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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
0 100 200 300
1
3
0
612
18
0 100 200 300
020406080
0 100 200 300
0
10
2030
0 100 200 300
05
101520
0 100 200 300
020406080
100
0 100 200 300
Freq
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D Blastocyst ICM (C) Emergence of ES cells ES P1 ES P12
96 well 24 well 25 mm
Freq
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Freq
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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
uenc
y
Freq
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0
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0 200 300100
02468
0 200 3001000
4080
120160
0 100 200 300
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100150
0 100 200 300
Telo
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(K
b)
020406080
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0
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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
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30
40
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60
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M Bl E 7.5 E 10.5
Telo
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(a. u
.)
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.
15208 | www.pnas.org/cgi/doi/10.1073/pnas.1105414108 Varela et al.
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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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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
8-cell morula
ES cells (GFP and hyper-long telomeres)
Blastocyst
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Control blastocyst
ES usedfor injection
05
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mer
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.)
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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121620
0 50 100 150
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MEF
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14212835
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0 50 100 150H3k9me3 fluorescence/cell (a.u.)
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MEF
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% o
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.
020406080
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108 cells
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359 cells
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n=2
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.)
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0
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101520
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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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9me3
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.u.) ***
359 cells
048
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MEF ICM (C) 96 well ESP9
108 cells130 cells109 cells
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Freq
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MEF ICM (c) 96 well ESP9
n=280 cells2148 tel.
n=20100 cells2633 tel.
n=1276 cells4906 tel.
n=2100 cells4423 tel.
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MEF ICM (c) 96 well ESP9
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n=2047 cells2133 tel. n=12
78 cells3706 tel.
n=1262 cells2599 tel.
0,0070,03
048
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MEF ICM (C) 96 well ESP9
***
109 cellsn=2
119 cellsn=20
89 cellsn=12
383 cellsn=2
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020406080
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MEF ICM (C) 96 well ESP9
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.)
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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.
0
5
10
15
20
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
f cel
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
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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
uenc
y
TRF1 intensity a.u.
Oct3/4 intensity a.u.
TRF1 Oct3/4 Merge + DAPI
ES
P9
ICM
(c)
ME
FR
D
E
F
Freq
uenc
y
G
H
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
0
12
24
36
48
MEF R ICM (c) ESP9
Oct
3/4
int.
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.)
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n=1170 cells
n=3060 cells
n=256 cells
* ***
*p<0,0001
Freq
uenc
y
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F1
inte
nsity
(a.u
.)
0
20
40
60
80
MEF R ICM (c) ESP9
**
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n=1170 cells
n=3060 cells n=2
* **p<0,0001*
015304560
0 40 80 1200
2
4
6
0 40 80 1200
2
4
6
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R² = 0,0459
012243648
0 5 10 15 20 25
MEF
TRF1
int.
(a.u
.)
Oct3/4 int. (a.u.)
y = 0,4813x + 98,94R² = 0,0178
0
50100
150200
0 20 40 60
y = 0,8564x + 27,986R² = 0,5167
020406080
100120140
0 40 80 120
ESP9
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
010203040
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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.
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