Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017
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2018-06-19T09:05:03Z 2018-06-19T09:05:03Z 2017 Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 234–256. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138533 en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Conference reports Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 Article published earlier |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 Conference reports |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 |
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normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. r.e. kavetsky institute of experimental pathology, oncology and radiobiology, national academy of sciences of ukraine, kyiv october 5–6, 2017 |
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Normal and cancer stem cells: discovery, diagnosis and therapy international scientific conference. R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv October 5–6, 2017 // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 234–256. — англ. |
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234 Experimental Oncology 39, 234–256, 2017 (September)
NORMAL AND CANCER STEM CELLS:
DISCOVERY, DIAGNOSIS AND THERAPY
INTERNATIONAL SCIENTIFIC CONFERENCE
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology,
National Academy of Sciences of Ukraine, Kyiv
October 5–6, 2017
ABSTRACTS
UNDERSTANDING THE ORIGIN OF INDUCTIVE
MESENCHYME wITHIN THE wOUND-INDUCED
de novo HAIR FOLLICLES
S. Abbasi1, n. Rosin1, S. Sinha1, W. Scott1,
A. Hagner1, M. Underhill2, F. Rossi2, J. Biernaskie1
1Department of Comparative Biology & Experimental
Medicine, Faculty of Veterinary Medicine,
University of Calgary, Calgary, Canada
2Faculty of Medicine, University of British Colombia,
Vancouver, Canada
sabbasi@ucalgary.ca
We recently demonstrated the existence of an adult
dermal stem cell (DSC) that functions to populate both
mesenchymal compartments within the hair follicle
(HF): the inductive cells within the dermal papilla (DP)
and the connective tissue sheath (CTS) which wraps
around the distal follicle (Rahmani et al., 2014). Depletion
of DSCs disrupts normal HF regeneration highlighting
their potential importance for restoration or prevention
of hair loss (alopecia). The loss of adult HF is considered
permanent but in 2007, it was shown that when the skin
excisions on the back skin of mice are larger than 1 cm2,
de novo HFs form in the middle of the wound bed after
re-epithelialization. This wound-induced HF neogenesis
(WIHN) parallels the development of HFs during em-
bryogenesis. It has been demonstrated that the nascent
HFs arise from epidermal cells outside the HFs and not
from the bulge stem cells within the pre-existing HFs.
However, the origin of the mesenchymal compartments
of these neogenic HFs is still unclear. We hypothesize
that the dermal cells in the neogenic HFs originate
from aSMAve+ cell populations within the dermis. To test
that, we made full-thickness excisions (> 1 cm2) in the
dorsal skin of αSMACreERT2ROSAYFP mice. The wounds
were harvested from 18 days post-wounding (dpw)
up to 140 dpw to assess the dermal cells in the neogenic
HFs. Our data reveal that YFP+ve DSCs integrate into ~90%
of the mesenchymal compartments of the nascent HFs.
The neogenic HFs successfully complete their hair cycle
and YFP+ve DSCs appear to have re-assumed their role
as DSCs within newly formed HFs.
ROLE OF USP1, CORTACTIN
AND Hsp27 PROTEINS IN MOLECULAR
MECHANISMS THAT AFFECT CML DEVELOPMENT
S.v. Antonenko, d.S. Gurianov, I.v. Kravchuk,
G.d. Тelegeev
Institute of Molecular Biology and Genetics,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
antonenkoimbg@gmail.com
Introduction. Development of chronic myeloid leu-
kemia (CML) is the result of reciprocal translocation be-
tween chromosomes 9 and 22 that leads to emergence
of Philadelphia chromosome. Role of BCR domains
in differentiation of leukemia stem cell remains unclear.
Investigation of protein-protein interactions can be one
of the effective ways to reveal those molecular events
that alter normal cellular processes and cause malignant
transformation. Previous research showed that USP1,
cortactin and Hsp27 may interact with PH domain.
The main function of USP1 protein is to deubiquitinate
proteins in the cell. Intracellular Hsp27 plays an anti-
apoptotic role and acts as a chaperone protecting in-
correctly formed proteins from aggregation. The main
function of cortactin is remodeling of actin cytoskeleton
during fission of clathrin-coated vesicles.
Aim. To design genetic constructs for mammalian
expression and determination of protein-protein inter-
actions between PH domain of BCR and target proteins
suitable for the analysis of important signaling pathways
involved in CML pathogenesis.
Materials and Methods. The standard molecular
cloning techniques and expression in E. coli strain Ro-
setta.
Results and discussion. Following PCR, we am-
plified target gene sequences USP1, CTTN, Hsp27.
Their sizes of 2,300 bp, 1,600 bp and 650 bp are in ac-
cordance with expected. Using the ligation reaction,
we produced pBluescriptSKII (+)-USP1, pBluescriptSKII
(+)-CTTN and рET42a-hsp27 genetic constructs.
By subcloning the CTTN gene from pBluescriptSKII
(+)-CTTN plasmid, we derived pFastFT-N1-CTTN,
pMediumFT-N1-CTTN, pSlowFT-N1-CTTN. Recom-
binant genetic constructs were successfully identified
by restriction analysis. The absence of mutations and
correct reading frame was confirmed by sequenc-
ing. We also produced a genetic construct рET42a-
hsp27 for expression of HSP27 in bacterial host system
and obtained effective bacterial expression of Hsp27.
Analysis of protein expression by polyacrylamide gel
electrophoresis confirmed that the expressed protein
corresponds to the expected size of about 55 kDa. The
purified recombinant HSP27 may be used to determine
presence or absence of interaction with PH domain
of BCR by far-Western.
Conclusions. All DNA constructs obtained can
be effectively used to study biological role of interactions
of PH domain of BCR with USP1, cortactin and Hsp27 for
better understanding of molecular mechanisms of CML
pathogenesis. The development of the genetic con-
structs with inserts corresponding to the coding se-
quences of CTTN, USP1 and PH domain of BCR that may
be useful for determining spatio-temporal resolution
of target protein inside the cell on subdiffraction level
is under way.
Exp Oncol 2017
39, 3, 234–256
CONFERENCE REPORT
Experimental Oncology 39, 234–256, 2017 (September) 235
THE CONTENT OF TUMOR STEM CELLS IN BRAIN
GLIOMAS OF DIFFERENT DEGREE OF MALIGNANCY
L.M. Belska, n.I. Lisyaniy, v.M. Semenova,
v.d. Rozumenko, L.P. Stayno, d.M. egorova
SI “A.P. Romodanov Neurosurgery Institute,
National Academy of Medical Sciences of Ukraine”,
Kyiv, Ukraine
adsg@ukr.net
Introduction. At the present stage of research the
possible relationship of cancer stem cells (CSCs) and the
development of various tumors including brain tumors are
actively studied. CSCs are responsible for metastasis, infil-
trative growth, recurrence of gliomas as well as resistance
to chemotherapy and radiotherapy. While the question
of relationship between content of CSCs and malignancy
of gliomas is controversial, it is known that a fundamental
feature of CSCs is their ability to form neurospheres in vitro.
Aim. The purpose of the study was to investigate
the neurosphere formation of CSCs from brain gliomas
of varying degrees of malignancy and analyze CD133+
content in such cells.
Materials and Methods. 51 biopsies of gliomas with
varying degrees of malignancy were obtained during neu-
rosurgery. Histological diagnosis of brain tumors was con-
ducted in accordance with International Histological Classifi-
cation of Tumors of the Central Nervous System (2007). The
cell phenotype cells was analyzed by immunofluorescent
method using monoclonal antibody to CD133 (a marker
of CSCs) by cytometry protocol on flow cytometry (Beck-
man Coulter, USA). The CSC ability to form neurospheres
was studied in vitro in suspension and dissociated cultures.
Results and discussion. It is established that the
number of CD133+ cells in glioblastomas was 8.92 ± 6.81%
and in anaplastic astrocytomas 7.98 ± 4.62%. In benign
gliomas of second degree of anaplasia, the content
of CD133+ cells was less by half than in malignant gliomas.
Upon culturing of glioma cells in DMEM in the presence
of EGF within 10–12 days, fluctuating neurospheres char-
acteristic of CSCs were formed in 86.3% of glioblastoma
samples, 78.5% anaplastic astrocytomas, 88.2% anaplas-
tic olihodendrogliomas and 66% benign astrocytomas.
Conclusions. The increased content of CD133+ cells
was detected in malignant gliomas of III and IV degree
of malignancy with accompanying increased percentage
of neurosphere formation upon in vitro culturing. Further
study of these populations of cells may provide a new under-
standing of the nature of tumors and the causes of treatment
failure in brain gliomas.
MESENCHYMAL STEM CELL AND ONCOGENESIS
n.M. Berezhnaya
R.E. Kavetsky Institute of Experimental Pathology, On-
cology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
berezh@onconet.kiev.ua
Mesenchymal stem cell (MSC), a non-hemopoietic
stem cell defined also as “mesenchymal stem/stromal cell”
or “multipotent stem cell” is one of the major components
of the connective tissue system. As early as in the beginning
of 1970s, A. Fridenstein demonstrated that MSC may dif-
ferentiate into myofibroblasts. Nowadays, the role of myo-
fibroblasts in the formation of microenvironment of tumors
has been well proven. MSCs reside in the bone marrow,
dental pulp, adipose tissue and several other tissues. These
cells possess the adhesive properties and do not express
the markers of the hematopoietic stem cell. MSCs have the
capacity to differentiate into the osteoblasts, chondrocytes,
adipocytes, myocytes and neuron-like cells. The wide range
of the biological features of MSC explains their implication
in tumor growth. To date, several mechanisms of MSC
involvement in the stimulation of tumor growth have been
elucidated: a) MSCs interact with tumor cells increasing
their metastatic potential and inducing EMT in tumor cells;
b) MSCs secrete various soluble factors and cytokines in-
cluding those stimulating tumor growth such as IL-6, IL-8,
IL-10, TGFβ, HGP; c) MSCs are involved in the formation
of chemoresistance that has been proved for many types
of tumors; sometimes chemoresistance is provided by in-
creasing expression of MDR, MRP, LRP; and MSC exosomes
are no less important for chemoresistance. The role of MSCs
in suppression of the immunological response cannot
be overemphasized. Such immunosuppressive effects are
displayed as follows: a) MSCs affect antigen recognition,
primarily by TGFβ production; b) MSCs interfere with interplay
between lymphocytes and tumor cells; c) MSCs promote
the escape of tumor cells from immunological surveillance;
d) MSCs inhibit cytotoxic potential of cells possessing killer
activity, in particular via ROS generation; e) MSCs are actively
involved in the formation of perivascular niches in all types
of tissues being responsive to various extra- and intracellular
signals. On the other hand, according to the general principles
considering the effects of microenvironment on tumor growth
one should be aware also of the MSC protective effects
involving several mechanisms: a) MSCs may enhance INF
gamma production followed by activation of lymphocytes and
stimulation of tumor growth; b) sometimes MSC vesicles may
counter-balance the negative effects of MSCs themselves;
c) MSCs may protect tumors from cytostatics by inhibition
of caspase activity that holds true in several tumors.
To sum up, MSCs represent the heterogeneous popu-
lation capable of various activities depending on the stage
of tumor growth, the source of MSCs and seve ral other
factors. The diversity of the biological effects of these cells
explains why MSCs may be involved both in stimulation and
inhibition of tumor growth.
IMPACT OF BONE MARROw STEM CELLS
AND SOLUBLE FACTORS ON BIOLOGICAL
PROPERTIES AND EPITHELIAL-MESENCHYMAL
TRANSITION IN BREAST CANCER CELLS
n.o. Bezdieniezhnykh, o.o. Lykhova,
R.A. Kocherga, Yu.I. Kudryavets
R.E. Kavetsky Institute of Experimental Pathology, On-
cology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
beznaliano@gmail.com
Background. Bone marrow is a tissue containing stem
cells and cells of various degrees of differentiation.
Aim. To study the crosstalk between breast tumor
cells with epithelial or mesenchymal phenotype and bone
marrow stem cells of breast cancer (BC) patients using
noncontact co-cultivation in vitro system in the pres-
ence of biological or chemical antitumor agents (IFN-α,
mitomycin C, zoledronic acid), which are epithelial-
mesenchymal transition (EMT) modifiers.
Materials and Methods. Human breast carcinoma cells
with epithelial phenotype (MCF-7 cell line), mesenchymal
phenotype (MDA-MB-231 cell line) and aspirates of bone
marrow (BM) of BC patients with a different clinical course
of the disease (groups “Stabilization” (BM-S) and “Progres-
sion” (BM-P)) were used in noncontact co-cultivation system
in vitro. BM stem cells were used as a source of soluble
236 Experimental Oncology 39, 234–256, 2017 (September)
factors of microenvironment for tumor cells ex vivo. Cell
culture methods, immunoenzyme and immunocytochemical
analysis have been used.
Results. It was shown that co-cultivation of MCF-7 cells
with BM-P did not affect the tumor cells growth and migration
activity and co-cultivation with BM-S stem cells was accom-
panied by significant inhibition of such biological properties
of tumor cells. At the same time, significant inhibition of the
same parameters of MDA-MB-231 cells was observed only
after their cultivation in the presence of BM-P cells.
Co-cultivation of MCF-7 cells with BM-P resulted
in 30% increase of the E-cadherin expression in BC cells.
The incorporation of IFN-α in such system leads to changes
in E-cadherin expression (increase in MCF-7 + BM-S and
decrease in MCF-7 + BM-P). The cultivation of MCF-7 cells
in the presence of BM-S resulted in a 2-fold decrease in the
number of Vimentin+ cells, and the addition of IFN-α or mi-
tomycin C enhanced the effect by inhibiting Vimentin ex-
pression in MCF-7 cells by 50% or 100%, respectively.
It was shown that soluble factors produced by BM stem
cells practically did not affect the number of E-cadherin+
MDA-MB-231cells, whereas the addition of IFN-α to this
co-culture system or the complex action of BM-S and mi-
tomycin C were accompanied by a statistically significant
increase in the level of expression of this protein in these
tumor cells. It was interesting to note, that a significant
decrease in the number of Vimentin+ MDA-MB-231 cells
by 50% was observed after their co-cultivation with
BM stem cells in the presence of zoledronic acid.
The analysis of the soluble factors produced by cells in the
experiment showed that co-cultivation of MDA-MB-231 cells
with BM cells did not change significantly the levels of VEGF
and IL-6 in the culture medium. Only IFN-α in such system
suppressed VEGF level by 50% compared to control. It was
shown that co-cultivation of MCF-7 cells with BM cells did not
affect significantly the VEGF level in the culture medium, but
the addition of mitomycin C or IFN-α resulted in a decrease
of VEGF level. The cultivation of MCF-7 cells with BM-S leads
to two-fold increase in the level of IL-6 in the culture medium,
and IFN-α in this system intensified the effect and the level
of IL-6 increased 4-fold compared to control.
It should be noted, that in such co-cultivation system,
regardless of the tumor cells type, the level of TGF-β1 did
not change significantly in any setting of the treatment and
TNF-α was not detected in the culture medium.
