Genotoxic damage of human adipose-tissue derived mesenchymal stem cells triggers their terminal differentiation.

Abstract

Human adipose tissue-derived mesenchymal (stromal) stem cells (AT-MSCs) and genetically modified to express cytosine deaminase:uracil phosphoribosyltransferase (CDy-AT-MSCs) were treated with hydrogen peroxide in order to induce DNA damage and subsequently evaluate their genetic stability by single cell gel electrophoresis. Both cells types (parental and transgene modified) did not differ in the sensitivity to DNA breaks induction. Potential tumorigenicity of AT-MSCs and CDy-AT-MSCs was tested by subcutaneous inoculation of cell suspension into flank of immunocompromised mice. Dose of 15x10(6) cells was not found to be tumorigenic in given experimental setup. AT-MSCs, CDy-AT-MSCs and MSCs isolated from human lipoma were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) in attempts to transform them. Surviving cells after genotoxic stress were not transformed but underwent replicative senescence. Irreparable DNA damage caused triggered adipogenic terminal differentiation, rather than apoptosis induction in all kinds of cells tested.

Full text

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542
Genotoxic damage of human adipose-tissue derived mesenchymal stem cells
triggers their terminal differentiation
V. ALTANEROVA1, E. HORVATHOVA2, M. MATUSKOVA1, L. KUCEROVA1, C. ALTANER1*
1Laboratory of Molecular Oncology, 2Laboratory of Mutagenesis and Carcinogenesis, Cancer Research Institute SAS, Bratislava, Slovakia, e-mail:
exonalt@savba.sk
Received April 26, 2009
Human adipose tissue-derived mesenchymal (stromal) stem cells (AT-MSCs) and genetically modified to express cyto-
sine deaminase:uracil phosphoribosyltransferase (CDy-AT-MSCs) were treated with hydrogen peroxide in order to induce
DNA damage and subsequently evaluate their genetic stability by single cell gel electrophoresis. Both cells types (parental
and transgene modified)didnotdifferinthesensitivitytoDNAbreaksinduction.PotentialtumorigenicityofAT-MSCsand
CDy-AT-MSCs was tested by subcutaneous inoculation of cell suspension into flank of immunocompromised mice. Dose
of 15x106 cells was not found to be tumorigenic in given experimental setup. AT-MSCs, CDy-AT-MSCs and MSCs isolated
from human lipoma were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) in attempts to transform them.
Surviving cells after genotoxic stress were not transformed but underwent replicative senescence. Irreparable DNA damage
caused triggered adipogenic terminal differentiation, rather than apoptosis induction in all kinds of cells tested.
Key words: human adipose tissue-derived mesenchymal stem cells; genotoxic damage, single cell gel electrohoresis; chemical
carcinogen 4NQO, terminal differentiation
Human adipose tissue-derived mesenchymal (stromal)
stem cells (AT-MSCs) are multipotent adult stem cells derived
usually from lipoaspirate. Similarly as bone marrow derived
mesenchymal stem cells (MSC), AT-MSCs have the ability to
migrate to sites of injury to fulfill their role in repair of the
damage tissues [1]. MSCs and AT-MSCs can be isolated by
their adherence to plastic tissue culture plates [2] Theyieldof
MSCs from adipose tissue is higher in comparison to the bone
marrow [1, 3, 4]. Theeasyandrepeatableaccesstosubcutane-
ous adipose tissue and the simple isolation procedures provides
a clear advantage over other sources [4]. Therefore AT-MSCs
have potential therapeutic use as autologous and allogeneic
products for tissue engineering.
Mesenchymal stem cells both MSCs and AT-MSCs possess
unique ability to selectively migrate to tumors, metastases and
contribute to the formation of tumor-associated stroma. This
property predetermines them to become vehicles for stem cell
based targeted cancer gene therapy [5, 6].
Previously we have prepared yeast fusion cytosine deami-
nase:uracil phosphoribosyltransferase expressing human
adipose tissue derived mesenchymal stem cells (CDy-AT-
MSCs) by retrovirus transduction. CDy-AT-MSCs exerted
their anti-tumor potential on human colon cancer cells, human
malignant melanoma cell line, and human metastatic prostate
cells in the presence of prodrug 5-fluorocytosine (5-FC) [3,
7, 8]. CDy-AT-MSCs in combination with 5-FC augmented
significantbystandereffectandselectivecytotoxicityontarget
tumor cells in co-culture in vitro. Moreover, strong inhibition
of subcutaneous tumor growth was achieved by systemically
administered CDy-AT-MSCs on xenotransplanted human
tumors grown on nude mice upon 5-FC treatment.
Therefore human mesenchymal stem cells derived from
adipose tissue are not only useful cells in regenerative medi-
cine, but when modifiedbyprodrugconvertinggenemayalso
serve as attractive delivery vehicles for stem cell-based targeted
cancer gene therapy. Genetic stability of AT-MSCs is an im-
portant attribute to asses, when they are intended to be used
in regenerative and cancer gene therapy clinical studies.
The aim of the present study was to evaluate genetic sta-
bility, of AT-MSCs and CDy-AT-MSCs, their sensitivity to
transformation induced by chemical carcinogen and potential
tumorigenicity. Here we demonstrate that irreparable DNA
damage caused by chemical carcinogen triggered adipose
* Corresponding author
NEOPLASMA 56, 6, 2009
doi:10.4149/neo_2009_06_542

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GENOTOXIC DAMAGE OF HUMAN ADIPOSE TISSUE MESENCHYMAL CELLS
derived mesenchymal (stromal) cells to adipogenic, terminal
differentiation.
Materials and methods
Chemicals. Ethidium bromide, 4-nitroquinoline-1-oxide
(4NQO) were purchased from Sigma-Aldrich Co. (St. Louis,
MO). 4NQO was prepared as 5 mmol/L stock solutions in
100% dimethylsulfoxide (DMSO). Aliquots were diluted with
complete medium just before they were applied to culture
cells.
Mesenchymal stem cells isolation from human adipose
tissue, culture, and retrovirus transduction. AT-MSC cells
were isolated and cultivated as we described previously [7].
Lipoaspirates for their isolation were obtained from healthy
persons undergoing elective lipoaspiration, who provided an
informed consent. Cells were plated in low glucose (1,0 mg/L)
DMEM supplemented with 5% MSC-stimulatory supplement
(StemCell Technologies), 5% of human platelet extract and
antibiotic-antimycotic mixture. AT-MSCs were cultured at
370C in a humidified atmosphere containing 5% carbon di-
oxide with medium changes twice a week. Cells were plated
at a density of 3x103 to 5x103 nucleated cells/cm2. Adherent
cells were split upon reaching confluenceandAT-MSCswere
used for the experiments up to passage 5.
To prepare AT-MSCs expressing cytosine deaminase
(CDy-AT-MSCs), subconfluent cultures of AT-MSCs were
transduced thrice in 3 consecutive days with virus-contain-
ing medium from GP+envAM12/pST2 cells supplemented
with 100 μg/mL protamine sulphate. Transduced cells were
selected by cultivation in medium supplemented with 0.5
mg/ml of G418.
Experiments in vivo. Six- to 8-week-old athymic nude mice
(Balb/c-nu/nu) were used in accordance with institutional
guidelines under the approved protocols. The potential tu-
morigenicity of AT-MSCs and CDy-AT-MSCs was tested
by subcutaneous inoculation of cell suspension of 15x106
AT-MSCs or CDy-AT-MSCs in 0.1 ml PBS into flank of each
nude mouse (n = 2 in each treatment group). The animals
were inspected for tumor growth during 60 days and then the
tumor presence was control by autopsy.
Hydrogen peroxide treatment and DNA-damage testing.
The method of single cell gel electrophoresis (SCGE; comet
assay) according to [9] was performed with minor modifica-
tions suggested in [10, 11]. Briefly: 2.5 x 104 AT-MSCs or
CDy-AT-MSCs in 85μl of 0.75% low melting point agarose in
PBS were spread on a base layer of 100 μl 1% normal-melting
point agarose in PBS, placed on microscope slides and covered
with a cover slip. When the gel solidified, the cover slip was
removed. The cells were then exposed to different concentra-
tions of hydrogen peroxide (0, 100, 200, 300, and 400 μM).
Hydrogen peroxide treatment (5 min on ice) minimizes the
DNA repair process [12]. The slides were washed with PBS
and placed in lysis solution (2.5 M NaCl, 0.1 M Na2EDTA, 10
mM Tris–HCl, pH 10.0, 1% Triton X-100) for 1 h at 4 0C. After
lysis the slides were transferred to an electrophoresis buffer
(0.3 M NaOH, 1 mM Na2EDTA, pH > 13.0) for unwinding
(40 min at 40C) and then subjected to electrophoresis at 25
V (current adjusted to 0.3 A) for 30 min at 40C. Theslideswere
neutralized with 0.4 M Tris–HCl (pH 7.5) and stained with
ethidium bromide (EtBr, 5μg/ml). For each sample, 100 EtBr-
stained nucleoids on triplicate slides were evaluated and scored
with an Olympus fluorescent microscope and computerized
image analysis (Komet 5.5, Kinetic Imaging, Liverpool, UK)
for determination of DNA in the tail, which is linearly related
to the frequency of single strand DNA breaks
Treatment of AT-MSCs, CDy-AT-MSCs, and MSCs from
lipoma with 4NQO.
Cells were exposed to single dose of 10 μM concentra-
tion of 4NQO. Survived fraction of cells (about ten percent)
was further cultivated in growth medium and when reached
confluence transferred by trypsin treatment. The cultivation
continued for several months. During this period of time signs
of senescence appeared, intervals of cell transfer was longer,
the number of passages never reach more than 10 before the
terminal differentiation appeared.
Statistics. Statistical comparisons of mean tail DNA values
in hydrogen peroxide treatment experiments were performed
using the Student’s t-test.

Results
Induction of single stranded DNA breaks. Treatment of AT-
MSCs and CDy-AT-MSCs with hydrogen peroxide resulted
in an increase of DNA breaks in both parental AT-MSCs and
transgene-modified CDy-AT-MSCs when concentration of
hydrogen peroxide reached 100 μM. Higher hydrogen per-
oxide concentration did not further increase the number of
DNA breaks (Fig. 1.) Percentage of tail DNA was comparable
at each hydrogen peroxide concentration tested in both kinds
of cells. Thereforethegeneticallymodifiedmesenchymalcells
did not differ from parental cells in the sensitivity to DNA
breaks induction.
Tumorigenicity of AT-MSCs and CDy-AT-MSCs testing.
Inoculums of cell suspension (15x106 of AT-MSCs or CDy-
AT-MSCs) in 0.1 ml PBS was injected subcutaneously into the
flankofnudemouse(n= 2ineachtreatmentgroup).Animals
were inspected for tumor growth during period of time two
months. The palpable cell inoculum persisted on the site of
injection several days and slowly disappeared during firsttwo
weeks. By day 60 the experiments were ended and animals were
sacrificed and ispected for tumor presence. No tumors were
found either on the site of inoculation nor elsewhere during
the autopsy. ThereforetheAT-MSCsandCDy-AT-MSCswere
found not to be tumorigenic in given experimental setup.
Cytotoxicity of 4NQO to AT-MSC and CDy-AT-MSC.
Mesenchymal stem cells are known for their higher natural
resistance to toxic agents. In order to elucidate the sensitivity

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544V. ALTANEROVA, E. HORVATHOVA2 M. MATUSKOVA, L. KUCEROVA, C. ALTANER
of AT-MSCs, CDy-AT-MSCs and MSCs isolated from lipoma,
potent chemical carcinogen and mutagen 4-nitroquinoline-1-
oxide (4NQO) was chosen for cell treatment. Concentrations
higher than 60 μM of 4NQO were found to be toxic to destroy
cells completely during 4 days of cultivation. At least 10 per-
cent of cells survived treatment with 10 μM concentration of
4NQO for 4 days. Recovered 4NQO-treated cells were further
cultivated and split upon reaching confluence. Cell prolifera-
tion declined with passages kept in culture. Cells became more
light-refractory, the cell morphology changed to cells more
prolonged (Fig. 2 B). Typical signs of aging-related phenotype
appeared (Fig. 2 C). Finally, the cells appeared to form drops of
fat, signs of mature differentiationtoadipocytesinallcells(Fig.
2 D). Accumulation of irreparable DNA damage in adipose
tissue derived mesenchymal stem cells apparently triggered
their terminal differentiation. Terminal adipogenic differen-
tiation after genotoxic stress caused by 4NGO was noticed in
AT-MSCs, in cytosine deaminase transduced CDy-ATMSCs
and also in MSCs isolated from human lipomas.
Discussion
Several properties of human mesenchymal stem cells de-
rived from adipose tissue designate them attractive vehicles
for cell therapies. Theyareabletodifferentiatealonga variety
of lineage pathways, including bone, cartilage, adipose, neu-
ronal-like, and muscle in vitro [see 1 for a review]. They are
nonimmunogenic upon transplantation into allogeneic host [4,
13, 14]. Thecellsexhibita lowimmunogenicprofileasshown
by negative expression of MHC class II molecules and absence
of costimulatory molecule expression [15, 16, 17]. Furthermore
it was found that they are immunosuppressive [18, 19] as it
was demonstrated by their ability to control graft-versus-host
disease (GvHD) in humans [20].
Moreover AT-MSCs possess unique ability to selectively
migrate to tumors and metastases. When AT-MSCs are modi-
fied by prodrug converting gene they are attractive delivery
vehicles for anti-tumor agents such as IFNβ to tumors [21]
and for stem cell-based targeted cancer gene therapy [3, 7, 8,
22]. Taken in account nonimmunogenic properties of MSCs,
AT-MSCs may have potential therapeutic use as allogeneic
products for tissue engineering and for gene therapy upon
genetic modifications as well. For all these reasons, to assess
genetic stability of these cells is rather important, when they
are intended to be used in clinical studies.
There were several contradictive reports in literature with
regards to involvement of adult MSCs in tumor formation
[23]. Increasing number of reports indicated that MSCs are
recruited in large numbers to the stroma of developing tumors
[21, 24], or they could act by enhancing the motility, invasion
and metastasis ability of adjacent cancer cells [25].
Experiments with human xenografts on nude mice have
shown that systemically administrated MSCs possess intrinsic
preferential migratory ability towards breast carcinoma cells,
lung metastasis of melanoma cells, intracranial glioma and co-
lon cancer cells [26, 27]. Moreover exogenously administered
MSCs could form a significantproportionoftumormass[27].
It was demonstrated that bone marrow derived MSCs may
also be involved in cancer metastasis forming pre-metastatic
niche [28]. Thisfindingsupportstheideathattherapeutically
engineered MSCs may be used to treat metastases. Therefore
the introduction of a transgene into autologous stem cells pos-
sesses an attractive cell based delivery strategy [8, 29]. On the
contrary, other studies observed that MSCs may inhibit tumor
growth in animal models [30, 31] and posses antitumorigenic
effects on a model of Kaposi’s sarcoma [32].
Few contradictory reports appeared about spontaneous
transformation of cultured bone marrow derived murine
MSCs [32, 33, 34, 35] and human MSCs [36, 37]. It was
Figure 1. DNA-damaging effectofhydrogenperoxideonparentalandcytosinedeaminasetransducedhumanadiposetissuederivedmesenchymalstem
cells. Details of experimental procedure and evaluation is described in Material and Methods.

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GENOTOXIC DAMAGE OF HUMAN ADIPOSE TISSUE MESENCHYMAL CELLS
reported that murine but not human mesenchymal stem
cells generated osteosarcoma-like lesions in the lung [38]. If
murine MSCs were implanted subcutaneously together with
porous ceramic, host-derived sarcomas developed. Sarcomas
developed only in syngenic and immunodeficient recipients,
but not in allogenic hosts or when the cells were inoculated
as suspension [39].
In our experiments we did not observed any tumor forma-
tion when a high dose of AT-MSCs, or CDy-AT-MSCs was
inoculated subcutaneously as cell suspension in nude mice.
However, when these cells are co-injected with tumor cells,
they can support the growth of engrafts of human melanoma
xenografts, but not human glioma cells on nude mice, due
to their production of various growth factors in a paracrine
fashion [40].
Mesenchymal stem cells as cells of vital importance for
organism are equipped with efficient DNA repair system as
well as high natural resistance to toxic agents [41]. It was
therefore not surprising that DNA damage in CDy-AT-MSCs
was not influenced by the transduction with cytosine deami-
nase as documented by our results obtained with single-cell
gel electrophoresis, which represents a sensitive method for
measuring DNA damage.
In order to find whether adipose tissue derived mesen-
chymal stem cells can be transformed in vitro by chemical
carcinogen, we have chosen water soluble highly effective
carcinogen 4-nitroquinoline-1-oxide (4NQO). 4NQO is
metabolized into 4-acetoxyaminoquinoline-1-oxide (Ac-
4HAQO), which can form covalent adduct to deoxyguanine
and deoxyadenine in DNA. Thecarcinogenicactivityof4NQO
consist in DNA damage production and formation of irrevers-
ible topoisomerase I cleavage complexes (Top1cc) inducing
recombinations. Top1cc produced by 4NQO accumulate
progressively after 4NQO addition and persist to following
4NQO removal. [42]. In addition 4NQO also produces oxida-
tive damage and DNA single-stranded breaks [43]. Results of
our study indicate that replicative senescence of AT-MSCs as
a consequence of cumulating DNA damage is a continuous
process including far reaching alterations in phenotype like
the terminal differentiation. Similar observation with human
Figure 2. Adipogenic terminal differentiationofAT-MSCafteraccumulationofDNAdamagecausedby4NQO. A – untreatedcontrolAT-MSCs;B – AT-
MSCs exposed to 4NQO after serial passages became bigger; C- Senescence of damaged AT-MSCs progressed during serial passages, cells being more
light refractory and sparse; D – All 4NQO-treated AT-MSCs differentiated to adipocytes. Cells were stained with Oil Red-O. Magnification 100 fold.

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546V. ALTANEROVA, E. HORVATHOVA2 M. MATUSKOVA, L. KUCEROVA, C. ALTANER
bone marrow derived MSC together with detail analysis on
molecular level was reported [44, 45]. The DNA damage,
a prime suspect in stem cell aging, causes graying and loss of
melanocyte stem cells by inducing premature differentiation,
without inducing apoptosis or senescence as it was reported
very recently [46]. The irreparable DNA damage, as caused
by ionizing radiation, abrogated renewal of MSCs in mice.
Surprisingly, the DNA-damage response triggers MSC differ-
entiation into mature melanocytes in the niche, rather than
inducing their apoptosis or senescence [46].
Several other reports support the conclusion that accumula-
tion of DNA damage may cause somatic stem cell depletion [47,
48]. Typical sign of aging in mammals is hair graying caused
by the incomplete maintenance of melanocyte stem cells with
age [47]. TheresultingMSCdepletionleadstoirreversiblehair
graying. Deficiency of Ataxia-telangiectasia mutated (ATM),
a central transducer kinase of the DNA-damage response,
sensitizes MSCs to ectopic differentiation,demonstratingthat
the kinase protects MSCs from their premature differentiation
by functioning as a “stemness checkpoint” to maintain the stem
cell quality and quantity [49].
We observed that genotoxic damage of human adipose
tissue derived mesenchymal stem cells caused by chemical
carcinogen 4NQO is not leading to cell transformation but
to process of ageing. The aged AT-MSCs are triggered to adi-
pogenic terminal differentiation.Thesedataareinagreement
with above mentioned observations of terminal differentiation
of melanocyte stem cells [46]. Whether the elimination of
DNA damaged stem cells is generally provided by terminal
differentiation rather than apoptosis remains to be proven.
Acknowledgements. The study was supported by APVV grant
0260/07 (awarded to L. K.); VEGA grant 2/0072/09 (awarded to E. H.);
and financialsupportsawardedtoC.A.fromSPPFoundation,FIDURA
Capital Consult GmbH, Munich, Germany; and the League Against
Cancer. We thank D. Guba M.D., Institute of Medical Cosmetics, Bra-
tislava, Slovakia for providing us with material for AT-MSC isolation,
M. Dubrovcakova and V. Frivalska for technical assistance.
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