1952 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
Defective telomere elongation and
hematopoiesis from telomerase-mutant
aplastic anemia iPSCs
Thomas Winkler,1 So Gun Hong,1 Jake E. Decker,1,2 Mary J. Morgan,1 Chuanfeng Wu,1
William M. Hughes V,1 Yanqin Yang,3 Danny Wangsa,4 Hesed M. Padilla-Nash,4
Thomas Ried,4 Neal S. Young,1 Cynthia E. Dunbar,1 and Rodrigo T. Calado1,5
1Hematology Branch, National Heart Lung and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA.
2Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. 3DNA Sequencing and Genomics Core Facility,
NHLBI, and 4Section of Cancer Genomics, Genetics Branch, National Cancer Institute, NIH, Bethesda, Maryland, USA.
5Department of Internal Medicine, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto, São Paulo, Brazil.
Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malig-
nant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of
human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or
hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT)
or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated
TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a
lower rate compared with healthy iPSCs, and the deficit correlated with the mutations’ impact on telomerase
activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation
varied among iPSC clones derived from the same patient and among clones from siblings harboring identi-
cal mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced
by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant
and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in
vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases
can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs
to model disease.
Telomeres are nucleoprotein structures at the end of linear chro-
mosomes consisting of repetitive, non–protein coding DNA
sequences that are coated by the protein complex shelterin (1).
Telomeres protect the end of the chromosomes from DNA dam-
age and prevent the activation of DNA-damage signaling pathways
and nonhomologous end joining. In humans, telomeric DNA is
composed of TTAGGG tandem repeats. Telomerase consists of a
reverse transcriptase enzyme (TERT), an RNA template (TERC),
and stabilizing proteins including dyskerin (encoded by DKC1)
and TCAB1 (2). The enzymatic complex adds nucleotide hexam-
ers to the 3′ end of telomeres in highly proliferative cells.
Disturbed telomere homeostasis is etiologic in several severe
human diseases (3). In X-linked dyskeratosis congenita, critically
short telomeres are due to mutations in the X-linked DKC1 gene.
Although clinically characterized by a triad of mucocutaneous
findings (nail dystrophy, leukoplasia, and reticular skin hypopig-
mentation), dyskeratosis congenita is a pleiotropic, multi-organ
disease, and the majority of the patients succumb to bone marrow
failure (80%), pulmonary fibrosis, or liver disease (4). The clinical
diagnosis of dyskeratosis congenita requires the presence of at
least 2 features of the mucocutaneous triad (5).
We and others have demonstrated that heterozygous mutations
in TERT or TERC result in very short telomeres of leukocytes, trans-
lating into phenotypes that are milder and clinically distinct from
those observed in dyskeratosis congenita; heterozygous TERC or
TERT mutations often affect a single organ, the clinical presentation
is later in life, and patients usually lack the physical abnormalities
typical of dyskeratosis congenita. Telomerase mutations are genetic
risk factors for apparently acquired aplastic anemia (6), idiopathic
pulmonary fibrosis (7, 8), liver disorders (9–11), and acute myeloid
leukemia (12, 13). The clinical phenotype of individuals within fam-
ilies harboring a given mutation is variable, extending from asymp-
tomatic carriers through mild laboratory findings to severe aplastic
anemia (clinically indistinguishable from acquired aplastic anemia
and without the accompanying physical examination features of
dyskeratosis congenita). Patients and other family members with
mutations may have subtle or severe organ involvement beyond the
marrow, especially pulmonary fibrosis or hepatic cirrhosis. In con-
trast to the high genetic penetrance observed in X-linked dyskerato-
sis congenita, other genetic, epigenetic, and environmental factors
appear to modulate disease phenotype in these telomeropathies (3).
Dyskeratosis congenita represents the most severe phenotype, but
probably represents a small portion of the expanding spectrum of
illnesses caused by telomere dysfunction.
In the adult organism, telomerase expression is tightly regulat-
ed and mainly restricted to stem and progenitor cells responsible
for replenishing actively proliferating tissues such as the bone
marrow. Investigating the molecular mechanisms and environ-
Authorship note: Thomas Winkler, So Gun Hong, and Jake E. Decker contributed
equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2013;123(5):1952–1963. doi:10.1172/JCI67146.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
mental modulators of telomerase expression that translate into
disease phenotype is difficult because the primary affected cells,
for instance, bone marrow stem and progenitor cells in aplastic
anemia, are markedly depleted.
The recent technology pioneered by Takahashi and Yamanaka
(14) of reprogramming somatic cells into a pluripotent, ES cell–
like phenotype offers a method for modeling telomere dynamics
in the telomeropathies and for investigating pathways resulting
in tissue dysfunction. High expression of telomerase in human
ES cells ensures maintenance of telomere length and helps define
their pluripotent phenotype (15). Telomerase is also upregulated
during iPSC generation (16, 17) and is likely responsible for telo-
mere elongation and maintenance in these cells (18, 19). Therefore,
reprogramming technology followed by differentiation could be a
valuable tool for studying telomere dynamics in a mutation- and
patient-specific manner. Based on their pluripotent phenotype
and ability to differentiate into virtually any tissue type, induced
pluripotent stem cells (iPSCs) may enable the study of potential
environmental or epigenetic factors responsible for the variable
penetrance of telomere diseases.
Recently, 2 studies investigating telomere dynamics in iPSCs
derived from an X-linked dyskeratosis congenita patient with a
loss-of-function mutation in the DKC1 gene came to contradictory
conclusions. Albeit at very low efficiency, both studies successfully
derived iPSCs from patients’ fibroblasts. Despite the presumable
loss of function of DKC1, Agarwal et al. reported telomere elon-
gation in iPSCs (20) and TERT and TERC upregulation, whereas
Batista et al. observed reduced telomerase activity and progressive
telomere erosion, associated with loss of self-renewal potential and
early senescence (21). Batista et al. also found in iPSCs derived from
dyskeratosis congenita patients with TERT heterozygous muta-
tions that telomerase activity was reduced and telomere elonga-
tion during reprogramming was impaired. However, neither study
explored the telomere dynamics in iPSCs from patients with more
common and milder telomere diseases than dyskeratosis congenita.