Conclusions. The significant changes in biological
properties of BC cells dependently on their ЕМT status were
detected after their interaction with BM cells of BC patients
with a different clinical course of the disease in noncontact
co-cultivation system. BM cells of BC patients had differ-
ent modifying effect on proliferative and migration activity
of tumor cells depending on the clinical course. It has been
shown that cytokines are important elements of tumor
microenvironment that influence EMT status of tumor cells.
CONTENT OF STEM-LIKE CANCER CELLS
OF VARIOUS DIFFERENTIATION DEGREE
AT DIFFERENT STAGES OF BREAST CANCER
(EXPERIMENTAL STUDY)
n.A. Bondarovich, M.v. ostankov,
o.v. Chelombytko, L.v. ostankova, A.n. Goltsev
Institute for Problems of Cryobiology and Cryomedicine,
National Academy of Sciences of Ukraine, Kharkiv, Ukraine
cryopato@gmail.com
Introduction. It is known that the manifestation
of malignant tumors including breast cancer (BC) is stipu-
lated by the presence of cancer stem cells (CSCs). In this
regard, the vital is the question of studying the content and
functional activity of CSCs in BC development. The ap-
propriate experimental model for the evaluation of these
indices is the C3H mice with high incidence of mammary
cancer.
Aim of research was to assess the proliferative potential
of CSCs varying in differentiation degree in BC develop-
ment in the mammary tissue itself, mammary cancer and
metastases in lung.
Materials and Methods. Experiments were per-
formed in 16-month-old female CBA and C3H mice,
weighing 18–20 g, kept under standard vivarium condi-
tions of the IPC&C of the NAS of Ukraine. The mice were
divided into 5 groups of 10 animals each: Group 1 — intact
CBA mice (control); Group 2 — C3H mice without tumors;
Group 3 — C3H mice with tumors (0.5–1.0 cm in diam-
eter); 4 — C3H mice with tumors (1.5–2.0 cm in diam-
eter); 5 — those with tumors (2.0 cm in diameter) and
lung metastases (0.5 cm in diameter). Content of CSCs
in tissue suspensions (mammary tissue, tumors and
metastases in lungs) was determined by flow cytometry
(FACS Calibur) using monoclonal antibodies (BD, USA)
to CD24, CD44 molecules.
Results and discussion. In tissue of murine mam-
mary gland of group 2, CD44hi cells were detected, which
were absent in the mammary gland of CBA mice. The
average content of CD44+/24- cells in mammary gland
in mice of group 2 exceeded the same index in intact
animals. The concentration of CD44+/24+, CD44-/24+ cells
was lower (p < 0.05) than in the control group. In Group
3 of animals with minimal tumor size, the content of CD44hi
cells in mammary gland was unchanged if compared with
group 2, while their number in tumor was twice decreased.
Thus, the content of CSCs (CD44hi) in tumors of the
group 3 mice was lower than in mammary gland, while
a number of more differentiated CD44-/24+ cells on the
contrary was higher than in the control group. The dif-
ference between the content of CD44hi, CD44+/24-,
CD44+/24+ was statistically significant in mammary
gland and tumors in mice of group 4. On the background
of increasing content of CD44hi, a significant difference
between the number of CD44-/24+ in mammary gland
and tumor was kept. At the same time, in mammary gland
the number of CD44+/24- cells increased and the number
of CD44+/24+ and CD44-/24+ cells decreased, in tumors,
in contrast, a decrease in CD44+/24- subpopulations
and an increase in CD44+/24+ and CD44-/24+ cells were
observed.
It should be noted that in mice of group 5 in mam-
mary gland the concentration of CD44hi, CD44+/24- cells
decreased and CD44+/24+ cells increased, while the
number of CD44-/24+ was unchanged. In mammary tu-
mors of this group the number of CD44+/24-, CD44+/24+
and CD44-/24+ cells increased significantly and ex-
ceeded the corresponding values for group 4. However,
the number of CD44+/24- cells in metastases did not
change, and the content of CD44-/24+ and CD44+/24+
cells decreased. At the same time, the number of CD44hi
cells significantly increased relative to the tumor values
in this group.
Conclusions. It has been found that in C3H mice,
the redistribution of subpopulation composition was ob-
served in mammary gland as far as the tumor was mani-
fested. The results emphasize the importance of CSCs
in BC pathogenesis.
Experimental Oncology 39, 234–256, 2017 (September) 237
STEM CELL CXCR4 RECEPTOR IN GASTRIC
CANCER
L.n. Bubnovskaya1,v.M. Mikhailenko1,
S.P. Merentsev2, I.I. Ganusevich1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
2City Clinical Oncological Center, Kyiv, Ukraine
osinskysp12@ukr.net
Introduction. Metastatic spread of tumor cells is the
main cause of cancer-related deaths. Chemokines are the
most important factors controlling cellular migration. Stem
cell receptor CXCR4 as a transmembrane chemokine re-
ceptor and its specific ligand CXCL12 (stromal cell-derived
factor 1, SDF-1α) play a vital role in dissemination of tumor
cells from primary sites, transendothelial migration as well
as homing of cancer stem cells. In the tumor microenviron-
ment under hypoxic condition there is possible mechanism
by which cells of a growing tumor are reprogrammed to ex-
press the CXCR4 receptor thereby enhancing the metastatic
potential of the tumor cells. Therefore in the current work
it was investigated how hypoxia in primary tumor of patients
with gastric cancer (GC) affects the CXCR4 expression.
Aim of research. The aim was to find the association
between CXCR4 expression in GC and bone marrow (BM)
cells with tumor hypoxia, disseminated tumor cells (DTC)
and clinical outcome.
Patients. All patients with primary GC were diagnosed
and treated at the City Clinical Oncological Center (Kyiv).
No patient received chemotherapy or radiation prior to sur-
gery. Histological types of tumor were evaluated by WHO
histological classification (2000). Tumors were classified and
staged according to the 2002 version of the UICC staging
system. All patients were thoroughly informed about the study
that was approved by the local ethics committee.
Methods. DTCs and CXCR4 expression were detected
using immunocytochemistry in BM cytospin preparations,
CXCR4 expression in tumor — using immunohistochemistry,
tumor hypoxia level was evaluated by 31NMR spectroscopy.
The statistical analyses were conducted.
Results. Overall, 78.5% of patients had tumors with
CXCR4+ cells. The direct correlation between CXCR4+ cells
and the level of hypoxia in primary tumor was shown (r =
0.492; р < 0.05); the probability of the appearance of high
number of CXCR4+ cells in tumor increased by a factor
of 5 (OR = 4.926, 95% CI 7.027–23.628, p = 0.046), when
tumors were characterized by severe and moderate hy-
poxia. Moreover, under such hypoxic condition appearance
of tumor cells in BM increased by a factor of 11.4 (OR 11.4,
95% CI 2.71–47.89, χ2 =12.3, p < 0.001). CXCR4+ cells
in tumor tissue were detected in 80% of the patients with
the DTC in BM. When tumors were characterized by positiv-
ity for CXCR4 cells, the probability of appearance of DTCs
in BM of patients increased by a factor of 4.0 (OR = 4.024,
95% CI 1.0597–15.2782, χ2 = 4.367, p = 0.0408) and the
relative risk of DTCs appearance was approximately 2.0 (RR
= 1.955, 95% CI 0.999–3.824, p = 0.05). The CXCR+ cells
in BM were observed in 62.1% of patients with CXCR4+
tumors. Risk of unfavorable outcome in all patients with
CXCR4+ tumors increased almost by a factor of 3.0 (HR =
2.82; 95% CI 1.162–6.832; p < 0.05). It was evaluated that
in patients both with M0 category and CXCR4+ BM, the risk
of unfavorable outcome increased by a factor of 3.4 (HR =
3.4; 95% CI 1.156–12.054; р < 0.03). Overall survival (OS)
was significantly longer in all patients with tumors character-
ized by CXCR4-negative status as compared to the patients
with CXCR4+ tumors (log-rank test, p = 0.0375), and in the
patients with M0 (log-rank test p = 0.0137). OS was longer
in all patients with BM characterized by CXCR4-negative
status as compared to the patients with CXCR4+ BM (log-rank
test, p = 0.0441).
Conclusion. Obtained results have clearly shown that
overexpression of homing-protein CXCR4+ in tumor, espe-
cially in the patients with M0 category, mediated by the level
of hypoxia in primary tumor, is associated with presence
of CXCR4+ cells as well appearance of DTC in BM and cor-
related with poor survival and unfavorable clinical outcome.
CXCR4+ BM is a risk factor of unfavorable prognosis for the
patients with GC especially in the patients with M0 category.
THE EFFECTS OF SUPEROXIDE RADICALS
AND NITRIC OXIDE IN STEM CELLS
А.P. Burlaka, І.І. Ganusevich, А.v. vovk,
n.B. narolsky
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
apburlaka@gmail.com
The balance between self-renewal and differentiation
is a critical function of stem cells (SC) during the develop-
ment of the organism and maintaining tissue homeostasis
throughout the life. Recent studies of SC in the resting
phase show that their redox state is regulated by superoxide
radicals (SORs) and nitric oxide (NO).
The main sources of SORs in the SC are the mitochon-
drial respiratory chain. It should be pointed out that SORs
and NO are integral components of the redox-state of the
cell and because of related functions, they affect each
other. Low levels of these molecules in the SC maintain
resting state and self-renewal. Increased levels of SC gen-
eration cause proliferation/differentiation, senescence,
apoptosis, or depletion. Thus, generation of SORs and
NO in the SC must be strictly regulated for carrying out
function of redox homeostasis support and repairing dam-
aged tissues. Generation of SORs and NO in the SC is regu-
lated by numerous internal and external factors, and their
deregulation leads to the development of pathological
conditions. The consequence of aberrant generation
of SORs in SC tumor is the initiation and progression of ma-
lignancy. So, today the urgent problem is the development
of redox homeostasis regulators in the SC, although the
mechanisms and signal pathways regulated by the SORs
and NO have been not yet studied clearly. NO can cause
nitrosylation of SH-groups in FeS-proteins and release
of Fe ions from mitochondria Fe-S centers and other Fe-
containing proteins, which lead to the impairment of their
functions. In physiological concentrations, NO modulates
the electron transport in the mitochondrial respiratory chain
depending on the concentration of intracellular oxygen,
redox-state of the electron transport chain (ETL) and
the level of cellular hypoxia. SORs and NO have different
chemical properties, different reactivity, time of half-life,
which determines the rate of their interaction with DNA and
other structural molecules in the cell. It is believed that stem
cell are located in niches, which are characterized by hy-
poxia and low levels of SORs and NO, and it is crucial for
maintaining the self-renewal potential in the SC. But, while
SC are being isolated for therapeutic or research purposes,
generation of SORs is enhanced and extracellular matrix
undergoes degradation. SORs and NO can alter gene ex-
pression and activity of key metabolic regulators, thereby
to determine the specific metabolic patterns of the cells.
238 Experimental Oncology 39, 234–256, 2017 (September)
Rising levels of SORs and NO cause oxidative damage
to DNA, proteins, lipids, carbohydrates, premature aging
and apoptosis. The accumulation of oxidation-induced
DNA damage in human cord blood SC leads to the loss
of its ability to restore hematopoiesis. NO inhibits SC pro-
liferation through blocking the cell cycle and induces
CD34 apoptosis. Decreased activity of NOS increases the
number of SC in the bone marrow and improves hemato-
poiesis. Selective reactivity of SORs and NO to biotargets
is the basis of the impact on specific signaling pathways
that are initiated or damaged by them. These reactions
cause allosteric or configurational changes in the target
protein, which alter their function and interaction with other
proteins, causing a variety of signaling events.
Thus, the safe use of SC in the clinic requires improve-
ment of the production technology with strict control
of SORs, NO, matrix metalloproteinases levels that will
promote their ability to self-renewal, proliferation and
differentiation.
CHARACTERIZATION OF CELLS ISOLATED
FROM A BREAST CANCER BEFORE AND AFTER
NEOADjUVANT CHEMOTHERAPY
I. Cakstina, v. Pirsko, M. nakazawa-Miklasevica,
M. Priedite, R. dortane, L. Feldmane,
M. Samovica, d. nitisa, A. Irmejs, d. Lapina,
Z. daneberga, e. Miklasevics
Institute of Oncology, Riga Stradins University, Riga,
Latvia
inese.cakstina@rsu.lv
Introduction. Breast tumors contain heteroge-
neous cell populations. There can be dormant cancer
cells among them that can survive therapy and give rise
to secondary tumors. Therefore, in-depth studies are
necessary for better understanding of the tumor patho-
genesis. In this study, a standardized cell isolation and
characterization methods were used to look at cell growth
and differentiation state at different time points (before
and after neoadjuvant therapy).
Aim. To isolate, characterize and compare cells isolated
from the same breast cancer tumor biopsies before and after
neoadjuvant chemotherapy. The main tasks were: (i) to iso-
late and culture cells from breast cancer biopsies before and
after neoadjuvant chemotherapy; (ii) to perform cell growth
and morphological analysis; (iii) to analyze expression
of breast cancer type related genes; (iv) to compare gene
expression profiles among various isolated cell fractions
before and after neoadjuvant chemotherapy.
Materials and Methods. Cells were isolated from core
biopsies by sequential enzymatic disaggregation collecting
cell fractions obtained after each disintegration step and
culturing them in four diverse cell growth media until the
fourth or fifth passage (the length of cultivation depended
on the numbers of cells) for further analysis and biobank-
ing. Cells were monitored in phase contrast microscope
and fed every two to three days. Several gene expression
panels were developed to characterize isolated cell cul-
tures: proliferation panel, tumor inducing cell marker panel,
epithelial-mesenchymal transition marker panel, epithelial
differentiation gene panel with subpanels for common dif-
ferentiation, basal and Her2++, basal cell regulator, luminal
cell, luminal progenitor and mature luminal cell regulators.
Results and discussion. All isolated cultures had
lower proliferation rate in comparison to reference fibro-
blasts (shown by growth data analysis and gene expres-
sion profile). Also, gene expression profile is different
in cells cultured in media supplemented with 10% FBS
from other three media that had 5% FBS and supplemen-
tal growth factors/hormones.
Patient in this study was diagnosed as luminal type
breast cancer. The results from analysis of differentiation
gene expression may suggest that obtained cell popula-
tions contain both luminal and basal cells. Elevated ma-
ture luminal cell regulatory gene expression may suggest
that isolated populations contain cells with active epithe-
lial differentiation program allowing them to differentiate
towards more luminal cell type.
The analysis of the cultures isolated after chemotherapy
is still ongoing and preliminary results will be presented
in the conference.
Conclusions. In this study, we isolated different cell
populations from the breast cancer core biopsies before
and after neoadjuvant chemotherapy. Isolated cells show
some degree of heterogeneity and gradual decrease in the
proliferation potential by each passage. Cell pools probably
contain both basal and luminal mammary cells capable
of differentiating towards more mature luminal state.
