Cell Stem Cell
Article
Telomeres Acquire Embryonic Stem Cell
Characteristics in Induced Pluripotent Stem Cells
Rosa M. Marion,1,4 Katerina Strati,1,4 Han Li,2 Agueda Tejera,1 Stefan Schoeftner,1 Sagrario Ortega,3
Manuel Serrano,2 and Maria A. Blasco1,*
1Telomeres and Telomerase Group, Molecular Oncology Program
2Tumor Suppression Group, Molecular Oncology Program
3Transgenic Mice Unit, Biotechnology Program
Spanish National Cancer Centre (CNIO), Melchor Ferna´ndez Almagro 3, Madrid E-28029, Spain
4These authors contributed equally to this work
*Correspondence: mblasco@cnio.es
DOI 10.1016/j.stem.2008.12.010
SUMMARY
progress, iPS cells seem to have the same properties as
embryonic stem (ES) cells and share with them a similar global
Telomere shortening is associated with organismal
gene expression pattern, including a correct genome-wide
aging. iPS cells have been recently derived from old
epigenetic reprogramming (Takahashi and Yamanaka, 2006;
patients; however, it is not known whether telomere
Wernig et al., 2007; Maherali et al., 2007; Mikkelsen et al.,
chromatin acquires the same characteristics as in
2008). Furthermore, iPS cells contribute to mouse embryonic
ES cells. We show here that telomeres are elongated
development and to the mouse germline (Takahashi and
in iPS cells compared to the parental differentiated
Yamanaka, 2006; Wernig et al., 2007; Takahashi et al., 2007;
Stadtfeld et al., 2008; Maherali et al., 2007; Okita et al., 2007;
cells both when using four (Oct3/4, Sox2, Klf4,
Nakagawa et al., 2008; Aoi et al., 2008), supporting the notion
cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming
that they are pluripotent and indistinguishable from ES cells. In
factors and both from young and aged individuals.
the case of regenerative transplantation therapies, these find-
We demonstrate genetically that, during reprogram-
ings open the possibility of using iPS cells derived from patients
ming, telomere elongation is usually mediated by
as a way to bypass the technical difficulties, as well as ethical
telomerase and that iPS telomeres acquire the epige-
controversies, associated with the alternative method of nuclear
netic marks of ES cells, including a low density of tri-
transfer to human oocytes, in vitro generation of preimplantation
methylated histones H3K9 and H4K20 and increased
embryos, and obtention of ES cells.
abundance of telomere transcripts. Finally, reprog-
Telomeres have been shown to shorten associated to
ramming efficiency of cells derived from increasing
increasing age (Harley et al., 1990) and contribute to organismal
generations of telomerase-deficient mice shows
aging by limiting the proliferative capacity of adult stem cells
(Blasco, 2007a; Flores et al., 2005, 2006a). Although telomerase
a dramatic decrease in iPS cell efficiency, a defect
activity has been found upregulated in both human and mouse
that is restored by telomerase reintroduction.
iPS cells (Takahashi et al., 2007; Stadtfeld et al., 2008) and iPS
Together, these results highlight the importance of
cells have been recently derived from very old patients (Dimos
telomere biology for iPS cell generation and function-
et al., 2008), it is not known whether telomeres are re-elongated
ality.
and whether telomeric chromatin acquires the same characteris-
tics as in ES cells. For example, telomerase is reactivated in
INTRODUCTION
human cancers; however, the telomeres of most cancer cells
are shorter than those of normal tissues (de Lange et al., 1990;
Induced pluripotent stem (iPS) cells derived from differentiated
Meeker et al., 2004).
