Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell’s basic antioxidant machinery.

1. Human mesenchymal stem cells with enhanced telomerase activity
acquire resistance against oxidative stress-induced genomic damage
VARVARA TRACHANA1
, SPYROS PETRAKIS2
, ZISIS FOTIADIS3
, EVANGELIA K. SISKA2,4
,
VASILEIOS BALIS3
, EFSTATHIOS S. GONOS5
, MARTHA KALOYIANNI3
&
GEORGE KOLIAKOS2,4
1
Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41500 Larisa, Greece,
2
Biohellenika Biotechnology Company, 57001 Thessaloniki, Greece, 3
Department of Zoology, School of Biology,
Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, 4
Department of Biological Chemistry, Medical School,
Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, and 5
National Hellenic Research Foundation, 48Vas.
Konstantinou Str, 11635 Athens, Greece
Abstract
Background. Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their
use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative se-
nescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies
on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has
a telomere-length independent function that promotes survival. Methods. Here, we studied the DNA damage accumula-
tion and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that
overexpress the catalytic subunit of telomerase (hTERT MSCs). Results. We showed that hTERT MSCs at high passages
have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less
damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the
control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular
nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation
of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superox-
ide dismutase and catalase, than control MSCs. Discussion. On the basis of these results, we propose that hTERT enhancement
confers resistance to genomic damage due to the amelioration of the cell’s basic antioxidant machinery.
Key Words: mesenchymal stem cells, telomerase, oxidative stress, genomic damage
Abbreviations: MSCs, human Mesenchymal Stem Sells; ASCs, Adipose Stem Cells; WJ, Wharton’s Jelly; hTERT, catalytic
subunit of human telomerase; ROS, Reactive Oxygen Species; SAC, Spindle Assembly Checkpoint.
Introduction
Several studies have demonstrated the therapeutic po-
tential of human mesenchymal stem cells (MSCs).Their
self-renewal ability and multilineage differentiation
(stemness) have made them a rather attractive candi-
date for cell based therapies. In fact, clinical trials are
currently taking place using MSCs as the main ther-
apeutic agent for different human diseases [1]. Even
though MSCs can be easily isolated from bone marrow,
adipose tissue, the umbilical cord and other tissues, their
expansion in vitro is not free of concerns [2,3]. It is ac-
tually well documented that MSCs reachreplicative
senescence rather fast in culture conditions [4,5] which
impairs their regenerative potential [6,7].
Replicative senescence is a common feature to all
somatic cells and most stem cells; after a period of rig-
orous proliferation the rate of cell division drops until
cells reach a state where they are still alive but unable
to divide further.The phenomenon is accompanied by
a number of characteristic changes, such as increase in
size and flattening, nuclear changes as well as several
changes in gene and protein expression patterns [8,9].
The telomere shortening hypothesis is currently the best
explanation for the limited proliferation potential of the
cells.Telomeres are hexanucleotide repeats which extend
Correspondence: Varvara Trachana, PhD, Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41500 Larisa,
Greece. E-mail: vtrachana@med.uth.gr
(Received 20 February 2017; accepted 25 March 2017)
ISSN 1465-3249 Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jcyt.2017.03.078
Cytotherapy, 2017; 19: 808–820

2. over several thousand base pairs (bp) in length, forming
functional “caps” at the end of the chromosomes.These
repeats due to the “end-replication-problem” are sub-
jected to erosion with each round of DNA replication.
Telomeric DNA is synthesized by a specialized reverse
transcriptase called telomerase [10].When telomerase
is not present or is expressed at very low levels, pro-
gressive shortening of the telomeres is taking place in
every cell division until the telomeres reach a crucial
length. At this point cellular senescence is triggered by
activating the DNA damage checkpoint that prevents
cells from further cycling [11].
For MSCs the proliferation limit determined by
the length of their telomeres bears another obstacle:
the age of the donor of the cells, since it has been
shown to be correlated with the initial telomeres length,
and therefore the time that MSCs can be main-
tained in culture [12]. The initial telomere length of
the donor’s MSCs could therefore be a limiting factor
of the MSCs therapeutic potential since it will affect
the survival and integration ability of the trans-
planted cells to the adult tissue [13–15]. In fact there
is increasing evidence that the age of the donor affects
several properties of MSCs [16].
On the other hand, it has been demonstrated that
the erosion of the telomeres is not only due to the way
DNA is replicated, but it can also be accelerated or even
induced by environmental factors, such as the oxida-
tive stress (OS) [17,18]. Different human cells subjected
to long-term exposure to mild OS have shown to
undergo accelerated telomere erosion that triggers pre-
mature senescence [17]. This phenomenon has been
collectively termed ‘stress induced premature senes-
cence’ (SIPS) [19]. It was also reported that hydrogen
peroxide (H2O2) can cause predominant DNA damage
at the 5΄ site of 5′-GGG-3′ in the telomere sequence.