THE USE OF BONE MARROw STEM CELLS
IN RADIATION ONCOLOGY
e.A. domina
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
edjomina@ukr.net
Despite the intensive implementation of the confor-
mal strategy in the practice of radiation oncology, cells
of normal tissues inevitably fall in the tumor irradiation
zone. These are microscopic tumor infiltrates in normal
tissues; tissue structures located at the inlet and outlet
of the therapeutic ionizing radiation (IR) beam; blood ves-
sels and, correspondingly, cells of the circulating blood
pool, exposed to radiation at the same dose as the tumor.
In the process of therapeutic irradiation of cancer patients,
the early negative effects are most common in the he-
matopoietic system. Their severity depends both on the
absorbed dose of IR and on the volume of irradiated red
bone marrow. The mechanisms of these effects are due
not only to the high radiosensitivity of the hematopoietic
stem cells, but also to the individual radiosensitivity of the
organism as a whole.
Earlier, in Ukraine, experience in bone marrow trans-
plantation has been already accumulated (L.P. Kindzelsky
et al., 1987, 1992, 1996, 2001) in the patients with acute
radiation sickness of II–III degree who took part in the
emergency operations after Chernobyl accident (April–May
1986). The purpose of this medical intervention consisted
in a temporary replacement of the “failure” in the hemato-
poiesis using the donor bone marrow so that during this
period the own pluripotent hematopoietic elements could
start the production of blood elements. The period of pas-
sage through the stages of differentiation is 12–15 days
for the elements of the myeloid and erythroid series. Al-
logeneic bone marrow was implanted 3–6 weeks after
irradiation, that is, after detoxification procedures and the
decay of short-lived radionuclides. First, during this time,
the remaining elements in the bone marrow continued the
production of blood cells; secondly, incorporated radio-
nuclides could destroy not only own, but also transplanted
myelocaryocytes. We have observed no early and late
complications in any of the victims after the bone marrow
transplantation.
Experimental Oncology 39, 234–256, 2017 (September) 239
At the present time, in order to minimize the radiation
damage to healthy tissues, stem cells are used after in-
tensive antitumor therapy. This method includes grafting
bone marrow (a mixture of hematopoietic and mesenchy-
mal stem cells), mesenchymal stem cells, the mobilization
of autologous stem cells by growth factors (A.G. Konoply-
anikow et al., 2007; J.M. Lombaert et al., 2008). Stem cell
transplantation is carried out in an oncological clinic with
the aim of replacing damaged and dead cells in the patients
with radiation-induced damage to the tissues of vital organs.
It should be noted that most of the experimental and pre-
clinical studies in this direction were carried out with a single
irradiation. Therefore, their results must be extrapolated
with great care to the conditions of fractionated irradiation,
when the factors of the formation of radioresistance, the
processes of repopulation, etc. are involved.
EPIGENETIC AND METABOLIC REPROGRAMMING
AS A TARGET FOR PROSTATE TUMOR
RADIOSENSITIZATION
A. dubrovska1,2,3
1OncoRay-National Center for Radiation Research
in Oncology, Medical Faculty and University Hospital
Carl Gustav Carus, Technische Universität Dresden
and Helmholtz-Zentrum Dresden — Rossendorf,
Institute of Radiooncology — OncoRay,
Dresden 01307, Germany
2German Cancer Consortium (DKTK) Dresden,
Dresden, Germany
3German Cancer Research Center (DKFZ) Heidelberg,
Heidelberg, Germany
anna.dubrovska@oncoray.de
Radiotherapy is a standard curative treatment for solid
cancers including head and neck squamous cell carcinoma
(HNSCC) and prostate cancer (PrCa). Although HNSCC and
PrCa might be curable at early stages, advanced diseases
have a poor prognosis. Tumor relapse after therapy is attrib-
uted to the population of tumor initiating, or cancer stem cells
(CSCs) which escaped or survived the treatment. It has been
proposed that CSCs in some types of tumors can be protect-
ed from the different treatment modalities by multiple intrinsic
and extrinsic mechanisms that, along with CSC plasticity and
heterogeneity, can be a driving force for tumor recurrence.
Analysis of the phenotypes and density of CSCs in tumor bi-
opsies prior the treatment and development of CSC-targeted
therapies might be an essential approach for optimization
and personalization of the treatment strategies. This lecture
will review the results of studies of the Group of Biomarkers
for Individualized Radiotherapy (OncoRay, Dresden) on the
development of CSC-based predictive biomarkers and ra-
diosensitizers for HNSCC and PrCa.
THERAPEUTIC RESPONSE IN BREAST CANCER
BY DNA CONTENT PROFILING
B.I. Gerashchenko1, K. Salmina2, J. eglitis3,
J. erenpreisa2
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
2Latvian Biomedical Research and Study Centre, Riga,
Latvia
3Faculty of Medicine, University of Latvia, Riga, Latvia
biger63@yahoo.com
Introduction and Aims. Although the histological
assessment of therapeutic response significantly helps
to determine prognosis in breast cancer treated with
neoadjuvant (preoperative) therapy, the problem caused
by discrepancies in interpretation of results obtained from
different pathologists remains actual mainly because
of lack of standardized assessment criteria and methods.
We propose that DNA content profiling of the affected (can-
cerous) tissue in combination with immunohistochemical
judgment of its aggressiveness before and after therapy
may facilitate interpretation of therapeutic response.
Patients and Methods. The study population con-
sisted of 12 breast cancer patients, 11 of those 12 patients
had locally advanced breast cancer. The patients under-
went diagnostic procedures at the Latvian Oncology Cen-
ter of the Riga East University Hospital in 2014 and 2015.
The tissue specimens were collected after the patients’
informed consent was obtained in accordance with the
Ethics regulations. Both diagnostic biopsy and opera-
tion material, such as primary tumors surgically removed
after 3–9 courses of neoadjuvant chemotherapy (NAC)
using standard doses of paclitaxel and doxorubicin, were
subjected to DNA content analysis with image cytometry.
DNA histograms were classified according to Auer et al.
(1980). Positions of DNA peaks were determined by cal-
culating DNA index (DI) values. Based on the presence
or absence of aneuploid DNA peaks and cells that poly-
ploidize (> 4.5C), it seems reasonable to expect that NAC
effects may vary from positive to negative (i.e., opposite).
Immunohistochemical staining was applied to evaluate
proliferation (Ki67), invasiveness (CD44), and self-renewal
factors characteristic for stem cells (SOX2 and NANOG).
Results and discussion. The analysis of DNA content
profiles in operation material from 12 patients revealed the
features of tumor non-responsiveness to NAC in 7 cases,
among which 4 cases showed enhanced polyploidization,
suggesting the negative NAC effect. Polyploidization is likely
to be attributed to the cycling of near-triploid cells (DI =
1.26−1.74). Polyploid cells were positive for Ki67, SOX2,
NANOG, and CD44. Notably, stress-induced polyploidiza-
tion of tumor cells (including breast cancer cells) expressing
self-renewal stem cell factors was also demonstrated in vitro
by several groups of researchers [Salmina et al., 2010; Ghisol-
fi L. et al., 2012; Lagadec C. et al., 2012] prompting us to sup-
pose that the polyploid cells and their descendants released
by de-polyploidization can possess stem cell characteristics.
Conclusions. DNA content profiling data that may not
necessarily correlate with histopathologic scoring data
provide the additional helpful information for interpreting
therapeutic responses in NAC-treated breast cancers. Since
in some cases NAC effects may be unfavorable, the use
of further treatment strategy should be carefully considered.
Therapy-resistant polyploid tumor cells possessing stem
cell features can be induced in vivo as well, assuming that
this process is not autonomous, but rather stipulated by the
tumor microenvironment and intra-tumor heterogeneity.
This work was supported by European Social Fund
2013/0023/1DP/1.1.1.2.0/13/APIA/VIAA/037.
LEUKEMIC BLAST CELLS AND REVISED SCHEME
OF HEMATOPOIETIC CELLS DIFFERENTIATION
d.F. Gluzman, L.M. Sklyarenko, M.P. Zavelevich,
S.v. Koval, T.S. Ivanivskaya, M.S. Shvidka,
A.S. Polischuk, n.I. Ukrainskaya
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
vals@onconet.kiev
Introduction. Both classical and contemporary
models of hematopoiesis are based on study of normal
240 Experimental Oncology 39, 234–256, 2017 (September)
stem cells and early progenitor cells (G. Mathe et al.,
I.L. Chertkov, A.I. Vorobiov; J.E. Dick et al.; I.L. Weiss-
man et al.). The new approach based on studying cy-
tochemical, immunophenotypic and molecular-genetic
features of leukemic blast cells seems to be advanta-
geous for better understanding of the hematopoiesis
under physiological conditions specifying and clarifying
the pathways for the development of terminally dif-
ferentiated blood cells of various lineages originating
from hematopoietic stem cell (HSC) (A.C. Schmidt,
G.K. Przybylski).
The clones of blast cells in acute myeloid leukemia
represent the progenies of leukemic stem cells (LSCs).
The populations of LSCs in most cases mirror normal
granulocyte-macrophage progenitors (GMPs) and pre-
viously uncharacterized lymphoid-primed multipotential
progenitors (LMPPs) (N. Goardon et al.).
Aim. The analysis of immunocytochemical features
of leukemic cells in patients with different types of leu-
kemia studied at the Department of Oncohematology
proved to be useful to put forward several suggestions
as to the possible links between progenitor cells in the
scheme of hematopoietic cell differentiation.
Methods. For immunocytochemical study of leuke-
mic blast cells (APAAP and LSAB-AP methods) we used
a broad panel of monoclonal antibodies (MoAbs) pro-
posed by WHO experts for classification of acute leuke-
mias that include markers of hematopoietic progenitor
cells (CD34, HLA-DR, CD45), B-lineage markers (CD19,
CD20, CD22, CD79a), T-lineage markers (CD2, CD3,
CD5, CD7), markers of myeloid lineage (CD13, CD33,
CD15, MPO, CD117) and megakaryoblasts (CD41,
CD61).
Results and discussion. Our findings suggest that
leukemic cells in pro-B-ALL and monoblastic leukemia
(AML M5a) may derive from the common LSC analogous
to the presumed progenitor for B cells and monocyte/
macrophages in normal human hematopoiesis. We also
demonstrated the similarities of immunophenotype
between blast cells in acute erythroid leukemia (AML
M6b) and megakaryoblastic leukemia (AML M7). Such
similarity hints to the existence of the common bipo-
tent progenitor cells in AML M6b and AML M7, which
is analogous to precursor cell common for megakaryo-
cytopoiesis and erythropoiesis.
The important data for understanding the hierarchy
in normal human hematopoiesis were obtained in study-
ing some types of acute leukemias of ambiguous lineage
(acute undifferentiated leukemia, mixed phenotypic
acute leukemias — B/myeloid and T/lymphoid). In some
cases of acute lymphoblastic leukemia (ALL) from earlier
B cell progenitors, co-expression of myeloid markers
(CD13, CD33, CD15) is observed. However, we have
never seen T cell lineage associated antigens in B-ALL
leukemic blasts. Also, in ALL of T cell origin we do not
find out B cell differentiation antigens. Our findings call
into question the real existence of common lymphoid
progenitors (CLP) in human hematopoiesis postulated
elsewhere.
Conclusions. The data on the immunophenotype
of leukemic blast cells in various forms of leukemia
may be useful for the analysis of hematopoiesis
under physiological conditions. Further studies may
challenge several conventionally accepted differ-
entiation pathways in existing schemes of human
hematopoiesis.
CONTENT OF CD133+ AND CD15+ STEM CELLS
IN GLIOMAS AND RATIO OF PERIPHERAL BLOOD
CELLS OF DIFFERENT HEMATOPOIETIC LINEAGES
IN PATIENTS wITH BRAIN TUMORS
I.A. Gnedkova, n.I. Lisyanyi, L.n. Belskay,
L.A. Kot, o.G. Chernenko, T.A. Malysheva,
v.v. vaslovich, M.A. Gnedkova
SI “A.P. Romodanov Neurosurgery Institute,
National Academy of Medical Sciences of Ukraine”, Kyiv,
Ukraine
Irinagned53@ukr.net
Introduction. In patients with brain tumors, changes
in the content of stem cells in gliomas and cells of periph-
eral blood are noted (neutrophilia, lymphopenia, throm-
bocytosis).
Aim. To compare the ratios of peripheral blood cells:
platelet to lymphocyte ratio (PLR), platelet to neutrophil ratio
(PNR), neutrophil to lymphocyte ratio (NLR) with the content
of stem cells in gliomas of varying degrees of anaplasia.
Methods. The hemogram indices of 105 patients with
brain tumors and 28 healthy persons were analyzed. The
blood indices were measured on the automatic hemato-
logical analyzer Mindray 3000 plus, the ratio of PLR, PNR
and NLR was determined in the peri pheral blood. The
expression of receptors for CD133 and CD15 in tumor cell
suspension was studied by flow cytometry on CYTOMICS
Fc 500 (Beckman Coulter USA) using anti-CD133 and
CD15-FITC antibodies (Millipore).
Results and discussion. In gliomas of the I–II degree
of anaplasia, the content of CD133+ cells was 3.3 ± 1.1%,
in anaplastic gliomas — 5.1 ± 3.2%, in glioblastomas —
8.1 ± 2.2%. CD15+ cells (embryonic cell receptor) were
detected in gliomas twice as often as CD133+ for each
degree of anaplasia. The content of stem cells CD133+
CD15+ in gliomas increased with an increase of the degree
of anaplasia. In this case, the percentage and absolute
content of lymphocytes in the blood decreased. In malignant
gliomas, an increase in PLR, NLR was observed in com-
parison with astrocytomas. It has been established that low
values of PLR and NLR are observed with a relatively higher
remission in patients with glioblastomas.
Conclusions. In malignant gliomas, the content
of stem cells in the tumor tissue increases, and in the
peripheral blood the va lues of cells PLR and NLR reliably
increases and PNR decreases. Hematological indices
can be used as prognostic markers of clinical course
of gliomas and the effectiveness of treatment.
CONTRIBUTION OF EHRLICH CARCINOMA
SUBPOPULATION COMPOSITION
IN MAINTAINING TUMOR GROwTH
A.n. Goltsev, n.A. Bondarovich, n.n. Babenko,
Y.A. Gaevskaya, T.G. dubrava,
o.v. Chelombitko, M.v. ostankov
Institute for Problems of Cryobiology
and Cryomedicine, National Academy of Sciences
of Ukraine, Kharkiv, Ukraine
cryopato@gmail.com
Introduction. Development of malignant tumors re-
sults from expansion of cancer stem cells (CSCs) possess-
ing unlimited potential of self-maintenance. There is a need
in further study of functional and structural characteristics
of these cells in various types of model systems.
Aim of the work was to identify the subpopulation
composition of Ehrlich carcinoma cells including those
with the signs of CSCs as well as their tumorigenicity.