somatic cells represent a new source of stem cells for custom-
Telomeres are ribonucleoprotein heterochromatic structures
ized transplantation therapies (Takahashi and Yamanaka, 2006;
at the ends of chromosomes that protect them from degradation
Wernig et al., 2007; Takahashi et al., 2007; Stadtfeld et al., 2008;
and recombination activities (Blackburn, 2001). Telomeres
Maherali et al., 2007; Okita et al., 2007; Nakagawa et al., 2008;
consist of tandem repeats of the TTAGGG sequence bound to
Aoi et al., 2008). In particular, a combination of a few factors
a 6 protein complex known as shelterin (Blackburn, 2001; de
related to stem cell pluripotency allowed reprogramming of
Lange, 2005). Telomere length is influenced by changes in the
differentiated mouse and human cells into iPS cells (Takahashi
activity of telomerase, the reverse transcriptase that elongates
and Yamanaka, 2006; Wernig et al., 2007; Takahashi et al.,
telomeres (Greider and Blackburn, 1985), as well as by the so-
2007; Stadtfeld et al., 2008; Maherali et al., 2007; Okita et al.,
called alternative lengthening of telomeres or ALT pathway,
2007; Nakagawa et al., 2008; Aoi et al., 2008). These factors
which relays in homologous recombination between telomeric
include Oct3/4 (also called Pou5f1), Sox2, Klf4, and cMyc; the
sequences (Dunham et al., 2000). In turn, these telomere-elon-
latter was found dispensable for iPS cell generation (Nakagawa
gating mechanisms are regulated by the epigenetic status of te-
et al., 2008). Although full characterization of iPS cells is still in
lomeric chromatin (Blasco, 2007b). In particular, both telomeric
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Cell Stem Cell
Reprogramming of Telomere Chromatin in iPS Cells
and subtelomeric repeats are enriched in histone methylation
during nuclear reprogramming. Interestingly, we show that
marks characteristic of repressed heterochromatic domains
telomerase activity is the primary mechanism to mediate telo-
such as trimethylation of H3K9 and H4K20, and they show
mere re-elongation during reprogramming and that a minimum
binding of the heterochromatin protein 1 (HP1) (Garcı´a-Cao
telomere length is necessary for iPS cell generation. In particular,
et al., 2004; Gonzalo et al., 2006; Benetti et al., 2007). In addition,
cells derived from telomerase-deficient mice fail to properly
the DNA of subtelomeric repeats is heavily methylated (Gonzalo
elongate telomeres during reprogramming, and telomeres
et al., 2006). Loss of these epigenetic marks results in derepres-
continue to shorten during iPS cell generation. Furthermore, an
sion of telomere recombination and telomere elongation (Garcı´a-
increasing number of generations of telomerase-deficient mice
Cao et al., 2004; Gonzalo et al., 2006; Benetti et al., 2007).
with progressive shorter telomeres show a dramatic impairment
Furthermore, telomeres are transcribed, and the resulting
in iPS cell generation. These findings highlight the importance of
UUAGGG-rich RNAs (TelRNAs or TERRAs) remain bound to
telomere biology for the generation and proper functionality of
the telomeric chromatin, where they are proposed to regulate
iPS cells.
telomere length (Azzalin et al., 2007; Schoeftner and Blasco,
2008). Telomerase is expressed during embryonic development
RESULTS
and in the stem cell compartment of several adult tissues
(Blasco, 2007a; Flores et al., 2005; 2006a; Liu et al., 2007);
cMyc Is Dispensable for Telomerase Activation in Mouse
however, telomerase levels in these tissues are not sufficient to
iPS Cells
prevent progressive telomere shortening with age both in hu-
Telomerase activity has been previously shown to increase
mans and mice (Harley et al., 1990; Flores et al., 2008). Reduced
during iPS cell generation using a combination of four reprog-
telomerase activity due to mutations in telomerase components
ramming factors (Takahashi et al., 2007; Maherali et al., 2007;
in the human diseases dyskeratosis congenita, aplastic anemia,
Zhao and Daley, 2008), namely Oct3/4, Sox2, Klf4, and cMyc,
and idiopathic pulmonary fibrosis (Mitchell et al., 1999; Yamagu-
the latter being a transcriptional activator of telomerase in the
chi et al., 2005; Tsakiri et al., 2007; Armanios et al., 2007) further
mouse (Wu et al., 1999; Flores et al., 2006b). Here, we address
accelerates telomere shortening and leads to premature loss of
the putative role of cMyc in telomerase activation during mouse
tissue regeneration, suggesting that telomerase levels in the
iPS cell generation by comparing telomerase activity levels in
adult organism are rate limiting and influence organ homeo-
mouse iPS cells generated with four factors (Oct3/4, Sox2,
stasis. Further evidence for a role of telomerase and telomere
Klf4, and cMyc; referred to here as 4F iPS cells) with those of
length in organ homeostasis comes from the study of telome-
iPS cells generated with three factors in the absence of cMyc
rase-deficient mice (TercÀ/À mice), which show premature aging
(Oct3/4, Sox2, and Klf4; referred to here as 3F iPS cells) (Naka-
and a decreased proliferative potential of adult stem cell popula-
gawa et al., 2008; Wernig et al., 2008). To this end, 3F and 4F
tions (Blasco et al., 1997; Lee et al., 1998; Herrera et al., 1999;
mouse iPS cells were generated from mouse embryonic fibro-
Samper et al., 2002; Ferro´n et al., 2004). Therefore, the function-
blasts (MEF) as previously described (Takahashi and Yamanaka,
ality of iPS cells could be limited in elderly patients or in patients
2006; Nakagawa et al., 2008) (Experimental Procedures). The re-
with a limited telomere reserve, and the resulting iPS cells could
sulting iPS cells showed expression of the endogenous factors
inherit telomeric defects, including suboptimal telomere length.