In an effort to develop an in vitro iPSC model for patients
with aplastic anemia who do not display the typical dyskerato-
sis congenita phenotype but have very short telomeres due to
heterozygous loss-of-function mutations in telomerase complex
genes, we derived iPSCs from patients with hematologic abnormal-
ities harboring mutations in the TERT or TERC genes. Given the
conflicting results in prior studies as to whether iPSCs accurately
model telomere dynamics in dyskeratosis congenita, we generated
several iPSC clones from each of multiple patients with muta-
tions in TERT or TERC and studied their telomere dynamics. We
measured telomerase gene expression and telomerase enzymatic
activity at multiple time points during the reprogramming process
and after prolonged passaging. Additionally, we investigated the
impact of environmental factors on telomere maintenance, specif-
ically hypoxia, which had been shown to increase reprogramming
efficiency and to reduce differentiation in ES cells (22, 23). We also
investigated whether telomere elongation during reprogramming
exclusively depends on telomerase or whether recombination-
mediated alternative lengthening of telomere (ALT) contributes
to telomere length maintenance in iPSCs.
Patient characteristics and iPSC derivation. Four adult patients with
aplastic anemia or markedly reduced bone marrow cellularity, age-
adjusted markedly short telomeres, and known mutations within
the telomerase complex genes were studied (Figure 1 and Table 1).
Three patients had TERT mutations, including 2 siblings with
a reverse transcriptase domain mutation abolishing telomerase
function (TERT[R889X]f and TERT[R889X]m) and a patient with
C-terminal extension domain mutation of TERT (TERT[R1084P]),
reducing activity by 70% (24). One patient had a heterozygous
TERC promoter mutation in the CCAAT box (TERC[-58C>G])
(25), resulting in significantly decreased gene expression. Control
iPSCs were generated from a normal volunteer with no family his-
tory or manifestations of telomere diseases (control 1) and com-
mercially available newborn foreskin fibroblast cells (control 2).
iPSCs were generated by overexpression of the transcription
factors POU5F1 (OCT4), SOX2, KLF4, and MYC individually from
retroviral vectors (26) (retroviral OSKM) or from a lentiviral-
based polycistronic vector (27) (polycistronic OSKM, STEMC-
CA) in skin fibroblasts. Multiple iPSC clones could be obtained
from each patient or control. All derived iPSCs had typical ES
cell morphology, stained positive for alkaline phosphatase and
the pluripotency markers OCT4, SSEA4, NANOG, Tra1-60, and
Tra1-81 by immunofluorescence (Supplemental Figure 1A; sup-
plemental material available online with this article; doi:10.1172/
JCI67146DS1), and formed teratomas in NSG mice and formed
teratomas in NSG mice (Supplemental Figure 1B). Additionally,
microarray mRNA expression studies on control (control 2) and
all telomerase mutant iPSCs confirmed an ES cell–like mRNA
expression profile (Supplemental Figure 1C; GEO GSE42869).
Supplemental Table 1 summarizes all iPSC characterization
studies performed. Sequence analysis documented that all iPSCs
retained the original telomerase mutation present in the parental
fibroblasts (Supplemental Figure 2). Spectral karyotyping (SKY)
was performed on selected iPSCs (28). The composite karyotypes
are presented in Supplemental Table 4.
Impaired telomere elongation in mutant iPSCs. Using quantitative
PCR (qPCR) (29), telomere length was measured for at least 20
passages in all clones. We found that telomeres elongated in all
Blood leukocyte telomere length (kb) as a function of age. Individuals
with TERC and TERT mutations have very short telomeres in peripheral
blood leukocytes. The line designates the 50th percentile of telomere
length for 298 healthy blood donors (25). The peripheral blood leuko-
cyte telomere lengths were measured by qPCR and T/S ratios trans-
formed to kilobases based on a linear correlation between qPCR and
Southern blot results (r2 = 0.86).
1954 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
iPSCs compared with their parental fibroblasts (Figure 2); results
were validated by standard Southern hybridization (Figure 2,
H and I). However, the telomere elongation rate was substan-
tially lower in the TERT- and TERC-mutant iPSCs as compared
with iPSCs derived from healthy controls. Telomere extension of
heterozygous telomerase-mutant iPSCs was approximately half
of that observed in healthy control iPSCs (Figure 2G). However,
the telomere elongation pattern was substantially heterogeneous
among iPSC clones from different patients and even among inde-
pendent clones derived from the same patient (Figure 2). All TERT-
and TERC-mutant iPSC clones, except for clone TERT[R889X]f
c4.1, elongated telomeres at various rates for the first 15 passages
and then reached a plateau.
TERT [R889X] mutation reduces TERT RNA expression. We investi-
gated how reprogramming affected TERT and TERC gene expres-
sion. All iPSC clones (from mutant patients and controls) had
marked and comparable increases in both TERT and TERC mRNA
expression levels in comparison with parental fibroblasts (Figure 3,
A and B). TERC expression was lower in TERT-mutant iPSC clones
in comparison with healthy controls (Figure 3C). Although TERT
mRNA expression was comparable between controls and iPSC
clones with TERT[R1084P] or TERC[-58C>G] mutations, TERT
expression was significantly lower in iPSCs derived from both
patients carrying the truncation mutation in TERT [R889X]
(P < 0.05; Figure 3C). We also observed variability in mRNA
expression of both TERT and TERC between clones derived from
the same donor and at successive passages even in an individual
clone (Figure 3, A and B).
Transgene excision does not modulate the expression of TERT and
TERC. It is possible that permanent or transient reactivation of
reprogramming transgenes contributes to the telomere elonga-
tion and the observed heterogeneity, especially after the recent
observation that the Wnt/β-catenin pathway activates the TERT
gene (30) via interaction with KLF4 in a murine ESC model and in
human cancer cell lines. However, in multiple iPSC clones derived
from controls and telomerase-mutant patients, analysis of the
β-catenin canonical pathway via gene expression profiling did not
correlate with the rate of telomere elongation (GEO GSE42869).