Furthermore, H2O2 induced the formation of 8-oxo-
7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), an oxidized
derivative of deoxyguanosine and major product of DNA
oxidation [20], in telomere sequences more efficient-
ly than that in non-telomere sequences [21]. Different
groups, however, have reported that SIPS could also
be independent of telomeric damage [22]. Despite the
controversies on the matter, it is widely acknowl-
edged that the progressive oxidative damage to the
macromolecules, and especially to DNA, can induce
cellular senescence [23]. Even though many different
groups have studied the involvement of OS in MSCs
senescence in culture [24–26], the exact underlying mo-
lecular mechanism remains unclear.
Interestingly, it has been recently reported that
telomerase has also non-canonical or extratelomeric
functions, that are involved in processes such resis-
tance to stress and especially to oxidative stress. In fact,
Borras et al. [27] showed that glutathione, a physio-
logical antioxidant regulates telomerase activity,
highlighting therefore the interplay between telomerase
and the cellular oxidative status. Moreover, emerg-
ing data demonstrate that telomerase could be directly
involved in the reduction of reactive oxygen species
(ROS) generation and therefore decreased oxidative
DNA damage [28–31].
In order to investigate the antioxidant properties
of telomerase, and their implication in the DNA
damage driven decline in the cellular proliferation po-
tential, in the present study we analyzed the oxidative
stress-induced DNA damage response of MSCs ge-
netically modified with the catalytic subunit of human
telomerase (hTERT). In the analysis we included
adipose derived MSCs (ASCs) from adult individu-
als as well as umbilical cord’s Wharton Jelly derived
cells (WJ-MSCs), in an attempt to determine possi-
ble differences that could be attributed to the donor’s
initial telomeres length. We believe that this ap-
proach provides insights regarding the recently
proposed antioxidant role for telomerase, which could
contribute in developing strategies that will over-
come the senescence-associated impairment of the
regenerative potential of MSCs.
Materials and methods
Construction of the hTERT transposon
An entry clone encoding the human telomerase reverse
transcriptase catalytic subunit (hTERT), (GeneID:
7015, clone ID: IOH36343) was shuttled into a pT2-
CAGGS-EYFP-GW plasmid [32] using LR clonase
(Life Technologies), according to manufacturer’s in-
structions. The reaction mixture was used for
transformation of Mach1 E. coli cells and transformants
were selected on agar plates supplemented with 100 µg/
ml Ampicillin. Plasmid isolation was performed using
NucleoSpin plasmid isolation kit (Macherey-Nagel).
The identity of pT2-CAGGS-EYFP-hTERT plasmid
was verified by BsrGI restriction digestion.
Generation of hTERT mesenchymal stem cells
MSCs were enzymatically isolated from adipose tissue
(ASCs) of healthy donors (four different individu-
als, n = 4, age range of 49–60 years) by lipoaspiration.
Informed consent was given for the collection of
samples from all donors and the collection was per-
formed in accordance with established guidelines. For
the isolation of ASCs from the adipose tissue, tissues
were treated with collagenase (2.7 mg/ml) and hyal-
uronidase (0.7 mg/ml) solution for 1h at 37°C followed
by incubation with trypsin (2.5%). Cell suspension was
diluted with equal volume of PBS, passed through a
sterile 0.2 µm filter and centrifuged at 500g for 30 min
at room temperature (RT).We also isolated and used
MSCs from theWharton Jelly of umbilical cords (WJ
Telomerase protects against genomic instability 809

3. MSCs) from term-gestation newborns after birth (two
different individuals, n = 2), having obtained consent
from the parents. The isolating method used in our
laboratory was previously described [33].
After isolation ASCs and WJ MSCs were diluted
in Dulbecco’s modified Eagle’s medium (DMEM,
Sigma-Aldrich, Saint Louis, MO, USA) supple-
mented with 10% fetal bovine serum (FBS, Sigma-
Aldrich), penicillin (100 IU/mL) and streptomycin
(100 µg/mL) and placed in a 6-well plates. 2x105
cells
at passage 4 were grown to 70–80% confluency and
then nucleofected with 7.5 µg DNA encoding Sleep-
ing Beauty 100X transposase [34], pT2-CAGGS-
YFP-hTERT and pT2-SV40-neoR transposon
plasmids (1:8:1 ratio) using a Nucleofector device
(Lonzabio). hTERT ASCs and hTERTWJ MSCs were
selected with 100 µg/ml G418. All following analy-
ses were performed in four individual cultures of ASCs
(four control ASCs and four hTERT ASCs) and in
two individual cultures ofWJ MSCs (two controlWJ-
MSCs and two hTERT WJ-MSCs.
Culture conditions
Cells were cultured under standard cell-culture in
DMEM (supplemented with 10% FBS, penicillin
(100 IU/mL) and streptomycin (100 µg/mL)) in a hu-
midified incubator set to 37°C and 5% CO2 and 20%
O2. Medium was changed twice a week and cells were
passed when reached confluency. Cell number was de-
termined in duplicates using an hemocytometer. For
exposure to hydrogen peroxide (H2O2), 200.000 cells
(hTERT or control MSCs) were seeded per well on a
6 well plate and at 65–70% confluence cells were
exposed to 300µΜ H2O2 for 30 min in serum-free
medium. The medium was then replaced with fresh
complete medium and cells were normally cultured for
48 h (recovery time) in order to recover from the stress.