and a similar morphology to ES cells; stained positive for alkaline
In this regard, previous studies with cloned animals by means
phosphatase; maintained the ES cell morphology upon expan-
of somatic cell nuclear transfer into enucleated oocytes have
sion; and expressed the stemness marker Nanog (Figures
rendered contradictory results regarding whether telomeres
S1A–S1E available online). Importantly, both 3F and 4F iPS cells
are elongated or not during nuclear reprogramming (Shiels
were able to contribute to mouse embryonic development as
et al., 1999; Vogel, 2000; Lanza et al., 2000). On one hand, the
indicated by important mouse chimerism when microinjected
cloned sheep Dolly showed abnormally short telomeres, some-
into C57BL6-Tyrc (albino) blastocysts (Figure S1F and Table 1)
thing that was attributed to the adult origin of the donor nucleus
or when aggregated with CD1 (albino) morulae (Table 1). In
(Shiels et al., 1999), whereas cloning of cattle or mice for six
both cases, around 30% of the pups born were chimeras as
generations had no notable effect on telomere length (Lanza
judged by the coat color, with iPS contribution to the coat in
et al., 2000; Wakayama et al., 2000).
a range of 60%–100% in the case of microinjected blastocysts
Finally, it is known that, soon after fertilization but previous to
and
5%–50%
in
the
case
of
aggregation
to
morulae
the formation of the blastocyst, telomeres are elongated by
(Figure S1F and Table 1). Furthermore, both 3F and 4F iPS cells
a telomerase-independent recombination-based mechanism
showed germline transmission (Table 1). These results indicate
(Liu et al., 2007). This opens several scenarios for the reprogram-
that the 3F and 4F wild-type iPS cells generated for this study
ming of telomeric chromatin, including dispensability of telome-
are pluripotent and are able to contribute to the mouse germline.
rase for telomere elongation, cooperation between telomerase-
In agreement with this, iPS cells showed a normal chromosomal
based and recombination-based mechanisms, and, finally,
ploidy (Figures S2A and S2B).
complete dependence on telomerase for telomere elongation
Telomerase activity was increased $9-fold in 4F iPS cell
during reprogramming.
clones, compared to the parental MEF, reaching slightly higher
Here, we show that both telomere length and telomere hetero-
levels than those of control ES cells in the same genetic back-
chromatic marks acquire ES cell features during iPS cell gener-
ground (Figures S3A and S3B) (Takahashi et al., 2007; Maherali
ation. Importantly, we show here that telomeres are also effi-
et al., 2007; Zhao and Daley, 2008). A similar 8-fold increase in
ciently elongated in iPS cells derived from old animals,
telomerase activity was detected in the 3F iPS cells generated
demonstrating that telomeres can be efficiently rejuvenated
in the absence of the cMyc reprogramming factor (Figures S3A
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Reprogramming of Telomere Chromatin in iPS Cells
Table 1. Generation of Chimeras and Germline Transmission with C57BL/6 WT iPS Clones Reprogrammed with Three (3F) or Four (4F)
Factors
Microinjection in B6-tyrC-2J Blastocysts
iPS clone
Blastocysts Injected
Cells Injected
Blastocysts Transferred
Pups Born
Chimeras Sex (% Pigmentation)
iPS (3F)-7 wt
52
5–8
52
5
1M (100%)a
iPS (3F)-9 wt
43
5–8
43
4
1F (60%)
iPS (4F)-4 wt
58
5–6
58
8
2M (80%)a 1F (100%)
Aggregation with CD1 Morulae
iPS Clone
Morulae Aggregated
Cells Aggregated
Pups Born
Chimeras Sex (% Pigmentation)
iPS (4F)-3 wt
171
4–8
15
1M (30%) 1M (5%) 1F (50%) 2F (30%)
3F and 4F WT iPS contribution to the germline
Chimeras 3 B6-tyrC-2J Females
iPS Clone
Chimera Sex (% Pigmentation)
Black/White Pups
iPS (4F)-4 wt
M (80%)
5/14
M (80%)
8/10
iPS (3F)-7 wt
M (100%)
11/0
a Germline chimeras.
and S3B). These results indicate that cMyc is not essential to up-
from wild-type iPS cells (Table 2). These results indicate that
regulate telomerase activity during iPS cell generation. In this re-
telomerase activity is not limiting for in vitro iPS cell proliferation
gard, MEF have high levels of cMyc and may not need additional
when telomeres are sufficiently long, such as in the case of G1
cMyc activity to re-express telomerase during iPS cell genera-
telomerase-deficient iPS cells. However, these cells are severely
tion (Takahashi et al., 2007).
impaired in their ability to generate viable mice compared to
Next, we addressed whether telomerase activation per se was
wild-type telomerase-proficient 3F and 4F iPS cells, most likely
necessary for efficient iPS cell formation by using cells derived
due to increased chromosomal instability (see Figure 2).