Experimental Oncology 39, 234–256, 2017 (September) 241
Materials and Methods. Ehrlich carcinoma cells were
transplanted intraperitoneally into BALB/c mice. Total
population of Ehrlich carcinoma cells was divided by mag-
netic separation method with BD IMagnet cell separation
system (USA) into CD44+- and CD44–-fractions. The growth
potential of total population of Ehrlich carcinoma cells,
CD44+- and CD44–-cell fractions was assessed after 7 days
following transplantation. The volume of ascitic fluid in the
peritoneal cavity was measured and cell concentration was
counted. Immune phenotypic analysis of the subpopulation
composition of tumors was performed using monoclonal
antibodies to CD4, CD24, CD117, Sca-1 molecules with
flow cytometer “FACS Calibur” (“BD”, USA).
Results. Phenotypic analysis demonstrated that Eh-
rlich carcinoma is a heterogeneous population of tumor
cells of various differentiation degree. Identification of the
Sca-1 structure in virtually all the cells of Ehrlich carcinoma al-
lows us to consider it as a versatile marker of this type of tumor.
The particular significance of the subpopulation of CD117+
cells in maintaining microenvironment for tumor growth was
demonstrated. Cells with CD44high phenotype, being the part
of the CD44+ CD24– population may be considered as the
candidates for CSCs in this model system. This assump-
tion was confirmed by a comparative study of tumorigenic
potential of the cells from the fractions CD44+ and CD44–.
It was shown that the CD44+-fraction cells had a much higher
ability to form a tumor with the predominant content of highly
potent tumor-inducing CD44high and CD44+CD24– cells. It was
demonstrated that the cells of CD44+ fractions were able
to grow even at a 100-fold lower concentration (104 cells/
mouse) used for transplantation as compared to unfraction-
ated Ehrlich carcinoma population (106 cells/mouse). On the
contrary, tumorigenic potential of CD44– fraction was much
lower. Upon administration of 106 CD44– cells/mouse, ascite
developed only in 50% of animals with total cell count in the
peritoneal cavity being 5 times less compared to transplanta-
tion of unfractionated Ehrlich carcinoma cells.
Conclusion. The subpopulation composition of inocu-
lated tumor cell line of Ehrlich carcinoma was studied and the
significance of CD44+ subpopulation in maintaining the growth
of this type of tumor was established. Cells with the CD44high
phenotype being the part of the population of CD44+CD24–
could be considered as CSCs in this model system.
THE EFFECT OF DEUTERIUM DEPLETED wATER
ON THE FUNCTIONAL CHARACTERISTICS
OF NORMAL AND CANCER CELLS IN CULTURE
In vITRo
v.v. Goncharuk1, A.v. Syroeshkin2, A.v. Zlatska3,4,
n.v. Antipova2,5, I.A. Zlatskiy1,2
1A.V. Dumanskiy Institute of Colloid and Water Chemistry,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
2The Peoples’ Friendship University of Russia,
Moscow, Russia
3Institute of Genetic and Regenerative Medicine, National
Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
4Biotechnological Laboratory ilaya Regeneration, Kyiv,
Ukraine
5M.M. Shemyakin and Yu.A. Ovchinnikov Institute
of Bioorganic Chemistry of the Russian Academy
of Sciences, Moscow, Russia
zlatskiy@ukr.net
Introduction. Deuterium depleted water (ddw), in its
physico-chemical characteristics, differs from the water
of natural isotope composition (V. Goncharuk, 2011). How-
ever, the biological activity of ddw is poorly understood. The
ddw protective properties are confirmed by toxicological
studies, from which it follows that ddw, due to its trans-
port properties, effectively removes toxins and metabolic
products from the body (R. Robins, 2012). Different con-
centrations of deuterium in water can differently affect the
proliferative activity of the cell culture of prokaryotes and
eukaryotes in vitro (G. Somlyai, 2010; O. Mosin, 2014).
Positive effect of ddw in studies with transplantable tumors
in laboratory animals and in other in vivo and in vitro experi-
ments (H. Wang, 2013) was noted.
Aim. To study the effect of ddw on the functional
characteristics of cells in vitro.
Materials and Methods. After preparing growth media
based on ddw and deionized water (miliQ), the final concen-
tration of deuterium in water was D/H = 30 ± 2 ppm and 150 ±
2 ppm, respectively. Skin biopsies were dissociated by enzy-
matic treatment for 1 h in 0.05% collagenase IA and 0.05%
pronase. The cells were cultured in DMEM:F12 supplemented
with 10% FBS, 2 мМ L-glutamine and 1 ng/ml FGF-2 in a multi-
gas incubator at 5% CO2 and 5% O2. The following assays
were done: clonogenic potential (CFU test) at 2nd and 4th
passages and counting cell doubling time (CDT) at 2nd, 3rd,
4th, 5th and 6th passages. Human cancer cell lines A549 (lung
carcinoma) and HT29 (colon adenocarcinoma) were used.
The method of scratching was used to study the “amoeboid”
movement of cancer cells within 12–24 h.
Results and discussion. It was established that
growth media based on ddw contributes to a significant
push to the colony formation of human dermal fibroblasts
in the early passages by 15–20% more than in water of the
natural D/H ratio. The population doubling times in colonies
of human dermal fibroblasts, in late passages, are shorter
by 4–6 h in ddw than in the water of the natural isotope ratio
D/H. This may indicate a larger reserve of culture dividing
cycles in a growth medium based on ddw than in the water
of the natural isotope ratio D/H. A549 and HT29 cancer
cells filled the monolayer damage zone 50–70% slower
with the addition of ddw than the water-based control
of the natural D/H ratio. The data obtained require ad-
ditional studies to determine the molecular mechanisms
of deuterium action on intracellular structures.
Conclusions. The effect of the isotope ratio of D/H in wa-
ter on the proliferative potential and the rate of colony-
forming units of human dermal fibroblasts in vitro was es-
tablished. It was shown that ddw contributes to a significant
slowing of the “amoeboid” movement of A549 and HT29 hu-
man cancer cells. However, according to the results of our
work, the role of deuterium in biological systems in vitro is not
completely determined, which requires additional studies
in the field of chemistry, physics and biology of water.
POSTNATAL EXTRA EMBRYONIC TISSUES
AS A SOURCE OF MULTIPLE CELL TYPES
FOR REGENERATIVE MEDICINE APPLICATIONS
o.S. Gubar1,2, A.e. Rodnichenko2,3,
R.G. vasyliev2,3, A.v. Zlatska2,3, d.o. Zubov2,3
1Institute of Molecular Biology and Genetics,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
2Biotechnology Laboratory ilaya Regeneration, Kyiv,
Ukraine
3State Institute of Genetic and Regenerative Medicine,
National Academy of Medical Sciences of Ukraine,
Kyiv, Ukraine
olga.gubar@gmail.com
Introduction. Placenta, amnion and umbilical cord
are usually discarded after childbirth whereas these
242 Experimental Oncology 39, 234–256, 2017 (September)
tissues may serve as a valuable source of different cell
types. These postnatal extra-embryonic tissues are read-
ily available and their potential use for cell therapy pos-
ses no ethical issues for either autologous or allogeneic
application.
Aim. We aimed to isolate and characterize the cell
types which could be obtained from postnatal extra-
embryonic tissues.
Materials and Methods. Fresh tissues (no more than
12 h after delivery) were used for enzymatic or explants
methods of cell isolation. Obtained cultures were further
maintained at 5% oxygen. At P3 cell phenotype was as-
sessed by FACS and the multilineage differentiation assay
was performed.
Results and discussion. We have isolated multiple
cell types from postnatal tissues. Namely, placental mes-
enchymal stromal cells (Pl-MSCs) from placenta chorionic
disc, chorionic membrane MSCs (ChM-MSCs) from free
chorionic membrane, umbilical cord MSCs (UC-MSCs)
from whole umbilical cord, human umbilical vein endothe-
lial cells (HUVECs) from umbilical vein, amniotic epithelial
cells (AECs) and amniotic MSCs (AMSCs) from amniotic
membrane. All isolated cell types displayed high prolif-
eration rate together with the typical MSCs phenotype:
CD73+CD90+CD105+CD146+CD166+CD34-CD45-HLA-DR-.
HUVECs constitutively expressed key markers CD31 and
CD309. All MSCs and AECs were capable of osteogenic
and adipogenic differentiation. The only exceptions were
the Pl-MSCs and isolated by enzymes UC-MSCs that failed
typical osteogenic differentiation. Surprisingly, UC-MSCs
isolated via explants qualitatively demonstrated osteogenic
differentiation. Thus obtained MSCs cultures fulfill mostly
all minimal MSCs criteria. Of note, AECs, initially possess-
ing typical epithelial morphology, progressively acquired fi-
broblastoid appearance at P2-P3 probably due to the EMT.
Conclusions. We have shown that a wide variety
of cell types can be easily isolated from extra-embryonic
tissues, banked and expanded ex vivo for regenerative
medicine applications. These cells possess typical MSCs
properties and can be considered an alternative to adult
MSCs, obtained from bone marrow or fat, especially for
allogeneic use.
THE EXPRESSION OF THE EPITHELIAL-
MESENCHYMAL TRANSITION MARKERS
IN 2D AND 3D CELL CULTURES OF MCF-7 UNDER
THE INFLUENCE OF C-MEDIUM FROM
MESENCHYMAL STEM CELLS
T.S. Herheliuk1, o.M. Perepelytsina1,
L.I. ostapchenko2, M.v. Sydorenko1
1Department of Biotechnical Problems of Diagnostics,
Institute for Problems of Cryobiology
and Cryomedicine, National Academy of Science
of Ukraine, Kyiv, Ukraine
2Educational and Scientific Centre
“Institute of Biology & Medicine”, Kyiv, Ukraine
vodolyb@ukr.net
Introduction. It is known that breast cancer (BC)
consists not only of the neoplastic cells but also of the
changed stroma. The stroma, which is associated with
metastases, includes the proteins of extracellular matrix
and the cellular components, in particular mesenchymal
stem cells (MSCs), the blood vessels and the immune
cells. The various studies have shown conflicting data
on the MSC role in the development of BC. Such con-
tradictions may depend on a wide range of the factors,
including differences in the setting of experiments, the
heterogeneity of MSC populations, the dose and the time
of injection of these cells, and also on the model. The study
of the influence of the humoral factors from MSCs on the
development of BC is very relevant in recent years. In ad-
dition, it is important to study the change in the epithelial
phenotype to the mesenchymal phenotype in the tumor
cells. EMT cells actively proliferate and self-renew, which
leads to an increase in the number of the heterogeneous
populations. It has been shown that the most adequate
model of the study of tumor population in vitro is a multi-
layer (3D) cell culture. The target effect on the expression
of the molecules that regulate EMT is promising for cancer
therapy, including BC.
Aim of the study was to compare the expression of the
major EMT markers in the tumor cells of breast adenocar-
cinoma (MCF-7 cell line) under the influence of c-medium
from MSCs in 2D and 3D cell cultures.
Materials and Methods. MCF-7 cell line (breast
adenocarcinoma) was chosen as an experimental model
in vitro. The monolayer cell culture was cultured in standard
conditions (37 °C, 5% CO2, humidity 95%). The initial density
of culture was 2×104 cells/cm2. To initiate the generation
of the spheroids, the monolayer cell culture was removed
off the substrate after four days of the incubation, using
0.25% Trypsin-EDTA, and placed in the nutrient medium
with 5% carboxymethyl cellulose, 5×105 cells/ml. Then the
plates were incubated on an orbital shaker at 50 rpm for
3–5 h. Human BM-MSCs were cultured with MCF-7 cells
and multicellular tumor spheroids by non-contact method.
Detection of the markers (EpCAM, vim, CKs) in 2D and
3D cell culture was performed using IHC method with the
primary monoclonal antibodies.
Results and discussion. Our results demonstrate the
differences in the expression of the epithelial-mesenchymal
transition markers in 2D and 3D breast cancer cell cultures.
Thus, the percentage of epithelial markers (cytokeratines
and epithelial cell adhesion molecule) in the tumor spheroids
is less than in the cells of monolayer, however, the spheroids
cells begin expressing a mesenchymal marker — vimentin.
C-medium from hBM-MSCs reduces the volume of the
spheroids by 61% compared with the control and promotes
an increase in the expression of the tumor-associated mark-
ers — cytokeratins and EpCAM in 2D and 3D cell cultures,
but only in 3D culture the vimentin expression increased.
ADAPTOR PROTEIN RUK/CIN85 ENHANCES
THE FEATURES OF CSCS IN BREAST CANCER CELLS
I. Horak1, G. Pasichnyk1, L. Knopfova2, L. Borsig3,
L. drobot1
1V.A. Palladin Institute of Biochemistry,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
2Masaryk University, Brno, Czech Republic
3University of Zurich, Zurich, Switzerland
Iryna.horak@gmail.com
Introduction. Cancer stem cells (CSCs) are known
as a population of tumor cells with high tumorigenic poten-
tial and self-renewal ability. These cells are characterized
by a number of properties, such as expression of specific
molecular markers, higher invasiveness and metastatic po-
tential, drug resistance, expression of “embryonic” genes.
Adaptor proteins play significant role in assembly of signal-
ing complexes mediating various cellular processes includ-
ing acquiring for the development of stem-like phenotype.
In this study we investigated the effect of adaptor protein
Ruk/CIN85 on the CSSs properties of breast cancer cells.
Experimental Oncology 39, 234–256, 2017 (September) 243
Materials and Methods. As a model we used two breast
cancer cell lines: weakly-invasive human MCF-7 cell line and
highly-invasive mouse 4T1 cell line. In order to analyze the role
of Ruk/CIN85 in the manifestation of CSCs features, we gen-
erated stable sublines with overexpression of Ruk/CIN85 and
with its downregulation. Proliferation rate was estimated
by MTT-assay and growth curves. Invasive ability was ana-
lyzed using Boyden chamber assay. Adhesion-independent
growth ability was estimated by mammospheres formation
assay. Gene expression was evaluated by RT-qPCR.
Results. In 2D cell culture, the transition of both hu-
man and mouse Ruk/CIN85 overexpressing breast cancer
cells from epithelial to rounded and mesenchymal phe-
notype was observed. It was also demonstrated that Ruk/
CIN85 negatively regulates MCF-7 and 4T1 cell prolifera-
tion and adhesion while substantially enhances invasive-
ness of Ruk/CIN85-overexpressing cells. It turned out that
in both cases cells with overexpression of Ruk/CIN85 pro-
duce more mammospheres than control ones. Analysis
of stemness related genes expression demonstrated that
in MCF-7 cells with Ruk/CIN85 overexpression NANOG,
ID-1, Oct4, KLF-4, and CCL-2 genes were up-regulated,
while in 4T1 cells Ruk/CIN85 overexpression resulted
in up-regulation of NANOG, KLF-4, SNAIL1, Lcn2 and
down-regulation of Myb genes.
Conclusion. The obtained data suggest that adap-
tor protein Ruk/CIN85 is a key regulatory factor involved
in the development of stemness features in breast cancer
cells.
This study was partially supported by SC STCU and
SCOPES grants.