Antibodies
Mouse monoclonal anti-γH2AX (05–636; phosphor
S139, clone JBW301), and anti-53BP1 (clone BP13,)
were from Millipore (MA, USA). Anti- 8-hydroxy-
2’- deoxyguanosine (8-oxo-dG, Clone 2E2), was
purchased from Trevigen. For the western blots anti-
hTERT (NB120-32020) was from Novus and anti-
β-actin antibody (sc-47778) from Santa Cruz.
Secondary antibodies for confocal microscopy, Alexa
Fluor 594 anti-mouse IgG (A11005) and AlexaFluor
488 anti-mouse IgG (A11001 were obtained from Mo-
lecular Probes (Invitrogen).
qRT-PCR
Total RNA was purified from hTERT or control MSCs
using the NucleoSpin RNA kit (Macherey-Nagel),
according to manufacturer’s instructions. cDNA gen-
eration and qRT-PCR reactions were performed using
the KAPA SYBR Fast 1-step Green qRT-PCR kit
(KAPA Biosystems) in a Rotor-Gene 6000 operat-
ing system. Each analysis was performed in triplicates.
The correct size of amplified qRT-PCR products was
verified by electrophoresis in a 2% agarose gel.
Western blot analysis
hTERT or control MSCs were lysed in 1% SDS in
PBS containing Protease Inhibitors Cocktail Set III
(Calbiochem) and Benzonase (Novagen). Protein con-
centrations were determined by the Bradford method
with bovine serum albumin as standard (Bio-Rad
Laboratories, CA, USA). Samples(<20 µg total protein)
were analyzed by SDS-PAGE and transferred onto ni-
trocellulose membranes. Membranes were blocked with
5% weight per volume (w/v) non-fat dry milk in PBS/
0.1%Tween20 probed with the appropriate antibodies.
Secondary antibodies conjugated with alkaline-
phosphatase were detected using nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl phosphate
p-toluidine salt (NBT/BCIP) substrate (Applichem).
Equal protein loading was verified by reprobing each
membrane with the antibody against β-actin.
Quantification of Telomerase activity
Real Time-PCR based telomeric repeat amplification
protocol (TRAP) assay was performed according to the
manufacturer’s protocol (TeloTAGGGTelomerase PCR
ELIZAPLUS-Roche Applied Science, Indianapolis, IN,
USA). In brief, 2 × 105
hTERT or control MSCs were
lysed in 200 µl lysis buffer and the cell lysate was
centrifuged at 16,000 g for 20 min at 4°C. Cell extract
(0.5 µg of total protein) was used for PCR reaction.
The amplification product (3 µl) was transferred for the
hybridization, and the ELISA assay was performed in
triplicates, in which two served as controls (one con-
tained a negative control, and a second containing the
high telomerase activity positive control). Telomerase
activity of each sample was calculated as a percentage
of the relative telomerase activity of the positive control
cells.
Flow cytometry
For phenotypic characterization, hTERT and control
MSCs were stained with phycoerythrin (PE)-
conjugated antibodies against hemopoietic (CD34, 45)
or mesenchymal (CD29, 73, 90, 105 and 146) stem
cell markers. Unstained cells or cells stained with IgG
isotype antibodies were used as negative controls. Cells
were measured in a Cytomics FC500 flow cytom-
eter (Beckman Coulter) using the CXP2.2 software.
810 V.Trachana et al.

4. Multilineage differentiation
For osteogenic or adipogenic differentiation, hTERT
and control MSCs were grown to 90% confluency and
cultured for 28 days either in StemPro Osteogenesis
or StemPro Adipogenesis medium (Life Technolo-
gies). Differentiation of cells into osteocytes or
adipocytes was monitored by Alizarin Red or Oil Red
staining, respectively.
Immunofluorescence
Immunofluorescence experiments were performed as
previously described [35]. Briefly, MSCs were grown
on coverslips and subjected to the above described oxi-
dative treatment or left to reach the desired passage
and then fixed in 4% paraformaldehyde. Fixed samples
were incubated with primary antibodies of interest (γ-
H2AX, 53BP1 or 8-oxo-dG) and the appropriate
secondary antibodies. Coverslips were embedded in
10 µl of Vectashield mounting medium for fluores-
cence with 4,6-diamidino-2-phenylindole (DAPI,Vector
Laboratories, CA, USA) to visualize the nuclei, and
analyzed on ZEISS Axio Imager Z2 fluorescent
microscope. Images were captured on confocal
microscope ZEISS LSM780 and ZEN 2011 program.
For calculations of damaged nuclei at least 5 ran-
domly selected fields were analyzed for each culture
condition. Cells with >5 γ-H2AX, 53BP1 or 8-oxo-
dG stained foci in their nuclei were counted as positive
for DNA damage by a single observer blinded to treat-
ment regimen.
Measurement of antioxidant enzymes activity
Total superoxide dismutase (SOD) and catalase ac-
tivities were measured in MSCs cell lysates using
commercially available kits (Superoxide Dismutase
Assay Kit 706002, Catalase Assay Kit 707002 Cayman
Chemical, Ann Arbor, MI) according to the manu-
facturer’s recommendations.
Statistical analysis
For the statistical analysis of the results the GraphPad
Prism 5 software (GraphPad Software, San Diego, Cal-
ifornia USA) was used. P values less than 0.05 were
considered significant and the significance is ex-
pressed with asterisks: * = P < 0.05; ** = P < 0.01;
*** = P < 0.001. Results are reported as mean ± stan-
dard error (means ± S.Ε.) unless otherwise stated.
Results
Genetically modified MSCs have increased expression of
hTERT and enhanced telomerase activity
Ectopic expression of hTERT in hTERT ASCs was
confirmed by qRT-PCR analysis (Figure 1A).The iden-
tity of amplified hTERT and actin beta (ACTB)
housekeeping gene fragments was verified by agarose
electrophoresis (Figure 1B). Production of recombi-
nant yellow fluorescent protein (YFP)-TERT was
evaluated by Western blotting. A protein band with a
molecular weight of ~160 kDa was detected in hTERT
ASCs corresponding toYFP-TERT, whereas endog-
enous hTERT was detected in all samples at the
expected molecular weight (127 kDa) (Figure 1C).
These results verify successful overexpression of
hTERT in ASCs.
hTERT WJ-MSCs showed similarly increased
mRNA levels of the telomerase catalytic subunit and
production of the recombinant YFP-TERT protein
(Supplementary Figure S1A and B respectively).
Finally, realTime-PCR basedTRAP assay (Roche
Applied Science) was performed according to the man-
ufacturer’s protocol.The assay confirmed that ASCs
genetically modified with the hTERT (ASCs1-ASCS4)
Figure 1. Genetically modified ASCs have increased messenger RNA (mRNA) levels of hTERT and produce recombinant YFP-TERT
protein. (A) mRNA levels of control and hTERT-overexpressing ASCs normalized to housekeeping β-actin (ACTB) gene. Values showed
are the means ± SE. *P < 0.05 versus control ASCs. (B) qRT-PCR–amplified fragments analyzed in a 2% agarose electrophoresis. (C)Western
blot analysis for hTERT in control and hTERT-ASCs.The analysis was performed in all control and hTERT ASC individual cultures and
a characteristic image is demonstrated. Molecular weight markers are run in the middle lane. β-actin was used as a loading control.
Telomerase protects against genomic instability 811

5. have increased telomerase activity when compared to
the control cells (Figure 2).
MSCs overexpressing hTERT retain their
mesenchymal properties
hTERT MSCs derived from adipose tissue were further
characterized in order to verify that the genetic mod-
ification does not affect their mesenchymal properties.
hTERT ASCs were plastic-adherent and had a similar
fibroblastic morphology as control cells. Flow cytometry
of hTERT ASCs showed that these cells do not express
the hemopoietic CD34 marker, whereas they express
typical mesenchymal stem cell CD markers 29, 73,
90, 105 and 146 at similar levels to control ASCs
(Table I). Similarly, hTERTWJ-MSCs do not express
the CD34 marker and express the typical mesenchy-
mal CD29 and CD105 markers (Supplementary
Table SI).
The multilineage differentiation potential of hTERT
ASCs was also assessed. Cells were differentiated
towards adipocytes and osteocytes, as indicated by Oil
Red and Alizarin Red staining, respectively.At early pas-
sages (p ≤ 8) both control as well as genetically modified
ASCs were able to differentiate towards both adipocytes
and osteocytes (data not shown). As shown in Figure 3,
control ASCs at passage 10 were able to differentiate
towards adipocytes but not osteocytes, whereas hTERT
ASCs of the same passage retained their osteogenic dif-
ferentiation capacity (Figure 3A, B).
Moreover, as recently demonstrated (in a paral-
lel work done by members of our team [36],) control
WJ-MSCs maintain their differentiation ability towards
adipocytes and osteocytes even at high passage (passage
40). Here, we demonstrate that hTERT overexpression
did not affect this differentiation ability ofWJ-MSCs,
as indicated in Figure S1C.
The above data prove that the genetic modifica-
tion did not alter the mesenchymal properties of adipose
derived or WJ-derived cells. It also implies that the
overexpression of hTERT may contribute in retain-
ing their mesenchymal properties for longer (at higher
passages).
hTERT overexpression offers protection against DNA
damage accumulation during long-term culture
MSCs were kept in culture until their proliferation rate
dropped to less than 2 cumulative population doublings
per 4 weeks. At this stage control MSCs had the char-
acteristic senescent-like morphology (large, flattened
vacuolated cells), suggesting that they have reached their
proliferation limit. In accordance with previously re-
ported data [37], control ASCs reached this state at
passage 20 ± 2.3, whereas hTERT ASCs at did not seem
to enter this state even at high passages (passage > 30).
Similarly, control WJ-MSCs reached their prolifera-
tion limit at passage 22 ± 1.3, whereas hTERT
overexpressing WJ-MSCs did not show any reduction
in their duplication time until at least passage 30.
Our observations, together with those previously re-
ported [37], reveal the beneficial effect of hTERT
overexpression on the growth potential of MSCs, which,
based on our results, is not limited to adipose-derived
cells but apply to MSCs from different sources.
In an attempt to explain the amelioration in the
growth potential of hTERT overexpressing cells and
given the importance of DNA damage accumulation
in limiting cellular proliferation capacity [38], we
analysed the DNA damage in the nuclei of growing cells.
We evaluated the amount of DNA damage in control
ASCs as well hTERT ASCs at early passage (p6),
middle passage (p12) and high passage (p20).The pres-
ence of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-
dG), major product of DNA oxidation as mentioned
earlier, was assessed. 8-oxo-dG lesions could lead to
reduced efficiency of the base excision repair process
leading, in turn, to persistent DNA single strand breaks
Figure 2. ASCs genetically modified with hTERT have increased
telomerase activity. RT-PCR–basedTRAP assay was used to measure
telomerase activity in hTERT ASCs at passage 7. Telomerase ac-
tivity of the hTERT ASCs is depicted as fold change relative to
the activity of the control ASCs of the same passage.The analysis
was performed in triplicate with four control ASCs and four hTERT
ASCs. Values showed are the means ± SE.
Table I. Flow cytometry analysis results for CD stem cell markers
in control ASCs and hTERT-overexpressing ASCs (hTERT ASCs)
at passage 7.
Stem cell marker
hTERT ASCs
passage 7
Control ASCs
passage 7
CD34 0% 2%
CD29 99.6% 99.8%
CD73 99.4% 99.7%
CD90 99.3% 99.3%
CD105 99.7% 97.5%
CD146 96.4% 93.8%
812 V.Trachana et al.

6. (SSB) intermediates; the latter could then act as double
strand breaks (DSB)-prone sites in subsequent rounds
of DNA replication [39]. DSB activate ataxia-
telangiectasia mutated/ataxia telangiectasia and Rad3-
related serine/threonine-protein kinases (ATM/ATR)
that bind to the DNA and induce phosphorylation of
many downstream proteins, such as H2A.X and 53BP1,
in order to both mark the location and initiate repair
of the damage. In order to evaluate DSB formation in
long-term cultures of MSCs, we therefore assessed
control ASCs as well as hTERT ASCs of different pas-
sages (p6, p12 and p20) for the presence of the
phosphorylated form (on Ser139) of the histone variant
H2AX (γH2AX) and the 53BP1 protein. Figure 4A
shows that growing ASCs accumulate DSB as well as
oxidative lesions, as indicated by the increased per-
centage of cells positive for γΗ2ΑΧ/ 53BP1 (i, ii) and
8-oxo-dG staining (iii), respectively. All nuclei bearing
>5 foci (8-oxo-dG, γΗ2ΑΧ or 53BP1) were counted as
positive. Importantly, as depicted in the same figure
(Figure 4A), our result demonstrate that cells geneti-
cally modified with the catalytic subunit of hTERT
accumulate significantly less damage than the control
cells in all passages tested. Figure 4B shows confocal
microscopy characteristic images of foci positive for
γΗ2ΑΧ, 53BP1 and 8-oxo-dG in the nuclei of control
as well hTERT overexpressing cells. It is obvious that
hTERT overexpressing cells accumulate a lot less DNA
damage associated foci in their nuclei at high passage
(p = 20).The latter suggest that the elevated activity of
telomerase protects cells against oxidative lesions and
DSB accumulation which could explain hTERT cells’
enhanced proliferation capacity.
Ectopic expression of hTERT results in ameliorated
response to exogenous oxidative stress-induced
DNA damage
The protection acquired by hTERT overexpressing cells
against DNA lesions accumulating during growth of
ASCs, prompt us to investigate whether similar pro-
tection occurs after exposure to acute external oxidative
stress.Therefore, control ASCs as well as hTERT ASCs
were treated with H2O2 for 30 min in order to evaluate
DNA damage accumulation. One hour after treat-
ment nuclei with >5 foci (for all markers) were counted
as positive. As Figure 5 shows, both control ASCs as
well as hTERT ASCs at passage 6 (early), passage 12
(middle) and passage 20 (high) treated with H2O2, ac-
cumulate similarly high amounts of DNA damage
associated foci (Figure 5A–C, p(6/12/20) + H2O2). Both
control ASCs and hTERT ASCs at early and middle
Figure 3. ASCs that overexpress hTERT retain their differentiation potential. (A, B) Passage 10 ASCs and hTERT ASCs cultured under
standard conditions (control medium) or under conditions to induce adipogenic/osteogenic differentiation (differentiation medium). Under
differentiation conditions, cells began to accumulate lipid droplets and acquired a foamy appearance with the characteristic staining for oil
red, typically seen in adipocytes (A), or exhibited calcium deposition, with the characteristic staining for alizarin red, which is typically
seen in osteocytes (B) (Scale bar: 10 µmol/L). (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Telomerase protects against genomic instability 813

7. passage were, however, able to, at least partially, repair
the damage when left to recover in fresh medium for
48 h (p6/12 + H2O2 recovery 48h), but not at high
passage (p20 + H2O2 recovery 48h). This ability was
found to be enhanced in hTERT ASCs, since signifi-
cantly less amount of DNA damage was measured in
these cells 48 h after the oxidative treatment at passages
6 and 12, than in control ASCs. The latter was grad-
ually diminished as cells reached higher passages (p20).
Overall these results suggest that ASCs of early and
middle passage genetically modified with the cata-
lytic subunit of hTERT have enhanced ability to repair
the externally provoked oxidative stress-induced DNA
lesions.
A
B
Figure 4. hTERT overexpression in ASCs offers protection against DNA damage accumulation in long-term culture. (A) Percentage of
control ASCs and hTERT-overexpressing ASCs (hTERT ASCs) with DNA damage measured by immunofluorescence using specific an-
tibodies against γH2AX (i), 53BP1 (ii) or 8-oxo-dG (iii) at the indicated passages. Nuclei bearing >5 foci stained with 8-oxo-dG, γΗ2ΑΧ
or 53BP1 antibody were counted as positive. Values shown are the means ± SE. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control
ASCs in the passage indicated. (B) Characteristic images of control and hTERT ASCs at low (passage 6) and high passage (passage 20)
with DNA damage evaluated by confocal microscope and after staining with γH2AX, 53BP1 or 8-oxo-dG antibody (green). Nuclei are
stained with DAPI (blue). Scale bar 20 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
814 V.Trachana et al.

8. Figure 5. hTERT overexpression in ASCs offers protection against external oxidative insult. Percentage of control ASCs and hTERT-
overexpressing ASCs (hTERT ASCS) with DNA damage measured by IF using γH2AX (A), 53BP1 (B) or 8-oxo-dG (C) antibodies at
low (passage 6), middle (passage 12) and high (passage 20) passages after treatment with 300 µmol/L H2O2 for 30 min (passage 6/12/
20 + H2O2) and after 48 h of recovery time (passage 6/12/20 + H2O2 recovery 48 h). Nuclei bearing >5 foci stained with 8-oxo-dG, γΗ2ΑΧ
or 53BP1 antibody were counted as positive. Values shown are the means ± SE. *P < 0.05 and **P < 0.01 versus control ASCs in each
passage indicated.
Telomerase protects against genomic instability 815

9. Enhanced TERT activity protects against oxidative stress
provoked nuclei malformations
It has been suggested that oxidative stress could lead
to aberrant nucleus morphology, such as giant nuclei,
donut-shaped nuclei, micronuclei or multilobular
nuclei, which is also considered a characteristic of se-
nescent cells [40]. Indeed, as shown in Figure 6A both
control as well as hTERT ASCs demonstrate an in-
crease in such malformations (i.e mitotic defects) in
passage 20 (senescence reaching) as compared to low
passage 6. Nevertheless, hTETR overexpressing ASCs
had significantly less nucleus defects than control cells
in both time points tested (p6, p20). Figure 6B shows
characteristic nuclei aberration of high passage 20
control ASCs, such as giant nucleus, donut-shaped
nucleus, micronucleus or multilobular nucleus
(Figure 6B, i, ii, iii, iv respectively).
Additionally, we tested whether the externally pro-
voked oxidative stress could lead to an increase of such
nuclei malformations. Both control ASCs and hTERT
ASCs at low (p6) and high (p20) passage treated with
300 µΜ Η2Ο2 for 30 min and left to recover for 48 h,
were DAPI-stained in order to evaluate their nuclear
morphology. Firstly, as indicated in Figure 6A, both
control ASCs and hTERT showed an increase in
nucleus malformations in both passages tested after
treatment with H2O2 (p6/20 + H2O2 recovery 48h) in
comparison with the same passages without treat-
ment (p6/20). However, significantly less aberrant
nuclei were present in hTERT ASCs 48 after treat-
ment with Η2Ο2 at both low and high passages in
comparison with the control cells. The latter indi-
cates that the hTERT overexpression was able protect
against oxidative stress-induced nuclei malforma-
tions, which, together with the previous observations,
suggests an overall ameliorated antioxidant response.
Genetically modified MSCs with hTERT demonstrate
increased activity of the basic antioxidant enzymes
Superoxide dismutases (SOD) are metaloenzymes that
catalyze the dismutation of the superoxide anion to mo-
lecular oxygen and H2O2 and thus form a crucial part
of the cellular antioxidant defense mechanism [41].
Catalase (CAT) is a ubiquitous antioxidant enzyme in-
volved in the detoxification of H2O2 by catalyzing its
conversion to molecular oxygen and water [42]. In an
A
B
Figure 6. hTERT-overexpressing ASCs show resistance to aberrant nuclei formation. (A) Percentage of cells with mitotic defects (*giant
nuclei, donut-shaped nuclei, micronuclei and multilobular nuclei) evaluated using DAPI staining of control ASCs and hTERT-
overexpressing ASCs (hTERT ASCs) at low (passage 6) and high passage (passage 20). Same nuclei aberrations were assessed at passages
6 and 20 after the cells were treated with 300 µmol/L Η2Ο2 for 30 min and left to recover for 48 h (passage 6/20 + H2O2 recovery 48 h).
Values shown are the means ± SE. *P < 0.05 and **P < 0.01 versus control ASCs in the passage indicated. (B) Characteristic images of
DAPI-stained (blue) giant nuclei (arrow in (i)), donut-shaped nuclei (ii), micronucleus (arrow in (iii)) and multilobular nuclei (iv) of control
ASCs at high passage (passage 20) (Scale bar 25 µm). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
816 V.Trachana et al.

10. attempt to explain the amelioration of the oxidative re-
sponse observed in hTERT overexpressing cells we
analysed the basic antioxidant capacity of ASCs. For
this reason, we measured the activities of both enzymes
(SOD and CAT) using commercially available kits
(Cayman, Chemical, Ann Arbor, MI). As shown in
Figure 7A ASCs genetically modified with hTERT
(hTERT ASCs) have similar activity of SOD with
control cells at low passage (p6). At high passage (p20)
however, control cells show a decrease in their SOD
activity whereas hTERT overexpressing cells main-
tain significantly higher levels, similar to the levels of
the low passage. Regarding CAT activity, we observed
that both control as well as hTERT overexpressing cells
have increased activity at higher passages (Figure 7B,
p20), which is in accordance with previously reported
data. In specific, it was reported that senescent cul-
tures of human lung fibroblasts have elevated H2O2
generation which stimulate cells to increase their cata-
lase activity [43]. Besides confirming that, we report
here, that hTERT ASCs have significantly higher CAT
activity levels than control cells, both at passage 6 as
well as in passage 20 (Figure 7B). The above results
reveal an enhancement of the basic antioxidant enzy-
matic defense of the cells due to hTERT overexpression.
Discussion
Endogenous oxidants generated by normal cellular oxi-
dative metabolism are a major obstacle to overcome
in cell based therapies that require ex vivo long-term
cell culture. The DNA damage that oxidative stress
(OS) provokes could lead to cellular senescence and
therefore limit the number of cells with the self-
renewal and multilineage differentiation ability
(stemness) that is required for successful transplan-
tation. Besides DNA damage, it has been reported that
OS -either normally occurred due to metabolism or
through exposure to external oxidants- leads to ab-
errant nuclear morphology. It was demonstrated that
a significant number of senescent or progeroid human
fibroblasts exhibit nuclei malformations, such as
multilobbular nuclei and one or multiple micronu-
clei that are attributed to mitotic slippage [40]. Mitotic
slippage is a known consequence of a dysfunctional
spindle assembly checkpoint (SAC) [44] and OS was
proposed to be a major cause of its impairment [45].
Moreover, it was recently demonstrated that, exog-
enously induced OS is able to promote aneuploidy in
MSCs [37] which could also be attributed to an im-
paired SAC. Actually, it was proposed that OS could
affect the functionality of the SAC by regulating the
expression of two key SAC mediators, BubR1 and
Mad2 [46]. All the above suggest that OS provokes
genomic instability, i.e. DNA damage, nuclear defor-
mations and aneuploidy that reduce the stemness of
MSCs in culture and limit transplantation success.
On the other hand, accumulating recent evi-
dence link the catalytic subunit of telomerase to
functions other than telomere length maintenance. As
a matter of fact, one of these extratellomeric func-
tions of telomerase that attracted a lot of attention
recently is its implication in the antioxidant defense
mechanism(s) of the cell [47]. The precise molecu-
lar mechanism of telomerase’s telomere independent
functions is becoming an important-still mostly
unanswered-question.
In order to shed light onto these issues, in the present
study we genetically modified human mesenchymal stem
cells derived from different sources (adult adipose tissue
and umbilical cord’s Wharton’s Jelly) with the cata-
lytic subunit of telomerase (hTERT) in order to
elucidate its contribution in overcoming endogenously
occurring or externally provoked OS. Firstly, we showed
Figure 7. ASCs genetically modified with hTERT have higher basic antioxidant enzymes activities. Analysis of the SOD (A) and CAT
(B) enzymatic activity of control ASCs and ASCS overexpressing hTERT (hTERT ASCs) at low (passage 6) and high (passage 20) pas-
sages. Values shown are the means ± SE. *P < 0.05 and **P < 0.01 versus control ASCs in the passage indicated.
Telomerase protects against genomic instability 817

11. that genetic modification with hTERT did not alter the
mesenchymal properties of adipose orWharton’s Jelly
derived cells, in agreement with previously reported data
on human bone marrow stromal cells [48] and mouse
adipose derived MSCs [49]. Furthermore, we report
that overexpression of hTERT in ASCs may contrib-
ute in retaining their mesenchymal properties for longer,
an observation which is in agreement with previous
results [50]. Importantly, we proved that the genetic
modification of MSCs with hTERT results in de-
creased DNA damage accumulation, that occurs either
as a result of long term culture or due to acute oxida-
tive external insult. It has been suggested that hTERT
overexpression ameliorates the DNA damage re-
sponse by either accelerating DNA repair kinetics
through increased levels of intracellular deoxynucleotides
(dNTP) and ribonucleotides (NTPs) [51], or by at-
tracting DNA repair proteins to the sites of DNA
damage [52]. Even though the hTERT participation
in DNA damage repair is a rather attractive notion and
some supportive data do exist, the exact mechanism
remains elusive. Based on our data it is, at this point,
safer to hypothesize that the protection against DNA
damage accumulation results from an hTERT driven
enhancement of the antioxidant cellular response. In
support, recent studies showed that the telomerase lo-
calizes to subcellular locations other than the nucleus,
such as the mitochondria. hTERT was shown to shuttle
dynamically from the nucleus to the mitochondria,
where it decreases mitochondrial reactive oxygen species
(ROS) generation [28–31]. Furthermore, strong evi-
dence has been provided that increased hTERT
expression not only reduces the total basal ROS levels,
but also significantly antagonizes the increase in cel-
lular ROS in response to exogenous ROS triggers
(H2O2) [53].
We also showed that MSCs genetically modified
with hTERT exhibit resistance to nuclei malforma-
tions that arise either through prolonged culture or
due to H2O2 treatment. Nuclei malformations, as men-
tioned above, could be the result of an OS-induced
SAC impairment.The resistance against OS-induced
aberrant nuclei formation demonstrated here is a novel
indication of telomerase’s ability to counteract OS and
promote cell survival.
We know that in order to resist ROS, the cells use
enzymatic antioxidants, with superoxide dismutase
(SOD) and catalase (CAT) being the major ones [54].
Our data demonstrate that hTERT overexpression
results in increased SOD and CAT activities. It was
previously reported that hTERT modified MSC dem-
onstrated elevated protein levels of the principal protein
scavenger of mitochondrial superoxide, MnSOD
(SOD2), suggesting that hTERT overexpression could
be a key regulator of the metabolic status [37]. Here
we demonstrate that hTERT overexpression could
directly lead to an enhancement of the antioxidant ma-
chinery of the cell by elevating the activity of the key
enzymes. In support, one previous study has shown
that mouse embryonic fibroblasts isolated from
telomerase-deficient mice, have decreased catalase ac-
tivity which results in redox imbalance in these mice
[55].
Finally, it has been reported that the donor’s initial
telomeres length might be a critical restriction for the
maintenance of the MSCs in prolonged cell culture
[56]. Following transplantation, the telomere length
might limit the therapeutic potential of MSCs, by de-
creasing either the duration of cell survival in the tissue
or their ability to integrate in the adult tissue [57,58].
Here, we prove that both adult adipose derived stem
cells as well as umbilical cord’sWharton’s Jelly derived
cells genetically modified with hTERT have an en-
hanced growth potential that could be attributed to
their improved antioxidant capacity. Therefore, this
genetic manipulation implemented by us, and others
[48,59], could overcome the obstacle of shorter initial
telomere length of the adult stem cells. Understand-
ing the restrains of this strategy we believe that synthetic
or natural compounds that activate the telomerase
might be a better approach.
As a matter of fact, Tichon et al. [60] demon-
strated that two novel compounds, designated AGS-
499 and AGS-500, when administrated to MSCs
increased the average telomere length, preserved
genome integrity and allowed MSCs to differentiate
into various lineage. The authors also demonstrated
that these effects were telomerase depended. Our study
confirms these previously reported results but also
demonstrates further novel effects of the hTERT
overexpression. We showed that hTERT enhance-
ment, via direct genetic manipulation, confers higher
growth potential to the cells, most probably due to
the amelioration of the cellular response to the OS that
either occurs normally during cellular senescence or
it is exogenously provoked. Moreover, we demon-
strated that this ameliorated OS response of the
hTERT overexpressing cells comes from direct en-
hancement of the cell’s basic enzymatic antioxidant
machinery, which in turn protects against OS-induced
genomic damage. In other words, our study pro-
vides novel data in support of the recently proposed
antioxidant role of telomerase. Besides that, we believe
that our analysis offers a comprehensive evaluation of
the outcomes of an augmented telomerase activity in
human MSCs that might help overcome the adult stem
cell-based therapies limitations.
Acknowledgments
This work was funded by a SYNERGASIA project
(11ΣΥΝ_1_1112). Authors wish to thank Dr. Z. Ivics
818 V.Trachana et al.

12. and Dr. Z. Izsvák for kindly providing the Sleeping
Beauty 100X transposon system, A. Kalkavouri and
A. Ntoga for technical assistance and Dr. E. Panteris
for his help with the microscopy techniques.
Disclosure of interests:The authors declare no com-
mercial or financial conflict of interest.
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Appendix: Supplementary material
Supplementary data to this article can be found online
at doi:10.1016/j.jcyt.2017.03.078.
820 V.Trachana et al.