from first generation (G1) telomerase-deficient TercÀ/À mice in
a C57BL6 genetic background (Herrera et al., 1999). We were
Telomere Elongation by Telomerase in iPS Cells
able to generate iPS cells from G1 telomerase-deficient MEF
Next, we addressed whether telomeres were elongated during
to a similar efficiency to wild-type MEF (Figures 2A and 2B), indi-
the reprogramming of 3F and 4F iPS cells (all at passage 8)
cating that activation of telomerase per se is not necessary for
compared to control ES cells by measuring telomere length in
iPS cell generation, at least in the presence of a sufficient telo-
individual clones of these cells using two independent
mere reserve, such as in the case of G1 TercÀ/À mice (Herrera
techniques, Southern telomere restriction analysis (TRF) and
et al., 1999). As expected, telomerase activity was negative
quantitative telomere FISH (Q-FISH) on metaphase spreads
both in parental MEF and in iPS derived from G1 telomerase-
(Experimental Procedures). Telomeres were elongated in wild-
deficient mice (Figures S3A and S3B). Interestingly, in spite of
type 4F iPS cells compared to the corresponding parental
a normal efficiency of G1 TercÀ/À iPS cell generation compared
wild-type MEF, both as determined by TRF (Figure 1A) and
to wild-type controls, we failed to obtain any viable chimeric
Q-FISH analyses (Figures 1B–1E). Telomeres were also elon-
mice from these cells. In particular, from a total of 11 pups
gated in 3F iPS cells but did not reach the length of 4F iPS cells
born, only one showed a high level of iPS contribution as judged
(Figures 1A–1E), although both had similar levels of telomerase
by eye pigmentation; however, these mice did not survive, in
activity (Figures S3A and S3B). Of notice, whereas the freshly
contrast to a 100% survival in the case of chimeras derived
emerged colonies of wild-type 3F and 4F iPS cells have
Table 2. Generation of Chimeras with C57BL/6 G1–G3 TercÀ/À Mutant iPS Clones
Microinjection in B6-tyrC-2J Blastocysts
iPS Clone
MEFs Genotype
Blastocysts Injected
Cells Injected
Blastocysts Transferred
Pups Born
Chimeras
iPS (3F) 718-7.1
G1 TercÀ/À
36
5–7
36
10 + 1 dead
1 deada
iPS (3F) 710-8.1
G2 TercÀ/À
40
5–7
40
11
0
Aggregation with CD1 Morulae
iPS Clone
MEFs Genotype
Morulae Aggregated
Cells Aggregated
Embryos Transferred
Pups Born
Chimeras Sex
(% Pigmentation
iPS (3F) 710-8.4
G2 TercÀ/À
96
4–6
96
15 + 1 dead
0b
iPS (3F) 62T1-1
G3 TercÀ/+*
153
4–12
153
15 + 4 dead
1M (100%) 1M (70%)
1F (30%) + 3 deada
a The dead pups had pigmented eyes.
b By eye pigmentation.
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telomeres that are not as long as the telomeres of control ES cells
rase during iPS cell generation. In contrast, this was not
in the same genetic background, upon a limited number of
observed in TercÀ/À iPS cells, which showed a further increase
passages in vitro, the iPS telomeres eventually reached full ES
in signal-free ends compared to the parental G1 TercÀ/À MEF
telomere length (Figures 1F and 1G), in accordance with
(Figure 1D). Finally, the fact that G1 TercÀ/À iPS cell telomeres
previous findings showing that telomerase needs a number of
shortened compared to the parental MEF argues against telo-
cell divisions in vivo in order to restore a normal average telomere
mere recombination mechanisms operating to elongate telo-
length (Siegl-Cachedenier et al., 2007). Furthermore, an
meres during iPS cell nuclear reprogramming, at least in early-
increasing number of passages of wild-type iPS cells retained
generation G1 TercÀ/À cells. These results are in agreement
a normal telomere-capping function as indicated by normal
with telomere recombination operating from zygote to blastocyst
ploidy and a low frequency of end-to-end fusions and of
and switching to telomerase at the blastocyst stage (Liu et al.,
signal-free ends (Figures S4A–S4C), in agreement with the fact
2007).
that they produced mice with a high degree of chimerism and
contributed to the mouse germline (Figure S1 and Table 1).
Impaired iPS Cell Generation from Late-Generation
Furthermore, telomere length in the tail skin of iPS cell-derived
TercÀ/À MEF with Critically Short Telomeres
mice was similar to that of age-matched, sex-matched wild-
To address whether telomere shortening in the absence of telo-
type C57BL6 controls (Figure 1H), indicating normal telomere
merase activity could eventually limit iPS cell generation, we
dynamics in these mice. Finally, by analyzing telomere length
compared the frequencies of iPS cell generation from G1, G2,
of single MEF and iPS cells at different passages, we observed
and G3 telomerase-deficient MEF. Again, whereas G1 TercÀ/À
the appearance of iPS cells with progressively longer telomeres
MEF yielded a normal efficiency of iPS cell generation compared
compared to MEF (Figure 1I). Furthermore, the fact that, at their
to wild-type MEF, we detected a dramatic decrease in the effi-
time of isolation (passage 8), iPS cell clones had telomeres only
ciency of iPS cell generation in G2 and G3 TercÀ/À MEF (Figures
moderately longer than MEF, which are further elongated with
2A and 2B), indicating that telomere shortening represents
increasing passages, suggests that most telomere elongation
a potent barrier against iPS cell generation in telomerase-defi-
occurs postreprogramming.
cient cells. In addition, similarly to that previously observed for
Next, we addressed whether telomere elongation in iPS cells
G1 TercÀ/À iPS cells, G2 TercÀ/À iPS cells failed to generate
was mediated by telomerase activity and/or by telomere-length-
viable chimeric mice (Table 2). Importantly, the decreased iPS
ening mechanisms alternative to telomerase, which are based on
cell generation was coincidental with shorter telomeres both as
recombination between telomeric sequences (Dunham et al.,
determined by TRF (Figure 2C) and Q-FISH analysis (Figures
2000) and are described to mediate telomere elongation in early
2D and 2E) and by an increase in signal-free ends and chromo-
cleavage embryos (Liu et al., 2007). To this end, we measured
some end-to-end fusions in the parental G1–G3 TercÀ/À MEF
telomere length in iPS cells (3F) derived from G1 TercÀ/À MEF.
compared to wild-type MEF, which was further aggravated in
As shown both by TRF and Q-FISH techniques, telomeres
the corresponding G1–G3 TercÀ/À iPS clones (Figures 2F and
were further shortened in iPS cells derived from G1 TercÀ/À
2G). Interestingly, both when using TRF and Q-FISH analysis,
MEF compared to the parental wild-type MEF (Figures 1A–1E),
we noticed that average telomere length was not further short-
indicating that telomerase is the primary activity responsible
ened in G2 and G3 TercÀ/À iPS cell clones compared to the
for telomere elongation in telomerase-proficient iPS cells.
parental
G2-G3
MEF
(Figures
2C–2E;
see
asterisk
in
Signal-free ends (chromosome ends with undetectable telomere
Figure 2C), suggesting the activation of telomerase-independent
signals or critically short telomeres) were decreased in telome-
telomere elongation mechanisms in some G2-G3 TercÀ/À iPS
rase-proficient 3F and 4F iPS cells compared to parental MEF,
cell clones (Figures 2A and 2B). Of notice, telomerase-indepen-
reaching similar levels to those of control ES cells (Figure 1D),
dent telomere elongation mechanisms typically lead to hetero-
in agreement with re-elongation of short telomeres by telome-
geneous telomere length distributions, with the presence of
Figure 1. Telomerase-Dependent Telomere Elongation in Mouse iPS Cells
(A) TRF analysis. Two representative TRF gels are shown. Note the longer telomeres in wild-type 3F and 4F iPS cells compared to the parental MEF. In contrast,
G1 TercÀ/À iPS cells show shorter telomeres compared to parental MEF. Numbers refer to two to three independent cell cultures per cell type. MEF passage
number = 2; iPS passage number = 8.
(B) Telomere length distribution as determined by Q-FISH on metaphases. Two to three cell cultures per cell type were used for the analysis. MEF passage
number = 2; iPS passage number = 8. A Wilcoxon-Mann-Whitney rank sum test was used to calculate statistical significance of differences in telomere length.
(C) Quantification of average telomere length in kilobases. n, number of telomeres. MEF passage number = 2; iPS passage number = 8. Error bars, SD.
(D) Quantification of the percentage of signal-free ends. MEF passage number = 2; iPS passage number = 8. Error bars, SD. Statistical comparisons using
chi-square test are shown.
(E) Representative images of metaphasic chromosomes after telomere Q-FISH. Yellow, telomeres; blue, DAPI. MEF passage number = 2; iPS passage
number = 8.
(F) TRF analysis of two independent 4F iPS cell clones at the indicated passages. Right panel shows the signal distribution in each TRF lane.
(G) Telomere length distributions at different passages as determined by Q-FISH on metaphases. Telomere length of iPS cells increases with the passages,
reaching that of control ES cells. A Wilcoxon-Mann-Whitney rank sum test was used for statistical calculations.
(H) Telomere length by Q-FISH on tail skins. Samples were obtained from three iPS cell-derived mice (two from 4F-reprogrammed iPS cells and one from 3F
reprogrammed iPS cells) and three age-matched, sex-matched wild-type C57BL6 mice. The number of telomeres and nuclei analyzed are indicated. Telomere
length is shown in arbitrary fluorescence units (a.u.f.). Statistical analysis was performed using the Student’s t test.
(I) Average telomere length in kilobases of individual MEF and iPS cells as determined by Q-FISH. ‘‘1’’ and ‘‘2’’ indicate independent iPS cell clones. Each dot
represents the telomere length of a single metaphase. n, number of metaphases. p, passage number.
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very short and long telomeres (see G3 TercÀ/À iPS cell telomeres
cell types (Figures 3A–3C). Together, these results suggest that
in Figures 2C and 2D).
DNA methylation of repeated regions in the genome such as
Elongation of short telomeres by telomerase in Terc-reconsti-
pericentric repeats, SINE elements, and subtelomeric repeats
tuted mice is sufficient to rescue telomere dysfunction, as well as
does not significantly vary during nuclear reprogramming. In
degenerative pathologies and decreased life span associated
contrast, we observed a decrease in the density of H3K9m3
with telomere dysfunction in TercÀ/À mice (Hemann et al.,
and H4K20m3 histone heterochromatic marks at the telomeres
2001; Samper et al., 2001; Siegl-Cachedenier et al., 2007).
of iPS cells (passage 8) and ES cells compared to differentiated
Here, we addressed whether telomerase reintroduction into G3
MEFs (Figure 4A). A similar decrease in these marks was also
TercÀ/À MEF by means of mouse crosses between Terc+/À
observed at pericentric repeats (Figure 4B). Importantly, this
mice and G2 TercÀ/À mice was sufficient to rescue critically short
decrease in heterochromatic marks cannot be attributed to
telomeres and iPS cell generation in MEF derived from the G3
differences in the input of telomeric or pericentric DNA or in
TercÀ/+* offspring compared to the G3 TercÀ/À* littermates. As
nucleosome density at these regions, as ChIP values were cor-
shown in Figures 2A and 2B, iPS cell efficiency was fully restored
rected both by telomere and centromere inputs, respectively,
to wild-type levels in G3 TercÀ/+* cells compared to G3 TercÀ/À*
as well as by H3 and H4 abundance at these domains (Figures
cells, both of which inherited short telomeres from the G2 TercÀ/À
4A and 4B). Of notice, we did not detect significant changes in
parent. This rescue of iPS cell generation when using G3
the density of the TRF1 shelterin protein at telomeres between
TercÀ/+* MEF was concomitant with the disappearance of
iPS cells, ES cells, and MEF (Figure 4A). As control, TRF1 was
critically short telomeres (signal-free ends) and with rescue
not detected at pericentric chromatin (Figure 4B).
of chromosome end-to-end fusions in the G3 TercÀ/+* iPS
We have previously described that heterochromatic marks at
cells compared to iPS derived from G3 TercÀ/À* littermates
telomeres repress homologous recombination events between
(Figures 2F and 2G). Of interest, average telomere length in
telomeric repeats (Gonzalo et al., 2006; Benetti et al., 2007).
G3 TercÀ/+* iPS cells was not fully restored to that of wild-type
Next, we determined telomere recombination frequencies in
iPS cells (Figures 2C–2E), in agreement with previous reports
ES cells, iPS cells, and parental MEF by using chromosome
showing that telomerase preferentially elongates the very short
orientation-FISH (CO-FISH) (Experimental Procedures), which
telomeres (Hemann et al., 2001; Samper et al., 2001) and that
measures frequency of chromatid exchanges between sister
average telomere length is only recovered with time (Siegl-Cache-
telomeres (T-SCEs). Interestingly, iPS cells (passage 8) showed
denier et al., 2007). These results indicate that presence of criti-
higher telomere recombination frequencies than parental MEF,
cally short telomeres, rather than average telomere length, is the
reaching similar values to those of ES cells (Figure S5). Together,
key molecular event restricting iPS cell generation in the absence
these results suggest that telomere chromatin adopts a less-
of telomerase enzymatic activity. In accordance with this finding,
compacted conformation in ES cells compared to differentiated
we were able to derive viable chimeric mice from G3 TercÀ/+* iPS
cells and that telomeric chromatin in iPS cells resembles that of
cells in contrast to G1–G3 TercÀ/À iPS cells (Table 2).
control ES cells.
Telomeric Heterochromatin in iPS Cells Acquires ES
Increased Telomere Transcription in iPS Cells
Cell Features
Telomere chromatin is transcribed, generating long noncoding
Epigenetic marks, such as DNA methylation of specific loci, are
UUAGGG-rich transcripts (TelRNAs or TERRAs) that remain
properly reprogrammed during iPS cell generation, reaching
associated to the telomeric chromatin (Azzalin et al., 2007;
a pattern of DNA methylation similar to that shown by ES cells
Schoeftner and Blasco, 2008), where they have been proposed
(Wernig et al., 2007). DNA methylation at pericentric repeats
to act as negative regulators of telomere length (Schoeftner
does not seem to undergo reprogramming for the trivial reason
and Blasco, 2008). In particular, abundance of TelRNAs is posi-
that it remains unaltered in ES cells, iPS cells, and differentiated
tively correlated with telomere length, and these RNAs can effi-
cells (Wernig et al., 2007). Using 4F iPS, here we extend these
ciently inhibit telomerase activity in in vitro TRAP assays
observations to interspersed repeats (SINE repeats) and to sub-
(Schoeftner and Blasco, 2008). In agreement with their longer
telomeric repeats, which are also fully methylated in these three
telomeres, we find that TelRNAs are more abundant in ES cells
Figure 2. Impaired iPS Cell Generation from Late Generation TercÀ/À MEF with Critically Short Telomeres
(A) Quantification of relative efficiency of iPS cell clone generation. Values are normalized to viral transduction efficiency (EGFP fluorescence). Efficiency of iPS cell
generation is decreased in G2 and G3 TercÀ/À MEF but is fully restored upon telomerase reintroduction into G3 TercÀ/À MEF (G3 TercÀ/+* cells). Error bars, SE.
Statistical analysis was performed using a Student’s t test. n, number of independent measurements.
(B) Representative images of alkaline phosphatase-positive iPS cell clones.
(C) Telomere length analysis by TRF. Note that a subset of the telomeres is elongated in G2 and G3 TercÀ/À iPS cell clones compared to the parental G2-G3 MEF
(asterisk). MEF passage number = 2; iPS passage number = 8.
(D) Telomere length as determined by Q-FISH on metaphase spreads. Statistical significance values are shown as assessed by the Wilcoxon-Mann-Whitney rank
sum test. MEF passage number = 2; iPS passage number = 8.
(E) Quantification of average telomere length in kilobases. n, number of telomeres analyzed. MEF passage number = 2; iPS passage number = 8. Error bars, SD.
(F) Quantification of the percentage of signal-free ends. MEF passage number = 2; iPS passage number = 8. Note the disappearance of signal-free ends in the G3
TercÀ/+* iPS compared to the parental G3 TercÀ/+* MEF. n, number of telomeres. Statistical comparisons using the chi-square test are shown.
(G) Frequency of end-to-end chromosome fusions. n, number of metaphases. The total number of fusions out of the number of chromosomes analyzed is also
indicated. MEF passage number = 2; iPS passage number = 8. Chi-square test was used for statistical analysis. Error bars, SE.
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Cell Stem Cell
Reprogramming of Telomere Chromatin in iPS Cells
Figure 3. Global and Subtelomeric DNA Methylation in Mouse iPS Cells
(A) Fraction of methylated B1 SINE repeat elements. n, number of independent cell cultures. MEF passage number = 2; iPS passage number = 8. Statistical anal-
ysis was performed using a Student’s t test. Error bars, SD.
148 Cell Stem Cell 4, 141–154, February 6, 2009 ª2009 Elsevier Inc.

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Cell Stem Cell
Reprogramming of Telomere Chromatin in iPS Cells
than in differentiated MEF, whereas iPS (4F) (passage 8) show
eled in iPS cells to a similar conformation as that shown by ES
intermediate levels (Figures 4C and 4D), probably because addi-
cells’ telomeric chromatin. In particular, both iPS and ES cells
tional rounds of cell division are needed to reach full telomere
show a significant decrease in the density of histone heterochro-
elongation by telomerase (Figures 1F and 1G). Intriguingly,
matic marks (H3K9m3 and H4K30m3) at telomeric regions
production of centromeric transcripts was lower in ES cells
compared to differentiated MEF cells and present higher telo-
compared to parental MEF, and a similar scenario was found
mere recombination frequencies than parental MEF cells, sug-
for iPS cells (Figure 4C), pinpointing fundamental differences in
gesting a more relaxed chromatin conformation associated to
the regulation of telomeric and centromeric transcription. In
pluripotent stem cells, in agreement with their higher transcrip-
summary, these results indicate that iPS cell telomeres show
tional activity. These results go in line with the recent findings,
higher transcriptional levels than differentiated cells, in agree-
suggesting a higher plasticity in the chromatin of pluripotent
ment with their decreased density of histone heterochromatic
embryonic stem cells compared to differentiated cells (Meshorer
marks (Figure 4A).
et al., 2006).
Importantly, we show that donor cells with short telomeres ob-
Telomere Elongation in iPS Cells Derived from Old
tained from old animals show telomere elongation and functional
Donors
telomere capping during reprogramming into iPS cells, predict-
Telomeres shorten with aging, and this telomere shortening is
ing full functionality and long-term regenerative potential of iPS
known to limit the proliferative capacity of stem cells. There is,
derived from individuals with a limited telomere reserve, such
therefore, an interesting question of whether telomere length of
as elderly individuals or patients suffering from diseases charac-
donor cells represents a potential barrier to the proper function-
terized by short telomeres (Blasco, 2005). However, these
ality of iPS cells and whether iPS cells derived from donors with
results also raise the potential problem of a less efficient reprog-
a limited telomere reserve, such as elderly individuals or patients
ramming of iPS cells derived from patients with germline telome-
with diseases characterized by short telomeres, would inherit
rase mutations, such as dyskeratosis congenita and some cases
telomeric defects, including suboptimal telomere length. To
of aplastic anemia and idiopathic pulmonary fibrosis, as telo-
address this question, we generated iPS cells from dermal skin
mere elongation during iPS cell generation requires an active
fibroblast obtained from young (22 weeks old) and old (121
telomerase complex. This is supported by our findings showing
weeks old) mice (Figure 5). Dermal fibroblasts derived from old
that MEF derived from increasing generations of Terc-deficient
donors showed shorter telomeres than those of young dermal
mice have a decreased iPS cell efficiency from the second
fibroblasts both when determined by TRF (Figure 5A) and by
generation onward, which is concomitant with the presence of
Q-FISH (Figures 5B and 5C), in agreement with telomere short-
short telomeres and increased chromosomal aberrations. This
ening with mouse aging (Flores et al., 2008). Importantly, telo-
parallels the decreased stem cell functionality of different adult
meres of iPS cells from old donors were elongated similarly to
stem cell compartments of telomerase-deficient mice, including
those of iPS cells from young donors as indicated by TRF
neural stem cells, epidermal stem cells, and hematopoietic stem
(Figure 5A) and Q-FISH (Figures 5B and 5C). The percentage
cells (Samper et al., 2002; Allsopp et al., 2003; Ferro´n et al., 2004;
of signal-free ends and the frequency of end-to-end fusions
Flores et al., 2005). Importantly, the fact that telomerase reintro-
were also similarly low in iPS cells derived from young and old
duction and re-elongation of short telomeres are sufficient to
dermal fibroblasts (Figures 5D and 5E), suggesting normal telo-
restore a normal iPS cell generation and iPS cell functionality,
mere functionality in these cells.
as determined by generation of viable chimeras in late-genera-
tion telomerase-deficient MEF, pinpoints to telomerase reactiva-
tion as a putative therapeutic strategy to derive functional iPS
DISCUSSION
cells from patients harboring telomerase mutations and a limited
telomere reserve.
We show here that mouse iPS cell telomeres adopt similar
features to those of ES cell telomeres. In particular, telomere
length was significantly increased in 3F and 4F iPS cells
EXPERIMENTAL PROCEDURES
compared to parental differentiated cells, reaching intermediate
Generation of Mouse iPS Cells
levels to those of control ES cells in early passages but reaching
Reprogramming of primary (passage 2–4) MEF derived either from wild-type or
telomere length comparable to control ES cells at later
different generation (G1–G3) telomerase null TercÀ/À embryos (MEFs of
passages. We interpret that reprogramming changes the acces-
C57BL6 genetic background) or skin fibroblasts derived from young and old
sibility of telomerase to telomeres, allowing their progressive
animals was performed essentially as described (Blelloch et al., 2007). Briefly,
elongation until telomeres reach the length characteristic of ES
retroviral supernatants were produced in HEK293T cells (5 3 106 cells per
cells.
100 mm diameter dish) transfected with the ecotropic packaging plasmid
pCL-Eco (4 mg) together with either one of the following retroviral constructs
A similar scenario is found for the generation of telomere
(4 mg): pMXs-cMyc, pMXs-Klf4, pMXs-Sox2, or pMXs-Oct3/4 (Addgene).
RNAs, in agreement with the abundance of telomere transcripts
Transfections were performed using Fugene-6 transfection reagent (Roche)
being directly correlated with telomere length (Schoeftner and
according to the manufacturer’s protocol. At 2 days later, retroviral superna-
Blasco, 2008). In addition, telomere heterochromatin is remod-
tants (10 ml) were collected serially during the subsequent 48 hr at 12 hr
(B and C) Abundance of methylated CpG dinucleotides as determined by bisulfite sequencing in the indicated subtelomeric regions. n = 17–55 clones were
analyzed from a total of two cultures per cell type. Yellow and blue represent the frequency of methylated and unmethylated CpG dinucleotides, respectively.
Gray corresponds to undetermined methylation. CpG, CpG position; U, unmethylated; M, methylated; n.p., not present. Chi-square test was used for statistical
analysis, and the significant differences are indicated. Error bars, SD. MEF passage number = 2; iPS passage number = 8.
Cell Stem Cell 4, 141–154, February 6, 2009 ª2009 Elsevier Inc. 149

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Cell Stem Cell
Reprogramming of Telomere Chromatin in iPS Cells
150 Cell Stem Cell 4, 141–154, February 6, 2009 ª2009 Elsevier Inc.

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Document Outline

• Telomeres Acquire Embryonic Stem Cell Characteristics in Induced Pluripotent Stem Cells
  • Introduction
  • Results
    • cMyc Is Dispensable for Telomerase Activation in Mouse iPS Cells
    • Telomere Elongation by Telomerase in iPS Cells
    • Impaired iPS Cell Generation from Late-Generation Terc-/- MEF with Critically Short Telomeres
    • Telomeric Heterochromatin in iPS Cells Acquires ES Cell Features
    • Increased Telomere Transcription in iPS Cells
    • Telomere Elongation in iPS Cells Derived from Old Donors
  • Discussion
  • Experimental Procedures
    • Generation of Mouse iPS Cells
    • TRF Analysis
    • Q-FISH Analysis
    • B1-SINE Cobra Analysis for Global DNA Methylation
    • Analysis of Genomic Subtelomeric DNA Methylation
    • ChIP Assay
    • Telomere Transcription
  • Supplemental Data
  • Acknowledgments
  • References

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