COMPARISON OF EXTRACELLULAR MATRIX
AND ADHESION MOLECULES EXPRESSION
PROFILES OF DIFFERENT ORAL MESENCHYMAL
CELL CULTURES
n. Ishkitiev1, R. Tsikandelova1, v. Mitev1,
Z. Mihaylova2, P. Stanimirov2
1Department of Medical Chemistry and Biochemistry,
Medical University-Sofia, Sofia, Bulgaria
2Department of Oral and Maxillofacial Surgery, Medical
University-Sofia, Sofia, Bulgaria
ishkitiev@gmail.com
Introduction. A set of mesenchymal stem cells from
oral tissues were described in the last decade. Detailed
characterization and comparison of the ECM and adhe-
sion profile of different mesenchymal stem cell cultures
has not yet been performed.
Aim of this research is to isolate primary mesenchymal
cell cultures from adult (DPSC) and deciduous tooth pulp
(SHED), periodontal ligament (PDL), dental apical papilla
(SCAP), bone marrow (BMC) and gingiva (GMSC) and
to characterize them for expression of specific extracellular
matrix and adhesion molecules using RT Real time PCR.
Materials and Methods. DPSC, SHED, PDL, SCAP,
BMC, GMSC were isolated from unadulterated dental, al-
veolar bone and gingival explants from patients undergoing
routine surgical operations in Oral and Maxillofacial Sur-
gery Clinic of Dental Faculty, Medical University — Sofia,
Bulgaria, after acquiring informed consent. Explants were
digested in 3 mg/ml Collagenase I/ Dispase and seeded
in culture flasks with DMEM complemented with 20% FBS
and antibiotics. Cells were passaged after reaching 70%
confluence. Cells up to the 3rd passage were used for the
experiments. Total RNA was extracted with RNeasy Plus
Mini kit, cDNA was synthesized with RT2 First Strand Kit and
analyzed with RT2 Prolifer PRC Array, Human Extracellular
Matrix & Adhesion Molecules Array (all QIAGEN, German-
town, MD, USA) following manufacturer‘s instructions.
Results and discussion. Analysis of adhesion and
ECM markers showed significant differences between dif-
ferent oral mesenchymal stem cell cultures. Comparison
of collagen isotypes expression showed also significant
differences in fibrillar collagen expression and particularly
Collagen I, with highest expression in BMC and lowest
in SCAP.
Analysis of integrins showed similarity for the most
abandoned β subunit is ITGB1 followed by ITGB5.
ITGB3 on other hand were mostly expressed by dental
mesenchymal stem cells.
Significant differences in expression of integrin α sub-
units were discovered. ITGA8 was the most abundant except
in PDL and BMC where ITGAV and ITGA5 prevailed respec-
tively. Stem cell related ITGA1 and ITGA6 were expressed
in DPSC, RDL and SCAP and ITGA6 expression was much
lower than ITGA1 in other three sell types.
Comparison of matrix-metalloproteinases (MMP)
showed the highest amounts of MMP2 and MMP14 in all
cells with significant difference in other minor expressed
types.
Cluster analysis of total examined gene expression re-
vealed similar expression profiles of SCAP and PDL cells,
differing significantly from BMC and GMSC, whereas
DPSC and SHED showed bigger similarities with BMC
and GMSC respectfully.
All these results suppose the differences in potential
for wound healing, cell migration, modulation of the im-
mune response and participation in carcinogenesis of the
mesenchymal stem cell subpopulations.
Conclusions. Oral tissues contain a set of different
mesenchymal stem cells, having different adhesion and
ECM profile, thus possessing different stem cell proper-
ties. Further analysis and comparison between mesen-
chymal stem cell population gene expression is needed
for better understanding of stem cell potential of these
adult stem cells. This understanding would refine the
knowledge about adult stem cells and their role in healthy
and pathologic conditions and may lead to development
of regenerative and cell therapy methods.
CYTOCHEMICAL AND IMMUNOPHENOTYPICAL
FEATURES OF CELLS IN BLAST CRISIS
OF CHRONIC MYELOID LEUKEMIA
T.S. Ivanivska, n.I. Ukrainska, M.S. Shvidka,
A.S. Polischuk, Yu.v. Schvets
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
vals@onconet.kiev
Introduction. Multistage process of hematopoiesis
is sustained by hematopoietic stem cells (HSCs) and pro-
genitor cells that are generated in embryonic development.
Being at the base of the whole hematopoietic hierarchy,
HSCs finally give rise to all blood lineages in the adult or-
ganism. The study of the upper hierarchy of hematopoietic
system is in fact the topical problem. The molecular biology
techniques, genetic engineering, and flow cytometry are
widely used nowadays with the aim of elucidating the origin
of specified hematopoietic lineages. The study of mor-
phocytochemical and immunocytochemical features
of leukemic blast cells of various origin and differentiation
levels originating by transformation of the correspond-
244 Experimental Oncology 39, 234–256, 2017 (September)
ing progenitor cells may be also useful as one of the
approaches to clarify some challenging aspects relating
to normal hematopoiesis.
Aim. To define linear origin and differentiation level of leu-
kemic cells in various forms of blast crisis of chronic myeloid
leukemia (CML BC).
Methods. Cytomorphological, immunohistochemical,
cytochemical, statistical.
Results and discussion. When CML BC of monocytic
type was compared to acute monoblastic leukemia origi-
nating de novo, no differences could be observed in cy-
tomorpholgy and cytochemical features of leukemic cells.
Nevertheless, immunophenotyping revealed expression
of CD19, CD36, and CD61 in CML BC contrary to de novo
acute monoblastic leukemia (AML M5). Furthermore, the
expression of CD7 and CD56 in CML BC was much more
intensive as compared to de novo AML M5.
Despite the common morphological features of blasts
in megakaryocytic CML BC and de novo acute megakario-
blastic leukemia (AML M7), some differences were evident
upon their cytochemical study. Namely, in CML BC activity
of acid phosphatase was manifested as the granules against
the background of the diffusely stained cytoplasm whereas
in de novo AML M7 cytochemical features of blast cells varied.
In cases of less differentiated cells in AML M7, the cytochemi-
cal reactivity of blast cells was rather inert (except for acid
phosphatase) similar to that in blasts of CML BC. In more
mature blasts in AML M7, some other enzymes were detected
by the cytochemical techniques (except for myeloperoxidase).
Analysis of immunophenotype revealed several stages of blast
differentiation in de novo AML M7. On the contrary, phenotype
of blast cells in CML BC was monomorphic in all patients. More-
over, contrary to CML BC, blast cells in AML M7 expressed
CD7 and CD36. When cytomorphological, cytochemical
and immunophenotypical features were compared in cells
of de novo acute myeloid leukemia with minimal signs of dif-
ferentiation and CML BC with minimal signs of differentiation,
no distinctions were evident.
Conclusion. The origin of different types of BC CML
indicates to the secondary leukemic transformation that
occurs at the level of multipotent progenitor cells, whereas
in de novo acute myeloid leukemia transformed leukemic
cells seem to originate from oligolineage hematopoietic
progenitor cells.
ETHICAL ISSUES OF RESEARCH, BANKING
AND CLINICAL APPLICATIONS OF HUMAN CORD
BLOOD HEMATOPOIETIC STEM CELLS
T.o. Kalynychenko
SI “Institute of Hematology and Transfusiology,
National Academy of Medical Sciences of Ukraine”,
Kyiv, Ukraine
kalynychenko_tetiana@ukr.net
Introduction. Umbilical cord blood hematopoietic stem
cells (UCB-HSCs) are in the category of the most studied
adult multipotent stem cells (M.S. Santos, P. Ventura-Junca,
2012). As a component of the already formed human body,
which is in the neonatal period of its own development, UCB-
HSCs have the property of replicating themselves, maintain
their differentiation potential and, consequently, are capable
of hematopoietic tissue regeneration (D.L. DiGiusto, 1996;
Q.L. Hao et al., 2001). In general, the study and use of adult
stem cells does not raise such serious ethical issues related
to the source of extraction, which are painful for human em-
bryonic stem cells. At the same time, a number of common
problems associated with all stem cells are actual for the
UCB-HSCs, in particular, issues related to clinical research
in this field. There are also the specific nature issues that are
inherent in this type of human stem cells.
Aim. To discuss specific and common ethical aspects
of research, banking and clinical applications of UCB-
HSCs.
Methods of research. Systemic causal-dialectical
analysis of practical research experience, world-wide
scientific literature data, international legal standards
and recommendations.
Results and discussion. Cord blood as an effective
alternative source of hematopoietic tissue is recognized due
to a number of important advantages over other unrelated
allogeneic hematopoietic stem cell transplants (E. Gluckman,
2009; A.R. Smith, J.E. Wagner, 2009). These benefits include:
lack of donor risks, availability of frozen samples, reduced
incidence of graft versus host and relapse with similar overall
survival (J. Munoz et al., 2014). Current research on the devel-
opment of methods for improving engraftment, restoring the
recipient immune system is accompanied by ethical issues
that are common to transplantology and regenerative medi-
cine: the inadmissibility of commercialization of the human
body, the balance between the desire for clinical progress
and scientific caution (С. Petrini, 2012; N. King, J. Perrin,
2014). The functioning of the two main options of umbilical
cord blood banks (public and private) raises specific ethical
and legal issues of control and equitable access to medi-
cal care (N.M. Fisk et al., 2005; N.M. Fisk, R. Atun, 2008;
C. Petrini, 2010; E. Gluckman, 2012; C. Parco et al., 2013;
G.M. Guilcher et al., 2015). Provided the absolute benefits
of altruistic cord blood donation in public banks for the sake
of public interest over autologous private storage, there are
problems for priority resolution, such as the need for clear
regulation of property rights, as well as the wording of the
rules for granting consent to the definition of the concept
of a donor, consideration of interests of the newborn and his
family, confidential safekeeping of medical and social infor-
mation data, development of information forms for the donor
family, which are connected with the prospects of material use
(E.A. Meyer et al., 2005; A. Mohr et al., 2012; C. Patrini, 2013;
R. Isasi et al., 2013; C.L. Stewart et al., 2013).
Conclusion. Considering the prospect of the UCB-
HSC scope expanding, the designated complex of ethical
issues requires regular monitoring and attention.
EFFECT OF CRYOPRESERVATION FACTORS
ON BIOLOGICAL PROPERTIES OF UMBILICAL
CORD BLOOD STEM CELL TRANSPLANT
T.o. Kalynychenko1, M.Yu. Anoshyna1,
A.I. Gordienko1, e.v. Shorop1, Zh.M. Minchenko ,
v.v. Balan2
1SI “Institute of Hematology and Transfusiology, National
Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine
2SI “National Research Center for Radiation
Medicine of the National Academy of Medical Sciences
of Ukraine”, Kyiv, Ukraine
kalynychenko_tetiana@ukr.net
Introduction. Umbilical cord blood (UCB) is widely used
as a source of stem cells for hematopoietic reconstitution
(J.E. Wagner, E. Gluckman, 2010; J.N. Barcer, J. Kurtzberg,
K. Ballen et al., 2017). The limit of cell content and the forced
use of cryopreservation technologies cause profound study
of opportunities for saving and expanding the tissue trans-
plantation potential (P.R. Motta et al., 2014; G. Chen et al.,
2015; J. Kieman, P. Damien et al., 2016). It is known that the
cryoprotection effectiveness consists in controlling the ice
Experimental Oncology 39, 234–256, 2017 (September) 245
formation, the dehydration development and the solution
effect with the help of cryoprotective substances, directly
proportional to their concentration (H.T. Meryman, 1971;
G.M. Fahy et al, 1984; M.J. Taylor et al., 2004; P. Mazur, 1977,
1984, 2004; J.O. Karlsson, M. Toner, 1996; M.S. Berrada,
G.S. Bischof, 2001; L.E. Ehrlich et al., 2015).
The effectiveness of umbilical cord hematopoietic tissue
cryopreservation under the protection of dimethyl sulfoxide
(DMSO) has been proved by numerous studies (R. Syme
et al., 2003; D. Bauwens et al., 2005; A.S. Chen-Plotkin
et al., 2007; A.M. Junior et al., 2007; A. Abdelkefi et al., 2009;
A. Michael et al., 2012). Therefore, maintaining a balance
between preservation of biological structures and reduction
of the potential threat from using of cryoprotective agents
is considered one of the biggest problems in the cryopreser-
vation of hematopoietic stem cell transplants (HSCT).
Aim. To investigate the changes in the biological prop-
erties of the UCB HSCT during cryopreservation in order
to determine the ways of their correction.
Materials and Methods. The isolated mononuclear
fraction was frozen under protection of 5 and 10%
DMSO. The evaluation of the cell state was carried out
by morphological, functional, biochemical (determina-
tion of the activity of lipid peroxidation processes (LPP)
of neutral lipids and phospholipids) methods. The study
of apoptotic stages was carried out by double annexin V
(Ann V) and propidium iodide (Pi) staining.
Results and discussion. After exposure with DMSO
(5% concentration) Ann V+/Pi+ — cell content and cells with
damaged membranes increased to 2.2 ± 0.3% (p < 0.001)
and to 1.1 ± 0.5% (p < 0.02), respectively. The exposition
with higher concentrations of DMSO — 10% caused the
increasing early stages of apoptosis to 16.1 ± 1.8% (p <
0.05). Provided that the cryoprotective concentration was
decreased, there was a reduction in the granulocyte-mac-
rophage progenitor cell (CFU-GM) number (p < 0.001) and
a decrease of cell viability (from 92.9 ± 0.8% to 85.9 ± 1.0%;
p < 0.01) in defrosted samples. The correlation of the LPP
activity with the apoptosis indices and the functional activity
of the CFU-GM number was identified.
Conclusion. The enhanced activation of the lipid
peroxidation in the UCB HSCT both indirectly through the
induction of apoptosis and as a result of the toxic effects
of the peroxidation products on the cell membranes leads
to a reduced quality of transplant material. The need for
correction of these changes should be considered at the
development of long-term storage technologies.
ARSENIC-INDUCED LIVER CARCINOGENESIS:
IS THERE A LINK TO IRON METABOLISM
DEREGULATION IN PROGENITOR CELLS?
I. Kindrat1,2, K. dreval2, I. Pogribny2
1Ivano-Frankivsk National Medical University,
Ivano-Frankivsk, Ukraine
2Division of Biochemical Toxicology, National Center
for Toxicological Research, Jefferson, Arkansas, USA
Irakindrat0603@gmail.com
Introduction. Inorganic arsenic is a common environ-
mental contaminant and known human carcinogen. Liver
is the main organ for iron storage and metabolism and
an important target for arsenic carcinogenesis. However, the
impact of arsenic on hepatic iron homeostasis is not clear.
Aim of this study was to investigate the effect of inorgan-
ic arsenic on iron metabolism in human liver HepaRG cells.
Materials and Methods. Gene expression in the human
HepaRG cell line was analyzed by whole genome microar-
ray and quantitative reverse transcription-PCR techniques.
Protein level of transferrin (TF) was determined by Western
blot analysis, and iron content was measured by inductively
coupled plasma mass spectrometry.
Results and discussion. The results showed that
treatment of human liver HepaRG cells with 1 µM of inor-
ganic arsenic (as NaAsO2) for 14 days increased the ex-
pression of iron metabolism-related genes, TFRC, FTL,
FTH1 and SLC40A1, while the level of TF mRNA and TF pro-
tein was decreased. Additionally, exposure of HepaRG cells
to arsenic resulted in an increase of cellular iron content.
These changes were accompanied by a decrease in a num-
ber of mature functional hepatocytes and expansion of liver
cancer stem cells, evidenced by the accumulation of pro-
genitor like cells (e.g., primitive biliary cells).
Conclusion. Our results demonstrate that chronic expo-
sure of human liver HepaRG cells to a low dose of inorganic
arsenic resulted in substantial iron metabolism alterations,
loss of functional hepatocytes, and activation of liver cancer
stem-like cells. These findings indicate that arsenic-induced
iron metabolism dysregulation may be one of the underlying
mechanisms of arsenic-related liver carcinogenesis.
THE RESULTS OF 5-YEAR OVERALL SURVIVAL
OF STANDARD AND HIGH-RISK NEUROBLASTOMA
PATIENTS TREATED wITH MYELOABLATIVE
THERAPY AND PERIPHERAL BLOOD STEM CELL
RESCUE (ONE CENTRE EXPERIENCE)
G.I. Klymnyuk, e.v. Shaida, S.v. Pavlyk,
o.v. Skachkova, M.v. Inomistova, n.M. Khranovska
National Cancer Institute, Kyiv, Ukraine
shaida@i.ua
Introduction. Intensification of consolidation therapy
with autologous stem-cell rescue after myeloablative
doses of chemotherapy could contribute to the improved
survival of children with high-risk neuroblastoma. How-
ever, long-term cure rates remain low, presumably be-
cause of the emergence of resistant clones or persistence
of minimal residual disease.
Aim. To investigate the 5-year overall survival of pa-
tients with high-risk neuroblastoma treated with myeloab-
lative therapy and peripheral-blood stem-cell rescue
(PBSCR) in the National Cancer Institute of Ukraine.
Methods. In 2007–2016, 143 patients with neuroblas-
toma (74 standard-risk (SR) patients and 69 high-risk (HR)
patients) received treatment in the Scientific and Research
Department of Pediatric Oncology at the National Cancer
Institute. The risk group of patients was determined on the
basis of the most common prognostic factors including
the child’s age, stage, genetic markers (N-MYC oncogene
amplification, DNA diploidy). All enrolled patients received
treatment according to NB-2004 and HR-NBL-1/ESIOP
protocols. During the late stage of treatment, 54 high-risk
patients were cured with high-dose chemotherapy (HDHT)
with autologous stem cell support; of these, 13 patients were
cured with tandem HDHT.
Results. The 5-year overall survival (OS) was 67% for
SR patients and 30.4% for HR patients. Depending on the
child’s age: the OS was 58.8% for patients under 1 year
of age, while the OS was 19.2% for patients aged 1 year
or over. We also analyzed the survival rate of high-risk
patients based on whether the patient has N-MYC gene
amplification. The OS was 49.8% for N-MYC negative
subjects and 24.3% for N-MYC positive subjects. Currently,
the OS is 69.2% for high-risk patients who were treated with
tandem HDHT with autologous stem cell support.
246 Experimental Oncology 39, 234–256, 2017 (September)
Conclusion. The obtained results show that the sur-
vival in high-risk patients aged 1 year or over positive for
N-myc oncogene was worse greatly than in patients with
normal N-MYC status. The results of treatment of HR pa-
tients who received tandem HDHT with autologous steam
cell support are encouraging.
HUMAN MESENCHYMAL STEM CELLS FOR BONE
TISSUE ENGINEERING: MODERN GENE DELIVERY
TOOLS IN OPTIMIZATION OF OSTEOGENESIS
o. Korchynskyi
Institute of Cell Biology, National Academy
of Sciences of Ukraine, Lviv, Ukraine
Medical Faculty, Rzeszow University, Rzeszow, Poland
olexkor@hotmail.com
Introduction. Human skeleton is completely remod-
eled by mesenchymal stem cells (MSC) within roughly four-
and-half years. Bone morphogenetic proteins (BMP) are
main inducers of bone formation and maintenance apart
of their crucial roles in morphogenesis of other tissues and
organs. Optimal activity of BMPs in bone usually requires
coordinate signals with the Wnt pathway. Consequently,
several bone diseases occur when BMP or Wnt pathways
are misregulated. Therefore, proper optimization of the
osteogenesis requires efficient gene delivery tools on the
way of modulation of osteoblast differentiation of MSC. Un-
fortunately, classical transfection with multiple transfec-
tion reagents triggers massive cell death in transduced
MSC. On the other hand, viral tools for gene delivery allow
to get 100% of efficiently transduced stem cells without
any detection of toxicity.
Materials and Methods. Human MSC cell were
isolated from bone marrow aspirates obtained under
patients’ consent from surgical material upon hip joint
arthroplasty or from fat using a liposuction. Adv5-based
vectors were used for efficient overexpression of re-
combinant genes into the mesenchymal precursor cells
and lentiviral vectors were used for small hairpin RNA
(shRNA)-mediated knockdown of gene expression.
Results and discussion. The set of adenoviral and
lentiviral vectors generated at our lab allows to overex-
press or correspondingly downregulate expression of dif-
ferent genes important in regulation of osteogenesis. Us-
age of the gene delivery tools generated at our lab allowed
us to describe a molecular mechanism that mediates
osteogenesis regulation by secreted phosphoprotein 2.
Conclusions. Modern viral gene delivery tools open
a wide window for biomedical studies aiming to investigate
the molecular mechanisms of human MSC differentiation
and to optimize the efficacy of regenerative skeletal tissues
engineering based on MSC incorporation into the grafts.
EPITHELIAL-MESENCHYMAL TRANSITION
IN HUMAN STEM CELL LINE 4BL
v.o. Kushniruk1, v.A. Shabliy2, d.o. Mykytenko3,
G.S. Lobyntseva2, L.L. Lukash1
1Institute of Molecular Biology and Genetics,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
2Institute of Cell Therapy, Kyiv, Ukraine
3Clinic of Reproductive Medicine “Nadiya”, Kyiv, Ukraine
kushniruk_v_o@ukr.net
Introduction. We obtained a new human stem cell
line from peripheral blood cells of adult healthy donor.
It successfully overcame the Hayflick limit and is stable
(immortalized) cell line. During morphological analyses
of stained cells at different passages we observed two
types of cells: epithelioid and fibroblast-like.
Aim. Presence of two morphological cell classes
is an interesting phenomenon, thus the aim of research
was to study epithelial-mesenchymal transition (EMT)
in 4BL cell line.
Materials and Methods. Cells were cultivat-
ed in DMEM with 10% FBS, 100 U/ml penicillin and
100 µg/ml streptomycin. For morphological studies cells
were stained with 1% neutral red. The immunophenotype
was analyzed by flow cytometry on BD FACS Aria (BD,
USA). Soft agar test was performed in 0.3% agar on Petri
dish. For array CGH analyses DNA from the cells was ex-
tracted with QIAamp DNA Blood Mini Kit and purified with
QIAquick PCR Purification Kit (Qiagen, UK). 1 µg of cell
DNA was hybridized on CytoSure Aneuploidy array slide
followed by the analysis by the Innopsys Innoscan 710 de-
vice with OGT CytoSure Interpret Software 3.3.2 due to the
recommendations of the Oxford Gene Technology.
Results and discussion. Maintenance of two mor-
phological cell types: epithelioid and fibroblast-like, first
of all is concerned with stem cell plasticity and EMT. Stem
potential of 4BL cell line was revealed in the previous stud-
ies by demonstrating adipogenic, osteogenic and myo-
genic differentiation using special induction mediums.
Presence of different cell types in cell population can
provide advantages of survival in changing culture condi-
tions. Thus, in soft agar test 4BL cells grew well, proliferated
without attachment to the substrate and formed colonies.
It is worth mentioning that ability to grow in semi-liquid
medium is characteristic both of mesenchymal stem cells
and cancer cells. We detected the expression of MSC
markers: CD73+ and CD105+ with flow cytometry. Usually
EMT is associated with acquisition of malignant state, but
we have not revealed the expression of Oct4, the marker
of embryonic stem cell, which is re-expressed in cancer
stem cells. By array CGH data we have detected partial
deletion of 12p11.1pter, where Nanog gene is located,
which is also secondarily activated in cancer cells.
Due to array CGH we have revealed the dupli-
cations of chromosomes with genes, which take
part in dif ferentiation in bone t issue — FRZB
(2 q31.1–q33.1) and FGFR3 (4p16.1-pter); in myo-
genic tissue — M-cad herin (16q22.1qter) and NODAL,
MYOZ1 myozenin 1 (10q22.1q22.2); stem cell potential
maintenance — MATK, KLF1, GDF15, BST2 bone marrow
stromal cell antigen 2 (19 р13.3–р12), and also PARD6A,
which plays role in asymmetric cell division and EMT
(16q22.1qter).
Conclusions. Population of the stem cell line 4BL
maintains two morphological cell types: epithelioid and
fibroblast-like, which is concerned with stem cell plas-
ticity. It is verified by cell differentiation into three types
of lineages and by easy adaptation of cells to microenvi-
ronment change such as growth in soft agar.
AGE-RELATED CHANGES OF BIOLOGICAL
PROPERTIES IN THE MULTIPOTENT
MESENCHYMAL STROMAL BONE MARROw
CELLS, FUNCTIONING OF THYMUS AND PINEAL
GLAND IN MICE OF DIFFERENT STRAINS
I.F. Labunets, A.e. Rodnichenko
Institute of Genetic and Regenerative Medicine,
National Academy of Medical Sciences of Ukraine,
Kyiv, Ukraine
irina_labunets@ukr.net
Introduction. The bone marrow multipotent mesenchy-
mal stromal cells (BM-MSCs) can differentiate into various
Experimental Oncology 39, 234–256, 2017 (September) 247
directions, produce trophic and immunomodulatory effects,
influence the differentiation of hematopoietic stem cells.
With age, the BM-MSCs properties undergo changes, while
the occurrence of malignancies rises (Gross, 2007; Anisi-
mov et al., 2015). Thymic hormone thymuline influences the
proliferative capacity of BM-MSCs (Labunets et al., 2012).
The thymus function is under influence of pineal gland and
its main hormone, melatonin, possesses geroprotective
and antitumor properties. In aging, bone marrow-immune-
endocrine interrelations may be linked with genotypes.
Aim. Comparative analysis of the biological properties
of BM-MSCs, functions of the thymus and pineal gland
in FVB/N and CBA/Ca mice, varying significantly in their
life duration, frequency of malignancies (leukemia/lym-
phomas) and general adaptability.
Materials and Methods. The studies were conducted
on adult (4–6 months) and old (20–22 months) mice.
FVB/N versus CBA/Ca mice are characterized by both, high
frequency of spontaneous bone marrow chromosomal
aberrations and leukemia/lymphomas in aging. These
mice are sensitive to the B strain of Friend leukemia virus
and reveal low immune response. In our study, the number
of bone marrow co lony-forming unit fibroblasts (CFU-Fs)
was measured by the bone marrow monolayer culture
technique. The number of granulocyte/macrophage colo-
ny-forming cells (GM-CFCs) in the semi-agar cultures was
assessed. The phenotyping of BM-MSCs was performed
using the monoclonal antibodies to mouse membrane
antigens labeled with fluorochromes. To determine the im-
munomodulatory properties of BM-MSCs, we performed
a joint cultivation of MSCs from mice of different age with
syngeneic splenocytes of young mice stimulated by T-cell
mitogen phytohemagglutinin. The melatonin and thymuline
levels were determined in the serum.
Results and discussion. The stromal progenitor cells
significantly increase in their number only in bone marrow
of old FVB/N mice, indicating age-associated elevated
ability of the BM-MSCs to proliferation. The number of GM-
CFCs also increased in old FVB mice. On the contrary,
changes of indices in the old CBA/Ca mice are slight. The
immunosuppressive effect of BM-MSCs from adult mice
on lymphocyte proliferation was higher than in old mice. The
blood thymuline level falls significantly in old CBA/Ca mice
and remains unchanged in FVB mice. The melatonin level
decreases more intensely in old FVB/N versus CBA/Ca mice.
Conclusions. The genetic differences in biological
parameters of BM-MSCs may be linked with particulari-
ties of interrelations between pineal gland and thymus
functions in aging mice (a), may contribute to the de-
velopment of certain type spontaneous tumors in old
organism (b).
STEM CANCER CELLS. A NEw CHALLENGE
IN STUDYING ONCOGENESIS
n.I. Lisyanyi, I.A. Gnedkova, A.n. Lisyanyi,
L.n. Belskaya, d.n. Stanetskaya
SI “A.P. Romodanov Neurosurgery Institute,
National Academy of Medical Sciences of Ukraine”,
Kyiv, Ukraine
Nimun.neuro@gmail.com
The last 20 years due to the great success in the study
of the biology of human stem cells, a new direction in on-
cology has been defined — the study of the nature of stem
tumor cells. The theory of stem cells is not recognized
by all, although there are indisputable facts proving their
existence, in particular, with glioblastomas.
Thanks to the discovery of cancer stem cells (CSC),
not only the theory of carcinogenesis has changed, but
also the concepts of infiltrated growth, the resistance
to adjuvant methods of treatment acquired new out-
lines. Such properties of CSC as angiogenic potential,
heterogeneity, differentiated mimicry, migratory and
tumor-inducing abilities have been proved experimentally.
CSC are ten times more potent in experimental induction
of tumors in mice than non-CSC tumor cells. It has been
established that CSC of different types of human tumors
have certain phenotypic markers, which allows using
them for diagnosis and prognosis.
At the same time, there are many challenging questions
related to CSC. There are no clear phenotypic markers for
tumor and non-tumor stem cells. Theoretical assumptions
about the possibility of anti-CSC therapy of carcinogenesis
have not yet been confirmed. Anti bodies against marker CSC
molecules (anti CD133+) can both stimulate and suppress
tumor growth in the experiment. Induction of tumor growth
in mouse experiments with tumor cells without phenotypic
signs of CSC (CD133- cells) casts doubt on the assumption
that CSCs are the only or basic cells capable of inducing
tumor growth.
Therefore, today the questions about the role of can-
cer-inducing cells in oncogenesis as well as the possibility
of controlling carcinogenesis with the help of CSC remain
unresolved.
CONTENT OF CANCER STEM CELLS IN GLIAL
BRAIN TUMORS
n.I. Lisyanyi, d.n. Stanetskaya, v.M. Semenova,
T.A. Malysheva, A.n. Lisyanyi, A.I. Potapova
SI “A.P. Romodanov Neurosurgery Institute, National
Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine
Nimun.neuro@gmail.com
Introduction. In recent years special attention has
been paid to the study of the role of cancer stem cells
(CSCs) in the pathogenesis of malignant growth. CSCs are
responsible for migratory ability, radio- and chemoresis-
tance, for induction of new tumors in the experiment, which
is important for determining the characteristics of tumors
and predicting the outcome of the disease. The available
data on the CSC content in brain tumors are contradictory.
Aim. To study the content of CSCs in brain tumors
of various histology.
Materials and Methods. 134 samples of biopsy
material of brain tumors were examined with the aid
of immunohistochemical, immunofluorescence and flow
cytofluorometry methods. The monoclonal antibodies
to CD133 (Millipore, USA) CD15 and CD34 (Becton Dis-
kinson, USA) were used.
Results and discussion. CD133+ CSCs have been
detected in various brain tumors. Their highest content
was found in glioblastomas and medulloblastomas. After
repeated surgery, the CSC content in the tumor tissue
increased. In addition to CD133+ cells, a small number
of CD34+ and CD15+ cells was also detected. It was found
that an increase in the content of CD133+ cells in tumor
tissue is accompanied by a decrease in lymphocyte count
in the peripheral blood. Even the CSC content in tumors
of the same histological type (glioblastoma or medul-
loblastoma) varied significantly.
Conclusion. Determination of CSCs in tumor tissue
can serve as an additional indicator for choosing meth-
ods of combined treatment and predicting the duration
of remission.
248 Experimental Oncology 39, 234–256, 2017 (September)
EXPRESSION OF CD133 BY GLIOMA C6 CELLS
UNDER THE INFLUENCE OF RAT FETAL BRAIN
NEUROGENIC CELLS CONDITIONED MEDIUM
L.d. Liubich, n.I. Lisyanyi, v.M. Semenova,
L.P. Stayno
SI “A.P. Romodanov Neurosurgery Institute, National
Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine
lyubichld@gmail.com
Introduction. Current state of development of neu-
rooncology is focused on searching the criteria of treat-
ment personalization on the basis of identifying a unique
set of molecular changes in tumor cells. The neuroepithelial
stem cell markers are considered among the targets for
brain glial tumors. CD133 (pro minin-1) molecule may
be considered as one of the most characteristic biomarkers
of brain tumor stem cells (BTSC). Cellular and molecular
similarities between BTSC and normal neurogenic stem/
progenitor cells (NSC/NPC) substantiates the search for
new methods of treatment of malignant glioma using NSC/
NPC (K.S. Aboody et al., 2013; R.A. Morshed et al., 2015;
J. Portnow et al., 2016). In particular, immunobiological
properties of NSC/NPC may be realized via the expres-
sion and production of immunoregulatory cytokines and
growth factors.
Aim. To study the influence of rat fetal brain neuro-
genic cells conditioned medium (NCCM) on the content
of CD133+ cells in rat glioma C6 and normal rat fetal brain
cell cultures.
Materials and Меthods. NCCM was obtained from
suspensions of neurogenic rat brain cells on the 14th (E14)
day of gestation. The cells of rat brain glioma (cell line
C6, n = 6) and cells of rat fetal brain (E14, n = 6) served
as the material for culturing. The study was performed
in control cultures (standard cultivation conditions without
NCCM adding) and research cultures (with adding NCCM
(0.10 mg/ml) for 48 h). Immunocytochemical staining for
CD133 molecule was performed using murine monoclo-
nal antibodies to CD133 (Millipore, USA). In cytological
specimens the changes in cellular composition and the
number of immunopositive cells were determined and
morphometric studies were performed.
Results and discussion. According to the immunocy-
tochemical staining, BTSC or CD133+ cells accounted for
7.2–16.8% of the total number of cells in C6 glioma cultures
and 25.0–49.7% in fetal rat brain (E14) cultures. CD133+ cells
were smaller in size than CD13− cells (average values of cell
sectional area, nucleus cell-sectional area) and had greater
nuclear-cytoplasmic ratio. CD133+ cells and their nuclei in cell
cultures of fetal rat brain were twice larger in size than such
cells in cultures of C6 glioma.
NCCM (0.10 mg/ml for 48 h) reduced 4-fold the
number of CD133+ cells in C6 rat glioma cell cultures
(p = 0.02 compared to control, U-Mann — Whitney test).
Cell cultures of fetal rat brain did not reveal such effect.
The functional role of CD133 in NSC/NPC re-
mains poorly understood. According to J.M. Angelas-
tro and M.W. Lame (2010), exogenous expression
of CD133 caused a 2–4-fold decrease in apoptosis
of tumor cells in response to therapeutic agents (doxo-
rubicin, kaptotecin) and CD133+ glioma C6 cells had
increased by 62% expression of one of ABC-transporters
(P-glycoprotein), leading to drug resistance, which sup-
port the hypothesis of antiapoptotic CD133 functional
role in protecting tumor cells, particularly from chemo-
therapeutic agents. Our results demonstrate that NCCM
reduces the number of CD133-expressing BTSC cells
in cultured glioma C6 cells but not in cultures of fetal
rat brain. Moreover, the size of CD133+ cells in cultures
of glioma C6 and fetal brain differ in size that suggests
that BTSC of glioma C6 may be qualitatively different
from NSC/NPC (probably express ligands for the active
molecular agents of NCCM).
Conclusions. Since CD133 is regarded as critical
therapeutic target for therapy of brain tumors, the results
of reducing the number of CD133-expressing cells (BTSC)
in C6 glioma cell culture under influence of the NCCM can
be the basis for a comprehensive study of preparations de-
rived from neurogenic fetal cells for the purpose of theoreti-
cal justification of their application in pathogenetic treatment
of patients with brain gliomas.
NEw TUMOR STROMAL CELL LINE wITH
CHARACTERISTICS OF STEM CELLS TO ASSESS
THE IMPACT OF MICROENVIRONMENT
COMPONENTS ON TUMOR CELLS ex vIvo
o.o. Lykhova, n.o. Bezdieniezhnykh,
R.A. Kocherga, v.G. Lupan, Yu.I. Kudryavets
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
alexxDNA@gmail.com
Introduction. The basis of the formation of metastatic
phenotype of tumor cells is a phenomenon of epithelial-
mesenchymal transition (EMT). The key regulator of this
process in tumor cells is their microenvironment and,
in particular, the cellular components of the tumor stroma.
Mesenchymal stem cells as key elements of the stroma
are important regulators of tumor progression and its
sensitivity to antitumor factors.
Aim. To obtain and characterize new stromal cell line
for studying the interaction between malignantly trans-
formed cells and their microenvironment ex vivo and as-
sessing new approaches to anti-stromal therapy.
Materials and Methods. Cell culture methods, im-
munocytochemical and statistical analysis.
Results. SEBR line of stromal cells from the spontaneous
breast tumor adenocarcinoma of the rat was established and
characterized by morphology, growth parameters, immuno-
phenotypical profile and cytogenetic features. SEBR cells
are characterized by fibroblast-like morphology, a nuclear-
cytoplasmic ratio is similar to normal cells, morphology and
number of chromosomes was as in normal rat cells (2n =
42). The cells are characterized by the dominance of mesen-
chymal cell antigens: high expression of N-cadherin (262 ±
33), Vimentin (280 ± 11), and a low number of cells with pan-
cytokeratin expression. A high level of the expression of stem
cell marker CD44 (> 200 points by H-Score system) was
found. The SEBR cells were used in the co-cultivation system
with malignant transformed rat mammary cells (MRS, MRS-
T5 cell lines) with different morphological characteristics
and the initial status of EMT associated proteins. The aims
of investigation were to assess the impact of stromal cellular
elements on the proliferative and immunophenotypical prop-
erties of tumor cells. In parallel, the effect of normal embryo
fibroblasts on tumor cells and additional exogenous factors
with antitumor activity (chemotherapy drugs, cytokines) were
studied. The conditioned culture medium (CM) was col-
lected and assayed at different phases of cell growth. It has
been shown that CM from stromal SEBR cells significantly
stimulates the proliferation of MRS-T5 cells (tumorigenic
mesenchymal phenotype) and does not stimulate MRS cells
(nontumorigenic epithelial phenotype). CM from normal
Experimental Oncology 39, 234–256, 2017 (September) 249
fibroblasts does not activate the cellular metabolism of MRS
and MRS-T5 cells. Moreover, under specific conditions (de-
pending on cell concentration and CM content), a significant
inhibition of cellular metabolism and proliferation (up to 50%)
is evident. CM from tumor cells was shown to inhibit SEBR
cell growth and metabolism (20 to 60%) and stimulate nor-
mal fibroblasts (from 10 to 80%). In the study of sensitivity
of stromal and tumor cells to drugs, it was found that cells
are characterized by similar sensitivity to zoledronic acid
(IC50 1–2 µg/ml), cisplatin (IC50 5–10 µg/ml) and paclitaxel
(IC50 0.1–0.2 µg/ml). At the same time, the response of these
cells to the TNF and mitomycin C was different, in particular,
the tumor cells were more sensitive to mitomycin C and less
sensitive to TNF (at doses lower than 2000 IU/ml, the stimu-
lation of cellular proliferation was observed) as compared
to SEBR.
Conclusion. A new cell line SEBR of stromal origin
with characteristics of stem cells was established and
characterized. A significant difference between the
responses of tumor cells with various EMT status on co-
cultivation with SEBR cells was shown.
EFFECT OF PDGF-BB ON PROLIFERATION,
MATRIX SYNTHESIS AND MINERALIZATION
OF HUMAN PDL STEM CELLS
Z. Mihaylova1, P. Stanimirov1, R. Tsikandelova2,
v. Mitev2, n. Gateva3, n. Ishkitiev2
1Department of Oral and Maxillofacial Surgery,
Medical University-Sofia, Bulgaria
2Department of Medical Chemistry and Biochemistry,
Medical University-Sofia, Bulgaria
3Department of Children’s Dentistry,
Medical University-Sofia, Bulgaria
zorrymihaylova@gmail.com
Introduction. PDGF is one of the main active mor-
phogenes in the platelet concentrates which have been
increasingly used to stimulate tissue healing at the injury
site. Although in recent years many clinicians apply autolo-
gous platelet derived products to enhance healing process
and regeneration of PDL, the effect of PDGF on stem cell
markers expression, enzymatic activity, ECM synthesis
and mineralization in PDL has not been studied in detail.
Aims. The purpose of the present study is to de-
termine the presence of cells in human PDL capable
of expressing stem cell and differentiation markers and
to determine the effect of PDGF-BB on cell proliferation,
marker expression, collagen synthesis, enzymatic activity
and mineralization.
Materials and Methods. PDL cells were isolated and
characterized using stem cell and differentiation markers via
immunofluorescence and flow cytometry and then cultured
in vitro and treated with different concentrations of PDGF-
BB. The effect of PDGF-BB on cell proliferation, stem cell
and differentiation markers expression, soluble collagen
production, lysyl oxidase (LOX) activity, alkaline phosphatase
(ALP) activity and calcium nodules formation was assessed.
Results and discussion. PDGF-BB stimulated the
proliferation of cells with the maximum effect at 50 ng/ml.
The growth factor increased the expression of stem cell
markers and SPARC; Col1a2 expression was decreased,
whereas the expression of Col3a1 and BSPII remained
unchanged. Soluble collagen production, ALP activity
and calcium nodules formation were also significantly
decreased by PDGF-BB; LOX activity was significantly
increased. Our results show that PDGF-BB initiates
self-renewal through maintenance of cell proliferation
and stem cell properties on the one hand, and inhibition
of abnormal mineralization in human PDL on the other.
Conclusions. PDGF-BB is a powerful promoter of cell
proliferation that increases the expression of stem cell
markers; inhibits collagen production and mineralization
but accelerates the maturation of collagen chains through
increased LOX activity and SPARC expression.
INTERPLAY BETwEEN MITOCHONDRIAL
RIBOSOMAL PROTEIN
S18-2 AND RETINOBLASTOMA PROTEIN
IN REGULATION OF CELL STEMNESS
AND DIFFERENTIATION
M. Mushtaq1, L. Kovalevska2, v. Kashuba1,
e. Kashuba2, 1
1Department of Microbiology, Tumor and Cell Biology
(MTC), Karolinska Institutet, Stockholm, Sweden
2R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
elena.kashuba@ki.se
The retinoblastoma (RB) pathway is inactivated
in several different human cancer forms, including RB,
osteosarcoma, melanoma, and neuroblastoma. These
tumors are derived from neural crest from cells that fail
to undergo terminal differentiation.
We have found that mitochondrial ribosomal protein
S18-2 can bind RB. Thus, we discovered a novel mecha-
nism of cell fate regulation, which depends on overexpres-
sion of S18-2 (MRPS18-2).
Noteworthy, overexpression of the human S18-2 im-
mortalized primary rat embryonic fibroblasts (REFs) that
showed properties of embryonic stem cells. Moreover,
S18-2 is expressed at the high levels in endometrial,
breast and prostate cancer tissues, as we shown by im-
munohistochemistry and q-PCR.
Moreover, analysis of the available data bases revealed
the elevated expression of S18-2 at the mRNA levels
in stem cells and many cancer types. This raises the ques-
tion of whether this protein co-operates with the RB protein
in differentiation and carcinogenesis. We also hypothesized
that simultaneous expression of RB and S18-2 proteins
at the high levels might support stemness.
Here we discuss the mechanisms of how S18-2 pro-
tein, together with RB, is involved in the maintenance
of cell stemness.
BONE MARROw MESENCHYMAL STEM
CELLS AND MORPHOFUNCTIONAL STATUS
OF THE TRANSPLANT
M.e. ospanova1, Z.K. Burkitbayev1,
S.A. Abdrakhmanova1, T.S. Baltabaeva1,
T.n. Savchuk1, M.B. Askarov2
1Scientific-Production Center of Transfusiology,
Astana, Kazakhstan
2National Scientific Medical Center of Astana, Astana,
Kazakhstan
omninpct16@mail.ru
Introduction. In the transplantation immunology, T-
and B-lymphocytes play an important role in maintaining
morphoregulatory processes and implementing an im-
munological surveillance to maintain cellular homeosta-
sis. The posttransplant period is affected by the immune
system imbalance, in particular, of T-cells (CD3), T-helper
cells (CD4), cytotoxic T cells (CD8), NK cells, plasma cells,
regulatory lymphocytes (CD4+CD25+FOXP3) and cytokines
produced by these cells.
250 Experimental Oncology 39, 234–256, 2017 (September)
Aim. The aim of the study was to investigate the
morphofunctional status of the transplant in the post-
transplant period and transplantation of the bone marrow
derived mesenchymal stem cells (BM-MSC).
Materials and Methods. In the study, two groups
of mice were involved: a control group (n = 30) with a skin
graft transplantation and main group (n = 30) with the skin
graft transplantation combined with BM-MSC. Paraffin
technique was used for morphological control of transplant
engraftment after 7, 14, and 21 days. Briefly, the tissue
was fixed in 10% neutral buffered formalin, and then the
tissue samples were cut and embedded in paraffin. Tissue
samples were stained with hematoxylin and eosin. Further
histological slides were studied by microscopy and photo-
graphed with a digital camera Axiocam color (Germany).
Results and discussion. According to the histologi-
cal analysis and macroscopic observations at day 7, 14,
and 21 significant differences were seen among the study
groups. Thus, at day 7, in the control group the state
of the transplant was unsatisfactory with necrotic graft
changes and edema occurred. This picture was observed
almost throughout the study period (at day 7, 14 and 21).
After fortnight, the transplant of the mice of the control
group appeared a dark pale in color. A half size of the
transplant of the control group was rejected, there were
eruptions of the surgical sutures. Necrosis and edema
of the graft occurred as well. At the end of the third week,
there was a complete rejection of the graft in the control
group. In the main group, during all observation periods
(at day 7, 14 and 21), the functioning of the transplant
was noted. It should be noted that after a week of surgi-
cal procedure there were marginal necrosis, edema and
a pale coloration of the skin. The mice of the main group
had engraftment of the graft and had no signs of trans-
plant rejection. Under the microscope, moderate edema
of the graft and lymphohistiocytic infiltration was noted.
Overall, the function of the graft was retained. At the end
of observation period, the mice of the main group had the
complete engraftment and the function of the transplant
maintained.
Conclusions. The study has allowed establishing
a connection between transplant engraftment and im-
munoregulatory properties of mesenchymal stem cells,
both inducers of immunological tolerance, and their
regenerative properties, which contributed to the full
engraftment of the graft.
MESENCHYMAL STROMAL CELL-BASED TISSUE
ENGINEERING
A.Yu. Petrenko, e.Yu. Rogulska, d.n. Tarusin,
S.P. Mazur, e.B. Revenko, v.v. Mutsenko,
v.P. Grischuk
Institute for Problems of Cryobiology
and Cryomedicine, National Academy of Sciences
of Ukraine, Kharkiv, Ukraine
alexander_petrenko@cryo.org.ua
Introduction. Due to ability to produce a variety of cell
types multipotent mesenchymal stromal cells (MSCs)
are promising cellular component for tissue engineering.
Aim. Development of tissue engineered constructs
on the base of MSCs derived from adult human tissues
and various 3-D scaffolds.
Materials and Methods. MSCs were isolated from
adult human bone marrow, dental pulp, adipose and
dermal tissues after the patient informed consent in ap-
pliance with local Ethical guidelines. MSCs were seeded
into alginate microbeads (AMB) and wide-porous scaf-
folds prepared on the base of alginate, gelatin, collagen,
demineralized chitinous skeletons of marine sponges
and dental osteoplastic composite materials. MSCs vi-
ability, proliferation and capacity to differentiation toward
adipogenic and osteogenic lineages were studied during
3-D culturing.
Results and discussion. MSCs isolated from adult
human bone marrow, dermal and adipose tissues after
expansion in monolayer culture had similar phenotype
and growth patterns as well as ability to induced multilin-
eage differentiation. After encapsulation in AMB, MSCs
had spherical shape and during 3-D culture they stopped
to proliferate, but possessed viability and capability to dif-
ferentiation toward adipogenic and osteogenic lineages.
After seeding in 3-D wide-porous scaffolds, cells attached
to porous surface, proliferated and filled pores during
culturing. In the presence of definite inducers, MSCs
were able to multilineage differentiation. The efficiency
of osteogenic and adipogenic differentiation depended
on scaffold type and composition.
Some approaches for storage, transportation and
cryopreservation of MSC-based tissue-engineered con-
structs are presented and discussed.
Conclusions. Varying the properties of MSCs and
scaffolds made it possible to generate different tissue
engineered constructs that can be applied for correction
of a wide range of tissue defects.
EXPERIMENTAL STUDY OF CELL AND TISSUE
THERAPY PROTOCOLS IN REHABILITATION AFTER
CHEMOTHERAPY-INDUCED OVARIAN FAILURE
v.Yu. Prokopіuk1, M.M. Kozub2, K.P. Skibina3,
o.v. Prokopіuk2
1Institute for Problems of Cryobiology
and Cryomedicine, National Academy of Sciences
of Ukraine, Kharkiv, Ukraine
2Kharkiv Medical Academy of Postgraduate Education,
Kharkiv, Ukraine
3Kharkiv National Medical University, Kharkiv, Ukraine
v.yu.prokopiuk@gmail.com
Introduction. Premature ovarian failure is observed
in 1% of women under 40 and 0.1% under 30 years;
among those 37% cases are due to iatrogenic causes
(mainly after cancer treatment). Problem of rehabilita-
tion after chemotherapy is more important for women,
because chemotherapy often leads not only to hepatic
and renal dysfunction, but to severe menopause, psycho-
emotional, sexual, disorders, cardiovascular diseases
(A.J. Fenton et al., 2015). In recent years, cell and tissue
therapy is considered as an alternative method of treat-
ment of iatrogenic menopausal disorders and premature
ovarian failure. The human placenta is a promising source
of stem cells and biologically active substances capable
of affecting the female organism. For reasons of biosafety,
only cryopreserved material is possible to use for clinical
application (O.S. Prokopyuk et al., 2017).
Aim of this study was to compare different methods
of tissue and cell therapy in the rehabilitation and recovery
after chemotherapy in female mice.
Materials and Methods. The research was per-
formed on BALB/c mice model of ovarian insufficiency
induced by cyclophosphamide and busulfan (G.Y. Xiao
et al., 2014). The dynamics of ovarian, sexual function,
morphology of liver and kidney, the general state of the
animals was studied after application of cryopreserved
Experimental Oncology 39, 234–256, 2017 (September) 251
explants, extract, placental MSCs, adipose tissue
MSCs obtained by the previously developed methods
(D. Pogozhykh et al., 2015).
Results and discussion. In the chemotherapy group
without rehabilitation, loss of weight, absence of regular
changes in vaginal cytology, atrophy of the ovaries and
uterus, cirrhotic changes in the liver, a sharp edema of the
cortical and medullar layers іn the kidneys was observed.
The most rapid and complete recovery of the reproduc-
tive system, liver and kidney was noticed using placental
explants, MSCs or extract. Application of adipose tis-
sue MSCs led to slower recovery, but prevented critical
changes (cirrhotic changes in the liver, kidney tubular
necrosis). Recovery of morphology of liver and kidney,
regular changes in vaginal cytology, sexual function, but
not fertility was observed since follicles were lost in this
model. Sensitivity to chemotherapy and treatment in some
mice depended on the individual characteristics of the
organism.
Conclusions. It was shown that the different meth-
ods of cell and tissue therapy are comparably efficient
in the rehabilitation after chemotherapy and treatment
of chemotherapy-induced ovarian failure. The applica-
tion of placental preparations leads to faster and more
complete recovery of the female reproductive system.
ARE CD44+/CD24– CELLS THE ASSUMED CANCER
STEM CELLS IN BREAST CANCER?
d.e. Ryspayeva1, I.I. Smolanka1,
A.S. dudnichenko2, A.A. Lyashenko1,
Yu.A. Grinevich1, v.G. Gurianov3,
M.v. Koshubarova1, A.A. Seleznev4
1National Cancer Institute, Kyiv, Ukraine
2Medical Academy of Postgraduate Education, Kharkiv,
Ukraine
3O.O. Bogomolets National Medical University, Kyiv,
Ukraine
4CSD Health Care Laboratory, Kyiv, Ukraine
ryspayeva1@gmail.com
Introduction. Identification and characterization
of the population of cancer stem cells (CSC) depends
on several cellular markers, which combination is specific
for the phenotype of CSC in the corresponding tumor.
Several markers of CSC have already been identified
in breast cancer (BC), but there are no universal indicators
that could specifically identify the CSC in BC.
Aim. To determine the validation of the CSC model for
cell surface markers such as CD44 and CD24 and their
clinical significance.
Materials and Methods. Primary tumor samples
of 45 patients with invasive BC without chemotherapy
prior to surgery were examined in paraffin blocks.
CD44 and CD24 expression was evaluated by the per-
centage of positive cells using different chromogens and
the MultiVision detection system (Thermo Scientific, USA)
by immunohistochemical (IHC) method. In this research
the evaluation was determined by the following criteria:
(−), negative — expression in < 10% of tumor cells; (+),
positive — expression in ≥ 10% of cells. The same scoring
system was applied for the expression of CD44+/CD24−.
Results and discussion. 62.2% of investigated
patients were older than 50 years and most of them had
stage II of the disease (71%) and luminal tumor subtypes
(68.9%). We analysed the expression of CD44 and CD24,
CD44+/CD24− for different patients by dividing them into
two groups. The group A consists of the patients with
unfavorable prognosis (relapses and metastases have
occurred in the first three years after diagnosis), and the
group B — with a favourable prognosis (the development
of metastases after three years). Median disease-free
survival in the group A is 19 months, in the group B —
46 months. The difference between the overall survival
(OS) curves in the groups A and B is statistically significant
(p < 0.001), the risk of death was higher in the group A
[HR 5.9; CI 2.3–15.2].
The content of CD44 cells did not differ statistically be-
tween groups A and B (p = 0.18), but there was a tendency
for increasing in OS with the existence of CD44+ cells (p =
0.056). The distribution of the expression of CD24 marker
did not differ between the groups (p = 0.36) as well as the
OS curves (p = 0.59).
Analysis of the expression of CD44+/CD24-, which
were considered as possible CSC, revealed a paradoxical
increase (p = 0.03) in the frequency in patients of the group
B (40.9%) compared to the group A (8.7%). Nevertheless,
the comparison of the clinical outcomes did not reveal
a statistically significant difference in the survival curves
in the groups with existence and absence of CD44+/CD24−
expression (p = 0.08). The analysis showed the increase
of the risk of worse clinical outcomes in the cases of ab-
sence of CD44+/CD24− expression [HR 2.8 CI 1.1–6.8].
Conclusions. As a result of our research, the analysis
of the percentage of assumed stem cells of the BC, which
were identified by IHC as CD44 and CD24 cells, failed
to detect a statistically significant relation between groups
of patients with different prognosis, and the identification
of their expression is not enough for the characteristics
of CSC.
The obtained data demonstrating the worst clinical
outcome in the cases of absence of CD44+/CD24− ex-
pression apparently require further investigations and
the validation of the IHC method with the determination
of the cut-off line in defining of CD44 and CD24 status.
EFFECT OF THE OLFACTORY BULB NEUROGENIC
STEM CELLS TRANSPLANTATION ON THE MOTOR
FUNCTION IN AN EXPERIMENTAL SPINAL CORD
INjURY
v.M. Semenova1, v.v. Medvedev 2, L.d. Liubich1,
Yu.Ya. Yaminsky1, L.P. Stayno1
1SI “A.P. Romodanov Neurosurgery Institute,
National Academy of Medical Sciences of Ukraine”,
Kyiv, Ukraine
2O.O. Bogomolets National Medical University, Kyiv,
Ukraine
seveme22@rumbler.ru
Introduction. Transplantation of neurogenic stem
cells is increasingly used in the treatment of a number
of degenerative diseases of the central nervous sys-
tem. Neurogenic cells of the olfactory bulb of humans
and mammals possess stem properties: when cultured
in a nutrient medium with mitogens they form neuro-
spheres, and in the presence of retinoic acid they dif-
ferentiate into neurocytes and gliocytes.
Aim. To study the effect of transplantation of olfactory
bulb cells (TOBC) on the motor activity of rats in experi-
mental spinal cord trauma.
Materials and Methods. Experimental groups of ani-
mals (white outbred male rats) included: 1) modeling
of the left-sided half-intersection (LHI) of the spinal cord
in the lower thoracic region (n = 24); 2) and 3) — TOBC
in the tissue of the spinal cord below the LHI at 7 (n = 4)
252 Experimental Oncology 39, 234–256, 2017 (September)
and 13 (n = 4) weeks after injury. Cells from the rat olfac-
tory bulb were cultured in the presence of growth factors
hEGF and hFGF for 10 days. Estimation of motor activity
of experimental animals in comparison with control ani-
mals was taken into account according to D.M. Basso,
M.S. Beattie and J.C. Bresnahan (BBB) scale and param-
eters of electroneuromyography (gastrocnemius of the
posterior ipsilateral limb) with direct stimulation of the
spinal cord above the LHI.
Results. TOBC at 7 weeks after modeling of LHI in rats
causes an increase in the rate of the hind limbs function
growth. After TOBC the magnitude of the maximum am-
plitude of the M-response of the muscle being studied
was significantly lower than that in group 1 compared
to group 2.
Conclusions. TOBC in the long-term post-traumatic
process provides a positive functional effect against the
background of a decrease in electrical activity in the ef-
ferent parts of the motor system of the spinal cord, which
reflects the antispastic potential of the method.
ABERRANT PLACENTAL ALKYLINE
PHOSPHATASE (ALPP) IS THE BIOMARKER
OF MYELOID LEUKEMIC STEM CELL NICHE
MICROENVIRONMENT
L.P. Shvachko, I.v. Kravchuk, G.d. Telegeev
Institute of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine, Kyiv, Ukraine
l.shvachko@ukr.net
Human ALP is classified into four isoforms: intestinal
(ALPI), placental (ALPP), germinal (ALPG) and tissue non-
specific (bone/kidney/liver) (TNAP). Intestinal, placental
and germinal ALPs have higher homology between them-
selves than TNAP. The first three ALP genes are located
on chromosome 2q34–37, in contrast the TNAP gene
that is located on chromosome 1p36.1–34. It is referred
that alkaline phosphatase is considered a non-specific
tumor-related marker. However, the origin and nature
of tumor-related alkaline phosphatase is not clear.
We have focused on the role of ALP in chronic myeloid
leukemia (CML) progression.
Hematopoietic stem cells (HSCs) possess the ability
to self-renew and to differentiate to mature progeny along
multiple different hematopoietic lineages. The function
of HSCs depends upon the signals from surrounding cells
found within the highly specialized microenvironment
termed the hematopoietic stem cell niche. HSC niche was
first conceptualized by Schofield in 1978 and was defined
as “an entity in which the stem cell’s maturation is pre-
vented and the properties of ‘stemness’ are preserved”.
In addition, we suggest that hematopoiesis taking place
in the bone marrow is orchestrated by HSC niche. Under-
standing and exploiting the HSC niche is a goal of basic
research of the hematological malignancies. Osteocytes
in bone marrow niche are essential for granulocyte
colony-stimulating factor (G-CSF), which is the major
cytokine regulator of G-CSF-induced hematopoietic
stem/progenitor mobilization (N. Asadaet et al., 2011).
Myelopoiesis is regulated by HSC niche’s osteoblast/
osteocytes signaling through HSCs granulocyte colony-
stimulate signaling (K. Fulzele et al., 2012). ALP activity
was detected widely in osteoblasts of HSC niche in the
bone marrow, about 20% of bone marrow cells expressed
ALP activity (D. Miao, A. Scutt, 2002). Taken together,
ALP is secreted by bone marrow HSC niche osteoblasts
and has role in regulation of myelopoiesis through G-
CSF signaling. As a result, the niche-associated ALP
gene expression was absent in blood cells of healthy
persons as we have shown. In addition to this, normal
HSC is marked by G-CSFR (granulocyte colony-stimulate
factor receptor), while leukemic hematopoietic cells lose
this marker. