyst)

Mean l ength ± SE (kb): 82,4 ±7,5Number of cells/blastocysts: 21/6Telomeres < 25kb: 0%Telomeres > 60kb: 100%

TE (blastocyst)

Mean l ength ± SE (kb): 30,4 ±1,3Number of cells/Blastocysts: 77/6Telomeres < 25kb: 28,5%Telomeres > 60kb: 20,7%

96 well

Mean l ength ± SE (kb): 88,9 ± 5,3Number of cells/colonies: 106/12Telomeres < 25kb: 8,4%Telomeres > 60kb: 62,26%

ICM (cultured)

Mean l ength ± SE (kb): 54,9 ±2,9Number of cells/ICM: 107/20Telomeres < 25kb: 20,5%Telomeres > 60kb: 42,9%

Telomere length (kb)

ESP11

Mean l ength ± SE (kb):121,7 ±1,42Number of cells/ES:954/2Telomeres < 25kb: 0,88 %Telomeres > 60kb: 85,6 %

ESP12

Mean l ength ± SE (kb):125,6 ± 2,16Number of cells/ES:434/2Telomeres < 25kb: 0,69 %Telomeres > 60kb: 93,7 %

ESP9

Mean l ength ± SE (kb): 120,5 ± 2,2Number of cells/ES: 556/2Telomeres < 25kb: 0,71%Telomeres > 60kb: 87,4%

ESP12

Mean l ength ± SE (kb): 123,8 ± 2,9 Number of cells/ES: 251/2Telomeres < 25kb: 0%Telomeres > 60kb: 92,4%

iPS P1

Mean l ength ± SE (kb): 55,9 ± 0,9Number of cells/IPS: 1885/2Telomeres < 25kb: 22,17%Telomeres > 60kb: 39,09%

iPS P29

Mean l ength ± SE (kb): 103,6 ± 0,7Number of cells/IPS: 1672/2Telomeres < 25kb: 0%Telomeres > 60kb: 96,35%

ESP5

Mean l ength ± SE (kb): 80,5 ±1,2Number of cells/ES: 1326/2Telomeres < 25kb: 6,7%Telomeres > 60kb: 64,1%

MEF

Mean l ength ± SE (kb): 43,6 ± 0,9Number of cells/MEF: 945/1Telomeres < 25kb: 34,97%Telomeres > 60kb: 13,5%

0

2

4

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1

3

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18

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020406080

0 100 200 300

0

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05

101520

0 100 200 300

020406080

100

0 100 200 300

Freq

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Freq

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D Blastocyst ICM (C) Emergence of ES cells ES P1 ES P12

96 well 24 well 25 mm

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ICM(Bl) TE ICM(C) 96 well ESP5 ESP9 ESP12 iPSP1 MEFiPSP29

**

020406080

100120140

12 cellsn=6

77 cellsn=6

120 cellsn=20

106 cellsn=12

1326 cellsn=2

556 cellsn=2

251 cellsn=2

1885 cellsn=2

945 cellsn=1

1672 cellsn=2* *

*

EFreq

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Freq

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y

Freq

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Freq

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0 200 300100

02468

0 200 3001000

4080

120160

0 100 200 300

050

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01020304050

0 100 200 300

ESP6

Mean l ength ± SE (kb):99,02 ±1,24Number of cells/ES:859/2Telomeres < 25kb: 0,93 %Telomeres > 60kb: 84,1 %

0

10

20

30

40

50

60

70

M Bl E 7.5 E 10.5

Telo

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.)

E 13.5 ESP9 MEF

*p<0.0001

n=613 cells

n=1242 cells

n=33894 cells

n=41901 cells n=2

6489 cells

n=2182 cells

n=2529 cells

* ** * *

* **

***

*

*

**

**

Fig. 1. Blastocyst ICM bears the longesttelomeres which further lengthen upon ex-pansion of ICM-derived ES cells. (A) Quantifi-cation of telomere length by telomappinganalysis of embryo sections at the indicatedstages of development, ES cells (passage 9)and primary MEFs (passage 2). Telomere-lengthquantification isgiven inarbitraryunitsof fluorescence (a.u.). n = number of embryosor independent cell cultures. (B) (Top) Repre-sentative image of a telomapping of a blasto-cyst section. Nuclei are colored according totheir telomere length and normalized by thetelomere lengthofEScells.BecauseEScells arederived from the ICM we reasoned that theyshould have equivalent telomere length. Thedivision of the CY3 intensity value of eachblastocyst cell by themeanCY3 intensity valueof ES cells should render the blastocyst cellswith the longest telomeres (values around orequal to 1). For the blastocyst map we grou-ped intensity values in three fractions to sim-plify the identification of the cells with thelongest telomeres. Note that the longesttelomeres localize to the ICMof theblastocyst.(Scale bar, 10 μm.) (Lower) Quantification oftelomere length of blastocyst, ES cells andMEFs, as indicated. n = number of embryos orindependent ES and primaryMEF cultures. (C)Telomere-length frequency histograms ofblastocysts, ES cells at the specified passages,and primary MEFs and mean telomere lengthfor the same samples. Note that the telomerelength of ES at early passages is similar to thatfound in the ICM. n = number of embryos orindependent ES or primary MEFs cultures. (D)Scheme of the process of isolation of ES cellsfrom blastocysts. In brief, zona pellucida isremoved from blastocysts and they are trans-ferred to a 60-mm dish. After 4 to 6 d the ICMhas divided to ∼1,000 cells. Individual ICMcolonies are transferred to a 96-well plate. Atthis step, ES colonies emerge and are trans-ferred to a 24-well plate for expansion. Fromthe 24-well plate, cells are transferred to 25-mm plates and are considered passage 1.Further passages are plate colonies or ES andprimary MEF cultures. (E) Mean telomerelength and telomere-length frequency histo-grams in ICM from the blastocyst, in vitro cul-tured ICM, emerging ES from the 96-wellplate, established ES cells (passages 5, 9, and12), iPS cells (passage 1 and 29), and primaryMEFs determined by telomapping. Note thattelomeres of the ICM at the blastocyst arelonger than those of the cultured ICM.

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oblasts in blastocysts, in cultured ICM, in the ICM-derived cellsgrown in 96-well plates, and in established ES-cell lines at passages5, 9, and 12 (see scheme in Fig. 1D). We also included iPS cellsat both early and late passages. We confirmed that telomereslengthen during in vitro expansion of ES cells (80 kb at passage 5compared with 123 kb at passage 12) (Fig. 1E). Similarly, iPS-celltelomeres increased with passages (Fig. 1E) (5). Interestingly,telomeres from the in vitro ICM (55 kb) were shorter than those ofthe blastocyst ICM (86 kb) but seemed to recover their length atthe 96-well plate (89 kb) (Fig. 1E), which showed similar telomeresto early passage (passage 5) ES cells (80 kb). We confirmed thesefindings by using an independent technique based on Southernblotting (telomere restriction fragment analysis, TRF) (Fig. S6).These results may suggest that the cells from the ICM are sus-ceptible culture-stress–induced telomere-length changes. Indeed,during the establishment of ES-cell lines, the transient ICM of theearly blastocyst is forced to artificially exist and divide for severaldays in vitro. Under culture conditions, most ICM cells differen-tiate (only 17% and 38.5% of cells express the pluripotency factorsSox2 and Oct3/4, respectively), which in turn may lead to telomereshortening compared with pluripotent stem cells (5, 6).To better understand the dynamics of telomere lengthening in

the cultured ICM, and to avoid contamination with feeder cells(irradiated MEFs), we analyzed telomere length after 4 and 7 d ofculture, in the absence of feeders, by using telomapping (Fig. S7).We did not find any statistically significant difference in the telo-mere length at 4 or 7 d of culture. We also ruled out that meantelomere length of the in vitro cultured ICMwas lower than that ofthe blastocyst ICMbecause of the contribution of irradiatedMEFs.Next, we set to confirm telomere shortening in the in vitro

ICM, as well as telomere lengthening of ES cell over in vitroexpansion, by using Q-FISH on metaphase spreads. Metaphasespreads allow analysis of every single telomere at chromosomes ofa given metaphase. We confirmed shorter telomeres in the cul-tivated ICM (50 kb), which increased in length with subsequentpassages from a mean telomere length of 112 kb in passage 5 toa mean telomere length of 144 kb in passage 12 (Fig. 2A and Fig.S8A; note that absolute telomere-length values were higher thanin the telomapping experiment, most likely because of differencesin acquisition and the software used to measure intensity).

To in vivo test whether established ES cells have longer telo-meres than those of the ICM of the blastocyst, we aggregated EScells with hyper-long telomeres expressing GFP with eight-cellmorulae (Fig. 2B and Materials and Methods). At the blastocyststage, development was stopped and combined telomere FISH/GFP immunofluorescence was performed (Materials and Meth-ods). We found that average telomere length in GFP-expressingICM cells (derived from aggregated ES cells) was higher than thatof non-GFP–expressing ICM cells (derived from the recipientmorulae) (Fig. 2 C and D and Fig. S8B). These results demon-strate that established ES cells have longer telomeres than thecells of the blastocyst ICM. In addition, these results rule outpossible effects of different developmental stages on telomere-length measurements, as we are comparing the same cell typewithin the blastocyst ICM. In summary, these findings stronglysupport the unique finding of active mechanisms, leading to verylong telomeres in the process of ES-cell line establishment, whichare likely to involve telomere elongation by telomerase (5).We reasoned that the increase in telomere length observed in

established and during the establishment of ES cells could belinked to the structure of chromatin and ultimately to the epi-genetic status of telomeres, which is different to that observed inMEFs (5, 22). To test this idea, we first measured the global- andsubtelomeric-DNA methylation (SI Materials and Methods). Be-cause pericentric and subtelomeric repeats remain unalteredbetween ES and differentiated cells (5, 16), we analyzed the in-terspersed repeats (SINE repeats) and found no substantial dif-ference in DNA-methylation between the passages of ES cellsand MEFs (Fig. S9A). We found subtelomeric DNA mostlymethylated with small variations between the passages, whichwere not statistically significant (Fig. S9 B–D). We next analyzedheterochromatic marks at telomeres by performing FISH witha telomere probe combined with immunofluorescence for bothtrimethylation at lysine 20 of histone H4 (H4k20me3) andat lysine 9 of histone H3 (H3k9me3) (5, 22, 26–29). H4k20me3average fluorescence was similar in primary MEFs, ICM, and 96-well cells, but very significantly decreased in established ES cells.However, histograms of the frequency of cells with a givenH4K20me3 fluorescence already show a population of cells withlow H4k20me3 abundance in the cultured ICM and the cells inthe 96-well plates. Indeed, the percentage of cells with H4k20me3

Telo

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ngth

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)

3259 tel.

020406080

100120140160

ICM (C) ESP5 ESP6 ESP7 ESP8 ESP9 ESP10 ESP11 ESP12 MEF

3064 tel.n=26

1444 tel.n=12 3352 tel.

n=22 2320 tel.n=11

4045 tel.n=19 3972 tel.

n=203513 tel.n=23 4466 tel.

n=202124 tel.n=20

n=22

A

*p< 0,0001** ****

* * **

** ***

10 µm

GFP positive cells

Telomere FISH GFP

DAPI

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Blastocyst

B

Injectedblastocyst

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10152025303540

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Bl TE ICM(Bl)

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Telo

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(a.u

.)

110 cellsn=3 367 cells

n=8

157 cellsn=8

48 cellsn=8

529 cellsn=10

214 cellsn=10

87 cellsn=10

42 cellsn=10

122 cellsn=1

153 cellsn=2

* * ** *0,39

*p< 0,0001*

C

D

Fig. 2. Telomere-length dynam-ics during establishment and ex-pansion of ES cell lines as wellas in vivo aggregation of ES cellsin morulae. (A) Mean telomerelength for ICM cultivated fromthe 60-mm tissue-culture plate,and successive passages of ES cells.Telomere length was analyzed bymetaphase Q-FISH. n = number ofICM colonies or independent ESand primary MEF cultures. (B)Scheme of the aggregation ex-periments. Established ES cells atpassage 16 expressing GFP weremicroinjected in eight-cell moru-lae. Blastocyst from injected andnoninjected morulae were fixedfor the analysis of telomerelength by telomapping. (C) Meantelomere length for primaryMEFs (passage 2), noninjectedand injected blastocysts, as well asGFP-ES cells before injection (pas-sage 16) and ES cells at passage 9.n = number of blastocysts or in-dependent clones of ES cells orprimary MEFs. (D) Representativeimages of an injected blastocyst.

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fluorescence below 7 arbitrary units increased from MEFs (16%)to the ICM (33.7%) and 96-well plate (36.9%), to reach 83.5% inestablished ES-cell lines (Fig. 3 A–D). Very similar results wereobserved for H3k9me3 (Fig. 3 E–H). A lower colocalization ofheterochromatic marks with telomeres was also observed duringthe process of ES cell generation (Fig. 3 C and G) (5). Together,these results indicate a decrease in bothH3K9me3 andH4K20me3heterochromaticmarks during the generation of ES cells comparedwith MEFs, starting in the in vitro ICM. These unprecedentedfindings suggest that telomere lengthening is concomitant with

lower density of heterochromatic marks during the process of ES-cell establishment. Alternatively, only cells with a more open/less-compacted chromatin structure are selected from the blastocyststage to obtain stable ES-cell cultures.Next, we reasoned that the mechanisms leading to telomere

elongation during the establishment of ES-cell lines might belinked to pluripotency (30–35). Indeed, adult stem-cell compart-ments bear the cells with the longest telomeres in mice (6). Asa marker for pluripotency, we first tested Nanog, which is requiredto maintain pluripotency in the mouse epiblast and ES cells (32,36). To this end we combined immunofluorescence using a Nanogantibody with FISH for telomeres (Materials and Methods). Again,ICM-cultured cells had shorter telomeres than those from the 96-well plate or established ES cells (Figs. S10 A–C and S11A). In-terestingly, Nanog showed very low expression in the cultivatedICM (3% Nanog-positive cells) (Fig. S10B, Lower graph), whichwas dramatically increased at late-passage ES cells (Fig. S10B).Accordingly, the best positive slope between telomere length andNanog was found only in established ES cells (Fig. S11B). Theseresults suggest that Nanog expression and telomere length do notcorrelate during early stages of establishment of ES-cell lines, andthis only occurs at later passages.Several lines of evidence suggest a link between pluripotency

and the telomere-binding proteins, known as shelterins (37–39).The shelterin protein TPP1 is essential for telomere elongation bytelomerase during reprogramming of MEFs into iPS cells (40). Inaddition, deletion of TRF1 causes lethality at the blastocyst stage(41), and adult tissues conditionally deleted TRF1, show severestem-cell defects (40, 42). Thus, we next explored the regulation ofTRF1 during establishment of ES cell lines. TRF1 binds andprotects telomeres (18, 37, 38) and is proposed to have a role intelomere length regulation (43–46). We observed high TRF1levels already in the cultured ICM compared with primary MEFs(Figs. S10D–F and S11D). TRF1 levels were also high in emerging(96-well) and established ES cells, and Nanog showed similar ex-pression to the previous experiment (Fig. S10B). Thus, high levelsof TRF1 were associated with high levels of Nanog expression inemerging or established ES cells, but not in the in vitro ICM. Wetherefore tested whether TRF1 levels in the in vitro ICM associ-ated to other pluripotency markers. Oct3/4 or Sox2 function in themaintenance of pluripotency in early embryos and established EScells (47–49) and are essential for the reprogramming of differ-entiated cells into iPS (14–16). To test this possibility, we per-formed immunofluorescence with TRF1 and Sox2 (Figs. S10 G–Iand S12A). Interestingly, the mean intensity value for Sox2 in thecultured ICM was twice higher than in MEFs, and further in-creased in emerging and established ES cell lines (Figs. S10H andS12A). Similar results were found when TRF1 and Oct3/4 anti-bodies were used (Fig. 4 A–C and Fig. S12D). Of note, the per-centage of positive cells for Sox2 and Oct3/4 in the in vitro ICM(17% and 38.5%, respectively) was higher than that of Nanog(3%). Despite the high levels of TRF1 associated to differentpluripotency markers at every stage of establishment of ES cells,correlations were poor (Figs. S11 B and C and S12 B and C). Tofurther study a possible correlation between pluripotency factorsand TRF1, we used a mouse antibody against Oct3/4 in combi-nation with our best TRF1 antibody. The mouse cell line L5178Y-R, which bears long telomeres but is not pluripotent, was includedin our analysis to discard that association of high levels of TRF1and pluripotency factors are coincidental. Our results show thatthe cells from the L5178Y-R line had a higher mean TRF1 in-tensity than MEFs, bur lower than the cultured ICM (Fig. 4 D, F,and H). Oct3/4 levels were basal in primary MEFs and L5178Y-R(Fig. 4 E,G, andH). Furthermore, we observed a clear correlationbetween TRF1 and Oct3/4 in established ES cells (Fig. 4I) and inthe in vitro ICM in those cells expressing high levels of Oct3/4.Together, these results indicate that high levels of TRF1 occur inthe presence of some pluripotency factors (i.e., Oct3/4) from theearliest step of derivation of ES cells. The unprecedented findingof elevated TRF1 levels before telomere elongation (culturedICM) could represent a previously unnoticedmechanism to enable

Telomere length H4k20me3 Merge + DAPI

Freq

uenc

y

ICM (C)

H3k9me3 int. ± SE (a.u.):12,1 ± 0,7Number of cells/ICM: 130/20H3k9me3 Int. < 7 a.u.: 30%H3k9me3 Int. > 15 a.u.: 42,3%

96 well

H3k9me3 int. ± SE (a.u.):14,31 ± 1,02Number of cells/colonies:109/ 12H3k9me3 Int. < 7 a.u.: 30,2%H3k9me3 Int. > 15 a.u.: 46,7%

ESP9

H3k9me3 int. ± SE (a.u.):6,7 ± 0,5Number of cells/ES:339/2H3k9me3 Int. < 7 a.u.: 71,5%H3k9me3 Int. > 15 a.u.: 18,6%

MEF

H3k9me3 int. ± SE (a.u.):14,6 ± 0,4Number of cells/MEF: 151/3H3k9me3 Int. < 7 a.u.: 6,6%H3k9me3 Int. > 15 a.u.: 41,72%

010203040

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Merge + DAPITelomere length H3k9me3

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MEF

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.

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359 cells

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n=20 n=12109 cells

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20m

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nce

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.)

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H4k20me3 int. ± SE (a.u.):12,2 ± 0,9Number of cells/ICM: 119/20H4k20me3 Int. < 7 a.u.: 36,1%H4k20me3 Int. > 15 a.u.: 36,9%

96 well

H4k20me3 int. ± SE (a.u.):12,5 ± 1,06Number of cells/colonies:86/ 12H4k20me3 Int. < 7 a.u.: 33,7 %H4k20me3 Int. > 15 a.u.: 40,7%

ESP9

H4k20me3 int. ± SE (a.u.):3,7 ± 0,23Number of cells/ES:483/2H4k20me3 Int. < 7 a.u.: 83,5%H4k20me3 Int. > 15 a.u.: 5,3%

07

14212835

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H4k20me3 int. ± SE (a.u.):11,8 ± 0,5Number of cells/MEF: 96/2H4k20me3 Int. < 7 a.u.: 16%H4k20me3 Int. > 15 a.u.: 16,9%

Freq

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Freq

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n=280 cells2148 tel.

n=20100 cells2633 tel.

n=1276 cells4906 tel.

n=2100 cells4423 tel.

% o

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n=280 cells

2148 tel.

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78 cells3706 tel.

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048

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89 cellsn=12

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109 cells119 cells 89 cells

483 cells

n=2n=20 n=12

n=2

Fig. 3. The loss of heterochromatic marks accompanies telomere length-ening. (A) Mean H4k20me3 intensity for primary MEFs (passage 2), in vitrocultured ICM, cells from the 96-well plate, and established ES cells at passage9. (Lower graphs) The H4k20me3 histograms for the same samples. Note thatin the ICM as well as in the 96-well plate there are cells with low H4k20me3signals. (B) Percentage of cells with less than 7 arbitrary units of H4k20me3fluorescence. Note the portion of cells with low methylation signal in boththe cultured ICM and the 96-well plate. (C) Colocalization of the H4k20me3heterochromatic mark with telomeres in percentage for the samples de-scribed in A. (D) Representative images of telomeres and H4k20me3 signalsfor the samples described in A. (E) Mean H3k9me3 intensity and histogramsfor the samples described in A. (F) Percentage of cells with less than 7 ar-bitrary units of H3k9me3 fluorescence. Note the portion of cells with lowmethylation signal in the cultured ICM and the 96-well plate. (G) Percentageof colocalization of the H3k9me3 heterochromatic mark with telomeres forthe samples described in A. (H) Representative images of telomeres andH3k9me3 signals for the samples described in A. n = number of ICM or 96-well plate colonies or independent ES, and primary MEF cultures. Arbitraryunits of H4k20me3 fluorescence is plotted. (Scale bars, 10 μm.)

15210 | www.pnas.org/cgi/doi/10.1073/pnas.1105414108 Varela et al.

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cells to protect telomeres as they are elongated. In this manner,limiting TRF1 amounts could also limit further telomere elonga-tion by telomerase.

DiscussionHere, we provide unprecedented evidence that telomeres arespecifically elongated in the ICM at the blastocyst stage, and thatin vitro cultured ICM cell telomeres undergo a further elongationduring the establishment of ES cell lines, which is coordinatedwith decreased levels of histone trimethylation marks. Thus, incontrast to the intuitive idea of ES cells inheriting long telomeresfrom the cells of the blastocyst ICM, we show here that there areactive mechanisms operating in the process of ES establishment,which act in an orderly manner.We first describe changes in chromatin structure, specifically the

loss of heterochromatic marks at early stages of ES cell estab-lishment. In this context, a limited action of the histone metyl-tranferases Suv39 and Suv420 at telomeres may facilitate thegeneration of hyper-long telomeres in established ES cell lines,similar to that previously shown by us for iPS cells (26, 27). Second,

our results provide evidence for high expression of TRF1 associ-ated to early stages of ES-cell generation (cultivated ICM cells)coincidental with high Sox2 andOct3/4 levels, and before telomereelongation and presence of high Nanog levels. High TRF1 ex-pression at early stages of ES cell establishment, even beforetelomere elongation occurs, may be a mechanism to ensure pro-ficient telomere capping, suggesting that the safeguard of chro-mosome stability could be coupled to pluripotency. Finally, theevents described here associated to ES cell establishment, in-cluding the loss of heterochromatic marks, high levels of TRF1,and the elongation of telomeres, could also operate in the contextof tumorigenesis to maintain cellular immortality.

Materials and MethodsCell Culture Conditions and Embryo Collection. Cells and embryos used in thiswork were from the C57BL6 genetic background, unless specified otherwise.ES cells were derived at the Transgenic Mice Unit of the Spanish NationalCancer Research Center (CNIO). IPS cells were reprogrammed from primaryMEF (5), which were obtained from 13.5-d embryos (50). Culture conditionsare described in SI Materials and Methods.

TRF

1 in

tens

ity a

.u.

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MEF ESP9ICM (C)96 well

n=4252 cells

n=20130 cells

n=12174 cells n= 2

807 cells

** *

*p<0,0001

Oct

3/4

int.

/cel

l a.u

.

n=4469 cells

n=20102 cells

n=12180 cells

n= 2794 cells

0

20

40

60

80

MEF ESP9ICM (C) 96 well

***

****p<0,0001

% o

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ls w

ith O

ct3/

4 flu

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e >2

0.a.

u.A

TRF1 Oct3/4 Merge +DAPI

ME

FIC

M (c

)96

wel

lE

SP

9

MEF ESP9ICM (C)96 well

n=4469 cells

n=20102 cells

n=12180 cells

n= 2794 cells

0

22

44

66

88

B

C MEF Mean TRF1 i nt. ± SE ( a.u.): 26,99 ± 0,5Number of cells/MEF: 103/2TRF1 Int. < 25 a.u.: 38,8%TRF1 Int. > 40 a.u.: 0%

ICM (c)Mean TRF1 i nt. ± SE (kb): 72,99 ± 0,3Number of cells/ES: 56/30TRF1 Int. < 25 a.u.: 0%TRF1 Int. > 40 a.u.: 92,3%

ES10P9Mean TRF1 i nt. ± SE (kb): 69,72 ± 3,1Number of cells/ES: 56/2TRF1 Int. < 25 a.u.: 0%TRF1 Int. > 40 a.u.: 90,9%

ES10P9Mean Oc t3/4 int . ± SE (kb): 68,72 ± 3,1Number of cells/ES: 56/2Oct3/4 Int. < 5 a.u.: 0%Oct3/4 Int. > 15 a.u.: 96,3%

Freq

uenc

yFr

eque

ncy

Freq

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Oct3/4 intensity a.u.

TRF1 Oct3/4 Merge + DAPI

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P9

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ME

FR

D

E

F

Freq

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RMean TRF1 i nt. ± SE ( a.u.): 42,78 ± 0,6Number of cells/MEF: 170/1TRF1 Int. < 25 a.u.: 11,7%TRF1 Int. > 40 a.u.: 2,25%

ICM (c)Mean Oc t3/4 int . ± SE (kb): 22,25 ± 3,5Number of cells/ES: 56/

High TRF1 int.

Low TRF1 int.

Oct3/4 Int. < 5 a.u.: 0%Oct3/4 Int. > 15 a.u.: 75,3%

RMean Oc t3/4 int . ± SE (kb): 9,59 ± 0,25Number of cells/ES: 170/1Oct3/4 Int. < 5 a.u.: 11,9%Oct3/4 Int. > 15 a.u.: 2,3%

MEFMean Oc t3/4 int . ± SE (kb):7,17 ± 0,4Number of cells/ES: 103/2Oct3/4 Int. < 5 a.u.: 43,8%Oct3/4 Int. > 15 a.u.: 3,2%

Oct3/4 intensity a.u. Oct3/4 intensity a.u. Oct3/4 intensity a.u.

I

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Oct

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Freq

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F1

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nsity

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* **p<0,0001*

015304560

0 40 80 1200

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R² = 0,0459

012243648

0 5 10 15 20 25

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Oct3/4 int. (a.u.)

y = 0,4813x + 98,94R² = 0,0178

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y = 0,8564x + 27,986R² = 0,5167

020406080

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ICMc low TRF1 int. ICMc high TRF1 int.

Oct3/4 int. (a.u.) Oct3/4 int. (a.u.)

Oct3/4 int. (a.u.) Oct3/4 int. (a.u.)

y = 0,1889x + 8,7217R² = 0,6915

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Fig. 4. Analysis of TRF1 and Oct4 ex-pression during isolation of ES cells. (A)Mean TRF1 intensity for primary MEFs(passage 2), in vitro cultivated ICM, 96-well plate emerging ES cells, and estab-lished ES-cell lines at passage 9 analyzedby telomapping. (B) (Left) Mean Oct3/4intensity for the same samples describedin A. (Right) Percentage of cells withOct3/4 intensity bigger than 20 a.u. Notethat at the cultured ICM stage, a 38% ofcells are Oc3/4 positive. (C) Representa-tive images of TRF1 and Oct3/4 expres-sion for the same samples described inA. (Scale bars, 10 μm.) n = number of ICMor 96-well plate colonies or ES and pri-mary MEF cultures. (D) Mean TRF1 in-tensity values for primary MEFs, the cellline L5178Y-R or R cells, cultured ICM,and ES passage 9. (E) Mean Oct3/4 inten-sity for the samples described in D. (F)TRF1 expression frequency histogramscorresponding for the samples describedin D. (G) Oct3/4 expression frequencyhistograms corresponding to the sam-ples described in D. (H) Representativeimages of TRF1 andOct3/4 expression forthe samples described in D. (Scale bars,10 μm.) (I) TRF1 intensity values plottedagainst Oct3/4 intensity values to ana-lyze correlation. Primary MEFs, culturedICM, and established ES cells at passage 9are shown in the Upper panels. (Lower)Cells from the cultivated ICM were di-vided in high or low TRF1 intensity forthe analysis. n = number of ICM coloniesor L5178Y-R, ES, primary MEF cultures.

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Isolation of ICM from Blastocysts. Embryos were harvested from E3.5-preg-nant females. The zona pellucida was removed by treatment with Tirode’ssolution and then transferred to a 60-mm plate containing feeder cells (MEFstreated with Mytomicin-C). Blastocysts were cultured in ES-cell medium for48 h. The outgrowth of the ICM were picked, usually 4 to 6 d after the initialplating, and transferred to a microdrop of trypsin for disaggregation.

Agregation Experiments. For ES cell microinjection, Hsd:ICR(CD-1) morulaewere harvested from superovulated females at E2.5 d of gestation. Sixty-three morulae at the eight-cell stage were microinjected with 6 to 10 EGFP-expressing R1 ES cells (of 129 × 1/SvJ × 129S1/Sv genetic background as inref. 5). Microinjected embryos were incubated overnight at 37 °C under oil.At the blastocyst stage embryos were fixed for analysis.

Quantitative FISH. ES cells and cultured ICM cells were blocked in metaphasewith colcemid for 3 h, swollen in hypotonic buffer for 10min at 37 °C, andfixedas described in ref. 51. Metaphases were dropped on slides and Q-FISH witha telomere or centromere probewas performed as in ref. 28. TFL-Telo software(52) was used to quantify the fluorescence intensity of telomeres from 5 to 10

metaphases for each datapoint. Microscope settings are described in SIMaterials and Methods.

Telomapping of Blastocyst Sections. Quantitative image analysis was per-formedon confocal RGB images using theDefiniens platform (versionXD) as inref. 6. For details, see SI Materials and Methods.

Immunfluorescence combined with FISH. Immunofluorescence was performedas in ref. 43 (SI Materials and Methods). Samples were fixed in 4% formal-dehyde, dehydrated and incubated with a telomere probe labeled with CY3(Panagene) as described in ref. (28).

Statistical Analysis. Statistical analyses were performed using the GraphPadPrism software version 5. Mean values reflect the arithmetic mean. Student ttest with “two tails” was used to obtain the P value.

ACKNOWLEDGMENTS. Work in the laboratory of M.A.B. is funded by grantsfrom the Minisetrio de Ciencia e Innovación (CONSOLIDER), the EuropeanUnion, the European Research Council, The Lilly Foundation, and the KorberEuropean Research Award.

1. Albert M, Peters AH (2009) Genetic and epigenetic control of early mouse de-velopment. Curr Opin Genet Dev 19(2):113–121.

2. Mattout A, Meshorer E (2010) Chromatin plasticity and genome organization inpluripotent embryonic stem cells. Curr Opin Cell Biol 22:334–341.

3. Shay JW, Wright WE (2010) Telomeres and telomerase in normal and cancer stemcells. FEBS Lett 584:3819–3825.

4. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells frommouse embryos. Nature 292(5819):154–156.

5. Marion RM, et al. (2009) Telomeres acquire embryonic stem cell characteristics ininduced pluripotent stem cells. Cell Stem Cell 4(2):141–154.

6. Flores I, et al. (2008) The longest telomeres: A general signature of adult stem cellcompartments. Genes Dev 22:654–667.

7. Flores I, Blasco MA (2009) A p53-dependent response limits epidermal stem cellfunctionality and organismal size in mice with short telomeres. PLoS ONE 4:e4934.

8. Wright DL, et al. (2001) Characterization of telomerase activity in the human oocyteand preimplantation embryo. Mol Hum Reprod 7:947–955.

9. Aoi T, et al. (2008) Generation of pluripotent stem cells from adult mouse liver andstomach cells. Science 321:699–702.

10. Maherali N, et al. (2007) Directly reprogrammed fibroblasts show global epigeneticremodeling and widespread tissue contribution. Cell Stem Cell 1(1):55–70.

11. Nakagawa M, et al. (2008) Generation of induced pluripotent stem cells without Mycfrom mouse and human fibroblasts. Nat Biotechnol 26(1):101–106.

12. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent inducedpluripotent stem cells. Nature 448:313–317.

13. Stadtfeld M, Maherali N, Breault DT, Hochedlinger K (2008) Defining molecular corner-stones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2:230–240.

14. Takahashi K, et al. (2007) Induction of pluripotent stem cells from adult human fi-broblasts by defined factors. Cell 131:861–872.

15. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell 126:663–676.

16. Wernig M, et al. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324.

17. Betts DH, King WA (1999) Telomerase activity and telomere detection during earlybovine development. Dev Genet 25:397–403.

18. Blasco MA, Funk W, Villeponteau B, Greider CW (1995) Functional characterizationand developmental regulation of mouse telomerase RNA. Science 269:1267–1270.

19. Liu L, et al. (2007) Telomere lengthening early in development. Nat Cell Biol 9:1436–1441.

20. Mantell LL, Greider CW (1994) Telomerase activity in germline and embryonic cells ofXenopus. EMBO J 13:3211–3217.

21. Xu J, Yang X (2001) Telomerase activity in early bovine embryos derived from par-thenogenetic activation and nuclear transfer. Biol Reprod 64:770–774.

22. Blasco MA (2007) The epigenetic regulation of mammalian telomeres. Nat Rev Genet8:299–309.

23. Jaco I, Canela A, Vera E, Blasco MA (2008) Centromere mitotic recombination inmammalian cells. J Cell Biol 181:885–892.

24. McIlrath J, et al. (2001) Telomere length abnormalities in mammalian radiosensitivecells. Cancer Res 61:912–915.

25. Schaetzlein S, et al. (2004) Telomere length is reset during early mammalian em-bryogenesis. Proc Natl Acad Sci USA 101:8034–8038.

26. Benetti R, et al. (2008) A mammalian microRNA cluster controls DNA methylation andtelomere recombination via Rbl2-dependent regulation of DNA methyltransferases.Nat Struct Mol Biol 15(3):268–279.

27. García-Cao M, O’Sullivan R, Peters AH, Jenuwein T, Blasco MA (2004) Epigeneticregulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 his-tone methyltransferases. Nat Genet 36(1):94–99.

28. Gonzalo S, et al. (2006) DNA methyltransferases control telomere length and telo-mere recombination in mammalian cells. Nat Cell Biol 8:416–424.

29. Vera E, Canela A, Fraga MF, Esteller M, Blasco MA (2008) Epigenetic regulation oftelomeres in human cancer. Oncogene 27:6817–6833.

30. Smith KP, Luong MX, Stein GS (2009) Pluripotency: Toward a gold standard for hu-man ES and iPS cells. J Cell Physiol 220(1):21–29.

31. Orkin SH, et al. (2008) The transcriptional network controlling pluripotency in ES cells.Cold Spring Harb Symp Quant Biol 73:195–202.

32. Mitsui K, et al. (2003) The homeoprotein Nanog is required for maintenance ofpluripotency in mouse epiblast and ES cells. Cell 113:631–642.

33. Liu N, et al. (2008) Identification of genes regulated by nanog which is involved in EScells pluripotency and early differentiation. J Cell Biochem 104:2348–2362.

34. Kuroda T, Tada M (2006) Molecular network of transcriptional factors controllingpluripotency of ES cells. (Translated from Japanese) Seikagaku 78(2):137–141.

35. Dong WZ, Shen WZ, Hua JL, Dou ZY (2007) Study on pluripotency and cultivation ofES-like cells derived from male germ stem cells of bovine fetuses. (Translated fromChinese) Sheng Wu Gong Cheng Xue Bao 23:751–755.

36. Chambers I, et al. (2003) Functional expression cloning of Nanog, a pluripotencysustaining factor in embryonic stem cells. Cell 113:643–655.

37. de Lange T (2005) Shelterin: The protein complex that shapes and safeguards humantelomeres. Genes Dev 19:2100–2110.

38. Blasco MA (2007) Telomere length, stem cells and aging. Nat Chem Biol 3:640–649.39. Blasco MA (2005) Telomeres and human disease: Ageing, cancer and beyond. Nat Rev

Genet 6:611–622.40. Tejera AM, et al. (2010) TPP1 is required for TERT recruitment, telomere elongation

during nuclear reprogramming, and normal skin development in mice. Dev Cell 18:775–789.

41. Karlseder J, et al. (2003) Targeted deletion reveals an essential function for thetelomere length regulator Trf1. Mol Cell Biol 23:6533–6541.

42. Martínez P, et al. (2009) Increased telomere fragility and fusions resulting from TRF1deficiency lead to degenerative pathologies and increased cancer in mice. Genes Dev23:2060–2075.

43. Ancelin K, et al. (2002) Targeting assay to study the cis functions of human telomericproteins: Evidence for inhibition of telomerase by TRF1 and for activation of telomeredegradation by TRF2. Mol Cell Biol 22:3474–3487.

44. Smogorzewska A, et al. (2000) Control of human telomere length by TRF1 and TRF2.Mol Cell Biol 20:1659–1668.

45. van Steensel B, de Lange T (1997) Control of telomere length by the human telomericprotein TRF1. Nature 385:740–743.

46. Muñoz P, et al. (2009) TRF1 controls telomere length and mitotic fidelity in epithelialhomeostasis. Mol Cell Biol 29:1608–1625.

47. Avilion AA, et al. (2003) Multipotent cell lineages in early mouse development de-pend on SOX2 function. Genes Dev 17(6):126–140.

48. Nichols J, et al. (1998) Formation of pluripotent stem cells in the mammalian embryodepends on the POU transcription factor Oct4. Cell 95:379–391.

49. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines dif-ferentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372–376.

50. Muñoz P, Blanco R, Flores JM, Blasco MA (2005) XPF nuclease-dependent telomereloss and increased DNA damage in mice overexpressing TRF2 result in prematureaging and cancer. Nat Genet 37:1063–1071.

51. Samper E, Flores JM, Blasco MA (2001) Restoration of telomerase activity rescueschromosomal instability and premature aging in Terc-/- mice with short telomeres.EMBO Rep 2:800–807.

52. Zijlmans JM, et al. (1997) Telomeres in the mouse have large inter-chromosomalvariations in the number of T2AG3 repeats. Proc Natl Acad Sci USA 94:7423–7428.

15212 | www.pnas.org/cgi/doi/10.1073/pnas.1105414108 Varela et al.

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