Furthermore, cre-excision of the transgenes expressed from the
polycistronic lentiviral vector (Supplemental Figure 3) did not
result in reduced expression of TERT and TERC (Figure 3D),
indicating that potential residual MYC or KLF4 expression was
not responsible for telomerase activation or the heterogeneity
in telomere elongation observed among iPSC clones. Indeed,
we observed a similar variability in the expression of both genes
among different clones and passages of the transgene-free mutant
iPSCs (Supplemental Figure 4, A and B).
Telomerase activity and ALT. We determined telomerase functional
activity in iPSCs. There was a significant increase in telomerase
activity in all iPSCs grouped by mutation type in comparison with
parental fibroblasts, comparable to the level seen in ESCs, but
also displaying some variation in activity at successive passages
as well as in different clones from the same individual (Figure 4A
and Supplemental Figure 5). However, telomerase activity did not
directly correlate with TERT or TERC mRNA expression.
The decreased telomere elongation rates and reduced telom-
erase function in heterozygous telomerase-mutant iPSCs, both
corresponding to the degree of telomerase deficiency predicted
by the gene lesion, suggested that telomere elongation in iPSCs
is directly dependent on a functional telomerase complex. Telom-
erase-independent ALT mechanisms have been reported in some
cancer cells (reviewed in ref. 31). The accumulation of C-rich
partially single-stranded circles of telomeric DNA (C-circles) is a
sensitive and quantifiable marker for ALT (32). In order to detect
any contribution of the ALT pathway to telomere extension dur-
ing reprogramming, we applied the C-circle assay, but found no
significant accumulation of C circles in healthy or mutant iPSCs
(Figure 4B). Additionally, increased telomere length heterogene-
ity is a hallmark of active ALT mechanisms. We did not observe
increased telomere length heterogeneity in healthy control or
Patient characteristics and iPSC selected for further analysis
Effect of mutation on
Clinical manifestation Family history iPSC clone Reprogramming method
53 years old, male
45 years old, female
55 years old, male,
(brother of 45 years
30 years old, female
22 years old, male
BJ-1, newborn foreskin
reduced by 70%
expression of TERC
marrow (<5%) with
normal PBC counts
Empty for telomere diseases
OSKM, OCT4, SOX2, KLF4, MYC; polycistronic, OSKM expressed from the single polycistronic lentiviral construct STEMCCA.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
Telomere lengths in iPSCs derived from controls and from patients with telomerase complex mutations followed over time. (A–F) Each panel
shows telomere lengths (represented as T/S ratios) of cells from an individual patient or control. Each line shows values in relation to passage
number from independent iPSC clones. In each panel, parental somatic cell lengths are shown in green. To quantify changes in telomere length
over time, slopes were generated by linear regression analysis of the T/S ratios during the first 10 passages. (G) Combined elongation slopes
from all clones derived from a particular patient or healthy control. Note: TERT[R889X]f and TERT[R889X]m clones are derived from a brother/
sister pair. The suffixes indicate either female (f) or male (m). Southern blot analysis of telomere length in early passages from control, TERC (H),
and TERT (I) mutated iPSC confirming qPCR results. Fib, fibroblasts.
1956 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
Heterogeneity in TERT and TERC mRNA expres-
sion. Expression of TERT (A) and TERC (B) was
compared at the mRNA level in the patient-derived
iPSCs and parental fibroblasts as a function of pas-
sage. Expression data were analyzed as average
expression from all clones and passages for each
mutation (C; 1-way ANOVA, *P < 0.05). TERT and
TERC expression are not altered after excision of
the transgenes in mutant iPSC (D). FB, fibroblasts.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
telomerase-mutant iPSCs (Figure 2, H and I). Taken together,
these results indicate that telomerase is the only major mechanism
responsible for telomere elongation during human cell reprogram-
ming to a pluripotent state.
Telomere maintenance in iPSCs is influenced by environmental and
genetic factors. Pluripotency and reprogramming efficiency may be
influenced by conditions that alter the intracellular redox state
(23), but the impact of ambient oxygen concentration on telomere
dynamics in human iPSCs has not previously been investigated.
We cultured iPSCs from healthy subjects and from patients with a
telomerase complex mutation in hypoxic (5%) or ambient oxygen
(21%). After 4 passages, telomeres were longer in both control and
telomerase complex mutant iPSC clones cultured at low oxygen lev-
els, in comparison with the same clones cultured in ambient oxygen
(P = 0.001; Figure 5A). In an independent reverse approach, iPSCs
previously kept at 5% oxygen were either maintained at hypoxic
conditions or transferred to 21% oxygen, and similarly, iPSCs
transferred to 21% oxygen had telomeres much short-
er than those maintained at 5% oxygen (Figure 5B).
Furthermore, iPSCs grown in low (5%) oxygen tension
had higher TERT mRNA expression in comparison
with TERT expression in iPSCs cultured in 21% oxygen
(Figure 5C). However, TERC expression was not altered
in response to different oxygen concentrations (data
not shown). These observations indicate that low oxy-
gen tension resulted in higher telomerase expression
in human iPSCs and enhanced telomere extension,
regardless of telomerase mutation status.
We observed that one clone carrying the TERT
truncation mutation R889X (clone 4.1) showed sud-
den erosion of telomere length beginning at passage
9, in contrast with 2 other clones derived from the
same patient’s fibroblasts, used to derive the iPSCs
(Figure 5D). This accelerated telomere attrition was
accompanied by an obvious increase in spontaneous
differentiation that eventually precluded further pas-
saging. SKY revealed an unbalanced translocation
between chromosomes X and 2 (46,XX, der[X],t[X,2]
[p22.3;p23]) at the time of accelerated telomere attri-
tion (p26) (Figure 5, D and E). Telomere attrition was
confirmed by Southern blot hybridization (Supple-
mental Figure 6).
Impaired hematopoietic differentiation in telomerase
mutant iPSCs. We differentiated control iPSCs and
mutated iPSCs toward hematopoietic progenitors
via a multistep differentiation procedure (Figure 6A).
iPSCs derived from patients with TERT or TERC
mutations and aplastic anemia showed embyroid
body (EB) formation comparable to control iPSCs and
ESCs (Figure 6B). However, in the telomerase mutant
iPSC, the percentage of hematopoietic progenitors
(CD34+CD45+) on day 20 after EB formation was
reduced (Figure 6C) as well as the numbers of hema-
topoietic CFUs (Figure 6D), indicating an impaired
hematopoietic differentiation capacity. Moreover,
the differences in CFU numbers reflected the differ-
ent severities of the patients’ clinical hematopoietic
phenotypes (Table 1), which was mostly apparent in
the brother/sister pair with the TERT[R889X] muta-
tion. The sister, with clinical severe aplastic anemia,
had profoundly depressed CFU formation from her iPSCs in com-
parison with her brother’s much more moderate defect in this
assay of hematopoietic progenitors.
In the present study, we show that adult somatic cells from
patients with bone marrow failure syndromes associated with
mutations in genes of the telomerase complex are reprogramma-
ble to pluripotency, but telomerase function and telomere elon-
gation remain defective after reprogramming and hematopoietic
differentiation is impaired. Impairment in telomere length main-
tenance in patient iPSCs may depend on the mutated gene and its
pattern of influence on telomerase activity, but telomere elonga-
tion may also be influenced by genetic abnormalities acquired dur-
ing reprogramming, clone heterogeneity due to factors involved in
iPSC generation and maintenance, and culture conditions such as
Telomerase activity and ALT. (A) Telomerase activity was measured and normal-
ized to the activity of HeLa cells. Reactions were performed in triplicate. Data were
analyzed as average activity of passages of all clones from a particular mutation
or control. Each circle represents 1 data point of measured telomerase activity. (B)
Dot blot of C-circle assay performed on genomic DNA from TERT and TERC mutant
as well as control IPSCs cultured either in 5% or 21% oxygen. Genomic DNA from
the ALT-positive cell lines Saos2 and U2-OS served as positive controls. **P < 0.01;
***P < 0.001, 1-way ANOVA.
1958 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
The ability to maintain telomeres is a necessary attribute
of pluripotent stem cells with unlimited replicative potential
(33). The induction of telomerase expression and resultant
telomere elongation occur following reprogramming of somat-
ic cells to pluripotency in both murine (18) and human (19)
iPSCs. MYC and KLF-4, 2 of the transcription factors commonly
used to derive iPSCs and endogenously upregulated following
reprogramming, directly or indirectly induce TERT expression
(30, 34). Reprogramming is associated with de novo changes in
methylation of subtelomeric chromosomal regions and eleva-
tion of telomeric-repeat–containing RNA (TERRA) expression,
indicating that significant changes in the architecture and
function of telomeric and subtelomeric structures take place
during this process (19).
Previous derivations of iPSCs from patients with X-linked dys-
keratosis congenita and a DKC1 mutation yielded conflicting
results. Agarwal et al. (20) reported significantly elongated telo-
meres and high TERC expression after reprogramming in single
clones derived from 2 patients, but Batista et al. observed telomere
erosion and low TERC expression in DKC1-mutant clones (21). The
Environmental and genetic factors influence telomere dynamics in iPSC. (A) Telomere length over time is shown in iPSC from normal (CTRL-1
c26), TERT (TERT[R1084P] c5.1), and TERC (TERC[-58C>G]c13) mutated iPSCs cultured simultaneously in either 5% or 21% oxygen. (B)
Reverse experiment: normal iPSC (CTRL-1 c3) and mutated iPSC (TERT (TERT [R1084P] c7.0, TERC (TERC[-58C>G]c13) previously cultured
in 5% oxygen were either continuously cultured in 5% or placed in 21% oxygen. Telomere lengths were measured at indicated passages. (C)
TERT mRNA is upregulated in iPSC cells cultured in reduced oxygen conditions (5%). (D) Telomere attrition in clone TERT[R889X]f c4.1 (orange
line) compared with other clones derived from the same starting population of patient fibroblasts. (E) Karyotype analyzed via SKY for clone
TERT[R889X]f c4.1, with arrow identifying the unbalanced translocation der(X)t(X;2).
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
mechanisms of telomere maintenance during reprogramming of
human telomerase-mutant cells remain controversial.
We observed that TERT mRNA expression levels in TERT- and
TERC-mutant iPSCs were generally increased upon reprogram-
ming as compared with parental fibroblasts, similarly to the
degree of upregulation observed during generation of control
iPSCs. However TERT mRNA levels were consistently lower in
iPSCs derived from 2 individuals carrying the TERT codon R889X
truncation mutation. This mutation results in a premature stop
codon, eliminating the enzymatic C-terminal domain from the
protein. How this premature stop codon results in lower mRNA
levels is not clear and may involve the mRNA nonsense-mediated
decay (NMD) mechanism (35). Analysis of TERT-mutant iPSCs
in the current study demonstrated that lower TERT mRNA levels
might be an additional mechanism for telomerase insufficiency in
some patients with truncation mutations and highlights the con-
tribution of the reprogramming technology to identify regulatory
mechanisms that would have been difficult to detect by conven-
tional knockin and knockout models.
During early passages, the telomere elongation rate was signif-
icantly higher in control iPSCs than in TERT- or TERC-mutant
iPSCs (Figure 2), and continuous elongation over the first passages
indicated the observed telomere elongation in our iPSCs was not
due to a selection of somatic cells with longer telomeres to begin
with. These findings are consistent with results reported by Batista
et al. in 2 patients with dyskeratosis congenita and heterozygous
mutations in TERT (21). However, patients with dyskeratosis con-
genita usually have multi-organ dysfunction (skin and mucous
Hematopoietic differentiation is impaired from telomerase mutant iPSC. (A) Schematic overview of the experimental procedure. EB were
cultured for 20 days in hematopoietic differentiation medium. After the dissociation of the EB, cells were plated in methylcellulose containing
a cytokine cocktail that favors the differentiation of human hematopoietic progenitors toward myeloid lineages, including erythroid and mega-
karyocyte progenitors. (B) Similar morphologies of EB from control iPSC, and mutant iPSC on day 20 before dissociation. Original magnifica-
tion, ×40. (C) Flow cytometry analysis for hematopoietic differentiation markers CD34 and CD45 from 3 independent experiments. (D) Qualita-
tive and quantitative analysis of hematopoietic colonies derived from control and mutant iPSC. CFU-GEMM, CFU–granulocyte, erythrocyte,
monocyte, megakaryocyte; CFU-GM, CFU–granulocyte, macrophage; BFU-E, burst forming unit–erythroid; CFU-E, CFU-erythroid; CFU-G,
CFU-granulocyte; CFU-M, CFU-macrophage.
1960 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
membrane findings and early bone marrow failure and liver and
lung manifestations) during the first decade of life, and the diag-
nosis requires at least 2 of 3 mucocutaneous features (3, 5, 36);
the diagnosis can be established by family history and the clinical
phenotype, and the prognosis is usually poor. In contrast, patients
bearing TERT or TERC mutations present with disease later in life,
often only a single organ is affected, and family members with
the same mutation may be clinically normal (3, 10, 37); they have
isolated aplastic anemia or pulmonary fibrosis without apparent
involvement of other organs. Clinically, these patients may not be
distinguishable from others with the same phenotype (aplastic
anemia, pulmonary fibrosis, hepatic cirrhosis) without a telomer-
ase mutation, and their prognosis is usually better than is that of
patients with dyskeratosis congenita.
We demonstrate that iPSCs from patients with TERT or TERC
mutations and aplastic anemia or marrow hypocellularity can
be utilized to model the hematopoietic defect, demonstrating
decreased production of hematopoietic colonies following differ-
entiation, with the degree of the in vitro defect corresponding to
the hematologic clinical status of the patient used to derive the
iPSC. This successful disease modeling could be utilized for drug
screening or investigation of the mechanisms linking telomere
shortening to impaired hematopoietic output and may be helpful
for uncovering the variable disease penetrance of telomeropathies.
Differentiation of the mutant iPSC toward hepatocytes or pulmo-
nary epithelial cells may in the future provide insights into the
genesis of dysfunction in these tissues in telomeropathy patients,
previously difficult to study due to lack of renewable tissue sam-
ples for experiments.
In families with TERT or TERC heterozygous mutations, clinical
features may manifest earlier and with greater severity in each sub-
sequent generation, a phenomenon termed disease anticipation
(38). Our findings provide a molecular mechanism during embryo-
genesis for disease anticipation in telomeropathies. Although telo-
meres are elongated during reprogramming of telomerase-mutant
somatic cells, the elongation is defective, resulting in pluripotent
cells with shorter telomeres in comparison with normal, suggest-
ing that telomere elongation during germ cell production and
early embryogenesis may also be insufficient. This could effectively
lead to embryos with shorter telomeres in each subsequent genera-
tion. We have recently directly demonstrated that telomere length
is inherited from parents using a murine model of telomerase defi-
ciency (39). In the offspring of animals deficient for telomerase,
telomeres are shorter than in their parents. In contrast, the pres-
ence of normal telomerase expression during embryogenesis after
several generations with deficient telomerase does not restore telo-
mere length to a “normal” set point, but rather maintains telomere
lengths comparable to those in their parents (40). Thus, telomer-
ase and telomere elongation are tightly regulated during embryo-
genesis, and disturbances in telomerase expression may result in
abnormal telomere attrition in the offspring.
Despite our demonstration of impaired telomere elongation in
telomerase-mutant iPSCs, there was marked heterogeneity among
individual iPSC clones derived from the same patient, between
clones from different patients with identical or similar mutations,
and even between individual clones measured at different passag-
es, regarding both telomere elongation rate and directly measured
telomerase activity. Of note, all cells were harvested at the same
time points during culture and had a similar cell density and spon-
taneous differentiation proportions. These findings raise at least 3
important issues. First, iPSCs are not a stable homogeneous clonal
population in culture, and culture conditions or stochastic events
may result in changes in telomerase expression and function.
Three different iPSC clones derived from the same starting popu-
lation from a single patient carrying a TERT mutation all showed
reduced telomere elongation during the first 5 passages, but 1
(TERT[R889X]f c4.1) eventually developed telomere attrition. In
this clone, we identified a derivative chromosome der(X)t(X;2) in
all metaphases analyzed in later passages. Apparently, acquisition
of a chromosomal abnormality may change iPSC homeostasis and
the ability to maintain telomeres. Genomic instability is a major
concern in reprogramming, and our knowledge of how large aber-
rations and less obvious point mutations affect iPSC behavior
is evolving (41). We also determined that oxygen concentration
affects the rate of telomere elongation, as iPSCs cultured in low
oxygen tension conditions consistently showed higher elonga-
tion rates and increased TERT expression regardless of telomerase
complex mutation status. In agreement with our findings, Cous-
sens et al. recently demonstrated that hypoxia-inducible factor 1 α
(HIF1-α) is a regulator of TERT expression in mouse ESCs (42).
In addition, oxidative damage may provoke telomere erosion in
human fibroblasts (43).
Second, heterogeneity in telomere length may, at least in part,
be responsible for the incongruent results of previous studies
examining telomerase complex mutations on iPSC derivation and
behavior. While Agarwal et al. observed telomere elongation in 1
of 2 clones derived from a DKC1-mutant patient (20), Batista et al.
did not observe any elongation of telomeres in iPSCs derived from
the same original DKC1-mutant fibroblasts. Instead, this study
found activation of TRP53 and CDKN1A/p21 and subsequent
senescence of the iPSCs (21). Agarwal et al. analyzed a very limited
number of clones from only 1 mutation and protein in the telom-
erase complex, which may not fully reflect a heterogeneous disease
or take into account the clonal heterogeneity described above, as
later acknowledged by the authors (44). Our results indicate clear
variability among and within clones by multiple measurements
and over time in culture. However, our results following transgene
excision indicate that this variability is not caused by residual
expression of the reprogramming factors. A careful analysis of
many clones is necessary to utilize iPSCs to study disease biology,
to screen for drug activity, and to ensure that artifacts of culture
are not being interpreted erroneously.
Third, heterogeneity in telomere elongation among clones may
provide the molecular basis for the wide spectrum in the clinical
presentation of telomere diseases, even within the same family.
In the brother/sister pair (TERT[R889X]m and TERT[R889X]
f) presented in our study, the clinical presentation was different;
while the sister had aplastic anemia, the brother, who was older,
was clinically healthy. These different phenotypes were mirrored
by differences in telomere elongation in iPSCs and hematopoietic
differentiation capacity between the siblings, consistent with addi-
tional genetic, epigenetic, and environmental factors that have an
impact on the clinical manifestations of these diseases in germ
cells and during early embryogenesis.
Finally, although telomere elongation was reduced in the repro-
gramming of TERT- or TERC-mutant haploinsufficient cells, it
was present and resulted in iPSCs with longer telomeres than
parental fibroblasts. The identification of additional cellular path-
ways involved may be helpful in the establishment of therapeutic
strategies to boost telomerase function and telomere elongation
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
further analyses, iPSCs were harvested and dispersed to single cells using
trypsin (0.05%) and purified away from residual MEFs via negative selec-
tion using MACS separation with antibodies directed against a murine
fibroblast surface protein (Miltenyi). iPSCs were cultured in 21% oxygen
tension unless otherwise designated.
In order to rule out the potential influence of the reprogramming trans-
genes on telomere behavior, the transgene cassette was excised using the
cre-recombinase–expressing vector pCL20i4rEF1-Puro-T2A-cre-GFP pro-
vided by Harry L. Malech (National Institute of Allergy and Infectious
Diseases, Bethesda, Maryland, USA). Briefly, iPSC were plated on Matri-
gel (BD), cultured in MEF-conditioned ES medium, and transfected with
pCL20i4rEF1-Puro-T2A-cre-GFP the next day using Lipofectamine LTX &
Plus Reagent (Invitrogen) according to the manufacturer’s recommenda-
tions. Transduced cells were positively selected for 3 days in Puromycine
(day 1: 3 μg/ml, day 2 and day 3: 2 μg/ml, Sigma-Aldrich) containing con-
ditioned ES medium. Approximately 10 days after the transfection, iPSC
colonies were picked, expanded, and analyzed by Southern blot for exci-
sion of transgenes. Excised iPSC subclones stained positive for common
pluripotency markers (Supplemental Table 3, data not shown) and were
maintained in culture for at least 10 passages after the excision.
Immunohistochemistry for pluripotency markers. Cells were cultured with
MEFs in 96-well plates for 3–5 days to allow for several colonies to estab-
lish growth. Cells were washed 1 time with PBS and fixed with 100 μl 4%
paraformaldehyde (Sigma-Aldrich) for 30 minutes at room temperature.
For intracellular staining, cells were permeabilized with 100 μl 0.2% Triton
X-100 (Sigma-Aldrich) for 30 minutes at room temperature followed by an
additional washing step with PBS and then incubated for 2 hours at room
temperature in 100 μl blocking buffer (3% BSA/5% donkey serum; Jackson
ImmunoResearch Laboratories). After blocking, cells were incubated with
100 μl primary antibody (1:100 in blocking buffer) overnight at 4°C. When
necessary, cells were incubated with 100 μl secondary antibody (1:500 in
blocking buffer) for 3 hours at 4°C. Following incubation with antibod-
ies, cells were washed with PBS, and nuclei were stained using Vectashield
with DAPI (Vector Laboratories). An overview of the antibodies used can
be found in Supplemental Table 3.
In vivo differentiation assay (teratoma formation). All mice were bred and
maintained at the NIH animal facility. Immune-deficient NOD.Cg-
PrkdcScid Il2rgtm1wjl/SzJ (NSG) male and female mice 6 to 16 weeks of
age were used as recipients for teratoma assays. Subcutaneous (nuchal
region) and intramuscular injections (left thigh) were employed. iPSCs
were harvested using collagenase IV as described above, spun down at 200 g
for 3 minutes, and resuspended in 115 μl (subcutaneous injection) or 65 μl
(intramuscular injection) of cold Iscove’s Modified Dulbecco’s Media
(IMDM; Gibco, Invitrogen) and complemented with the same volume of
cold Matrigel (BD). Approximately 2.0 × 106 cells were injected per site.
Following development of visible tumors between 7 and 12 weeks, mice
were euthanized and tumors were removed, fixed in Bouin’s solution
(Sigma-Aldrich), and processed (cutting and H&E staining) by the NHLBI
pathology core facility.
Hematopoietic differentiation assay. Hematopoietic differentiation of iPSCs
was performed according to a previously described procedure (47). Briefly,
undifferentiated iPSCs were harvested using collagenase type IV and incu-
bated in ultra-low attachment plate (Corning) with EB formation medi-
um KnockOut DMEM (Gibco, Invitrogen) supplemented with 20% non–
heat-inactivated FBS (Atlanta Biologicals), 0.5 mM l-glutamine, 0.1 mM
nonessential amino acids, and 0.1 mM 2-mercaptoethanol. After 24 hours,
medium was replaced with hematopoietic differentiation medium (EB
formation medium supplemented with 10 ng/ml IL-3, 10 ng/ml IL-6,
25 ng/ml bone morphogenetic protein 4 [all R&D Systems], 300 ng/ml
flt3 ligand [Flt3L] [Miltenyi], 50 ng/ml granulocyte CSF [G-CSF] [filgras-
in patients’ hematopoietic stem cells or other tissues in vivo. For
example, androgens and estrogens can induce telomerase expres-
sion in hematopoietic cells and may be of therapeutic benefit, but
their effects are limited and many patients eventually fail hormone
therapy (45). Our observation that hypoxia can increase telomere
length, even in telomerase-mutant cells, is of interest, and inves-
tigation of the mechanisms and pathways involved may result in
novel therapeutic strategies.
In conclusion, iPSCs derived from patients with telomere dis-
eases and heterozygous telomerase mutations showed reduced
and variable, but clearly present, telomere elongation. Pathways
that engage telomerase activation and telomere elongation during
reprogramming are potential therapeutic targets for the treatment
of patients with aplastic anemia with short telomeres. iPSCs could
indeed serve as a valuable tool to test these modalities in vitro, but
the variability of this model should be considered carefully.
Derivation of skin fibroblast cells from patient and healthy volunteer. Patients were
selected according to the following requirements: (a) heterozygous muta-
tion in either TERT or TERC, (b) decreased telomere lengths compared with
age-matched controls, and (c) a family history of telomere disease (bone
marrow failure, lung disease, or liver disease). The healthy male control-1
had normal telomere length and no family history of these diseases. Der-
mal fibroblasts were obtained by punch biopsy from the upper medial arm.
Biopsies were sectioned into approximately 1 mm3 pieces and placed in
10 ml PBS containing 100 mg/ml collagenase II, 2.5 U/ml dispase, and
10 U/μl DNaseI (all Gibco; Invitrogen). Skin fragments were incubated for
50 minutes on an orbital shaker (200 rpm) at 37°C. Following a wash-
ing step with complete culture medium (DMEM; Gibco, Invitrogen), 10%
FBS (Sigma-Aldrich), and 1% penicillin-streptomycin–glutamine (PSG)
(Gibco, Invitrogen), the skin pieces were placed in a 6-well tissue culture
plate, covered with a glass cover slip, and cultured for approximately
7–10 days in complete culture medium. After initial fibroblast-like cells
migrated from the tissue, the cover slip was taken off and the cells were
enzymatically (Trypsin 0.05%; Gibco, Invitrogen) passaged to a fresh tissue
culture dish. Following the initial derivation procedure, all the cells were
passaged according to standardized procedure, with a starting cell number
of 10,000 cells/cm2.
Generation of iPSCs and culture of pluripotent cells. iPSCs were generated from
human dermal fibroblasts by forced expression of the reprogramming fac-
tors OCT4, SOX2, KLF4, and MYC using either retroviral vectors expressing
the 4 factors individually (pMIG-OCT4, pMIG-SOX2, and pMIG-KLF4,
Addgene; pMIG containing MYC was provided by George Q. Daley, Chil-
dren’s Hospital, Boston, Massachusetts, USA) or all combined form the
polycistronic lentiviral vector STEMCCA provided by G. Mostoslavsky
(Boston University, Boston, Massachusetts, USA) (27). The production
of the viral particles was done as described (26, 27). Five days following
transduction, fibroblasts were transferred to plates coated with mouse
embryonic fibroblasts (MEFs) and grown in ES cell medium (ES medi-
um, DMEM/F12, supplemented with 20% knockout serum replacement
[KSR], 0.1 mM nonessential amino acids, 1 mM l-glutamine [all Gibco,
Invitrogen], 10 ng/ml recombinant human fibroblast like growth factor–
basic [Peprotech], and 0.1 mM 2-mercaptoethanol [Sigma-Aldrich]). From
day 5 to day 12, 0.5 mM valproic acid (Stemgent) was added to enhance
efficiency of iPSC generation in some cultures (ref. 46 and Supplemental
Table 1). Between days 14 and 30, individual, GFP-negative (for pMIG vec-
tors) ES cell–like colonies were picked and expanded on MEFs. iPSC clones
were enzymatically passaged using collagenase IV (Gibco, Invitrogen). In
order to avoid any influence of MEFs used in the coculture system on
1962 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
Real-time RT-PCR. RT-PCR was performed using the Rotor-Gene Multi-
plex RT-PCR kit (QIAGEN) according to the manufacturer’s recommen-
dations. Briefly, cell lysates were homogenized using the QIAshredder
Kit (QIAGEN) and total RNA was extracted using the RNeasy Mini Kit
(QIAGEN). RNA was quantified by Nanodrop analysis. Multiplex reac-
tions consisted of the kit-supplied master mix and reverse transcriptase
as well as primer-probe mixes for TERT (Fam), TERC (Tye), and ACTIN
(MAX) (each primer, 0.4 μM; each probe, 0.2 μM), and 50 ng of RNA.
Standard curves were generated using serial dilutions of TERT, TERC, and
actin plasmids. Primer sequence information can be found in Supplemen-
tal Table 2. Each reaction was performed in triplicate using a Rotor-Gene
Q 5plex Platform (QIAGEN). The final result given for a sample represents
the mean and SEM.
Telomeric repeat amplification protocol for telomerase activity. In vitro telom-
erase activity was assessed using the TRAPeze XL Telomerase Detection
Kit (Millipore) according to the manufacturer’s instructions. Protein
from cell pellets was extracted using CHAPS lysis buffer. Reactions
consisted of 300 ng of extracted protein, telomeric substrate, fluores-
cent primers, dNTPs, and Taq polymerase. The mixture was incubated
at 30°C for 30 minutes to allow for telomerase-mediated elongation of
telomeric repeats and then subjected to Taq polymerase–mediated PCR
amplification of the telomeric products generated. Quantification was
achieved using fluorescence spectroscopy. Each reaction was performed
in triplicate and normalized to the activity of an equivalent amount of
HeLa protein extract. Each experiment included a positive (HeLa protein
extract) and 3 negative controls (no template, no Taq-polymerase, and
parental fibroblast protein extract). The final result given for a sample
represents the mean and SEM.
Expression array. ST-cDNA from samples was fragmented, biotin labeled,
and hybridized on Affymetrix Human Exon 1.0 ST microarray according
to the manufacturer’s protocol (Affymetrix). The raw data were prepro-
cessed using the gene-level extended RMA-sketch method by Affymetrix
Expression Console (EC) Software (Affymetrix). The annotation of 133,672
extended transcript clusters was based on the current version annotation
file from Affymetrix (HuEx-1_0-st-v2.na32.hg19.transcript.csv). Hierarchi-
cal cluster analysis was performed by the similarity matrix (squared Pear-
son’s correlation coefficient) on normalized intensities on extended gene
level summarization crossing all the samples.
C-circle assay. We performed the C-circle assay developed by Jeremy Hen-
son and Roger Reddel as described by the authors (51), except that DIG-
labeled telomeric probes were used (Roche).
Statistics. Prism 5 software (GraphPad) was used in statistical analyses
and graph creation. ANOVA and Student’s t test (2 tailed) were used in
comparing rates of elongation. Nonparametric statistics (Kruskal-Wallis
and Dunn’s post test) were used in comparing grouped samples for telom-
erase expression and activity. A 2-tailed Student’s t test was used to analyze
the TERT and TERC expression of excised and nonexcised iPSC clones.
P < 0.05 was considered statistically significant. Data are presented as
mean ± SEM in all figure panels in which error bars are shown.
Study approval. All human subject material was collected at the NIH Clini-
cal Center under approval of the NHLBI institutional review board (04-H-
0012 and 07-H-0113), following written informed consent. All mice were
enrolled in protocols approved by the NIH Heart Lung and Blood Institute
Animal Care and Use Committee (H-0084R2).
This research was supported by the Divisions of Intramural
Research at the NHLBI, the National Human Genome Research
Institute, the National Cancer Institute, and the National Center
for Regenerative Medicine at the NIH. R.T. Calado was supported
tim; Amgen], and 300 ng/ml SCF [Amgen]). Medium was replaced with
fresh hematopoietic differentiation medium every 4–5 days. On day 20,
EBs were dissociated into single cells and analyzed by flow cytometry or
plated in CFU assays. For flow cytometry, cells were stained for viability
using LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen) and
CD34 and CD45 (BD). Data were acquired on a BD LSRII flow cytom-
eter (BD) and analyzed using FlowJo software (TreeStar). For CFU assays,
50,000 EB-derived cells were plated in MethoCult H4435 medium con-
taining SCF, IL-3, IL-6, G-CSF, GM-CSF, and erythropoietin (EPO) (Stem
Cell Technologies) for 12–14 days.
Mutation analysis. Total genomic DNA was extracted from cell pellets
using the automated Maxwell 16 LEV Blood DNA Purification Kit and
quantified by Nanodrop. PCR amplification of TERT and TERC genes
was performed as previously described, and PCR products were purified
with the QIAquick PCR Purification Kit (QIAGEN) (6). Direct sequenc-
ing was performed with BigDye Terminator version 3.1 and products
analyzed in an automated genetic sequence analyzer (ABI Prism 3100;
Applied Biosystems). The list of primers used for this analysis can be
found in Supplemental Table 2.
SKY. SKY analysis was performed as previously described (28). All aber-
rations described were in accordance with the International System for
Human Cytogenetic Nomenclature (48). Clonal aberrations were defined
as the presence of a minimum of at least 2 cells sharing the same chromo-
somal gain or structural alteration. A loss of chromosome was defined as
clonal if this abnormality was detected in at least 3 cells.
Southern hybridization for telomere length. Southern blot analysis was per-
formed using 275 ng of genomic DNA for each sample, following digestion
with Hinf1 and Rsa1. Using the TeloTAGGG Telomere Length Assay kit
(Roche), DNA digestion, Southern analysis, and determination of mean
terminal restriction fragment (TRF) length were performed according to
the manufacturer’s protocols.
Southern blot analysis to detect the transgenes expressed from the
STEMCCA provirus were performed as previously described (49). Briefly,
10 μg of genomic DNA of each sample was digested with BamHI (Fermen-
tas). After blotting, a 541-bp, p32-labeled DNA fragment complementary
to the proviral wpr-element was used to detect the nonexcised, integrated
proviral DNA. The primer information to generate the probe can be found
in Supplemental Table 2.
qPCR for telomere length. qPCR was performed as described by Cawthon
(29, 50), with several modifications. PCR reactions were pippetted in trip-
licate using the Qiagility robot (QIAGEN) and consisted of SYBR Green
PCR Master Mix (QIAGEN), forward and reverse primers, and 1.6 ng of
DNA per reaction. Amplification and quantification using the Rotor-Gene
Q (QIAGEN) was performed twice: first using primers for telomeric repeats
(T) and second using primers for the single-copy gene 36B4 (S). Primer
sequences can be found in Supplemental Table 2. The average telomere
length for each sample was calculated as a relative T/S ratio and normal-
ized to a standard control DNA (average telomere length by Southern blot,
8.6 kb; 2ΔΔCt). The final result for a given sample represents the mean and
SEM for at least 3 independent assays. In each run, a standard curve was
generated using the standard control DNA at various quantities (10, 5,
2.5, 1.25, and 0.625 ng/reaction). Two validation samples were run twice
in each plate (the standard control DNA and an umbilical cord blood sam-
ple), and data were only accepted if the results for the 2 validation samples
were within 5% of expected values. Based on control and validation runs,
the intra-assay variation of the method was 1.2% coefficient of variation
and the inter-assay variation was 3.1% for the validation samples. Samples
for each clone at different passages were run in the same experiment and
in at least 2 independent runs. In control experiments, the correlation
between Southern blot and qPCR was r2 = 0.86.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013
Received for publication October 4, 2012, and accepted in revised
form February 14, 2013.
Address correspondence to: Cynthia E. Dunbar, Hematol-
ogy Branch, NHLBI/NIH, 10 Center Drive, Bldg. 10/CRC,
Rm. 4E-5132, Bethesda, Maryland 20892-1202, USA. Phone:
301.402.1363; Fax: 301.496.8396; E-mail: firstname.lastname@example.org.
by a FAPESP (grant 98/14247-6). The authors have no conflicts
of interest to declare. We would like to thank Jichun Chen, Marie
Desierto, and Zu Xi Yu from the NHLBI pathology core facility for
their support during the teratoma assays. We thank Dara Wangsa
for providing the probes for SKY analysis and Vicky Guo for assist-
ing with the iPSC culture. We are also grateful to Sachiko Kajigaya
for her excellent technical support.
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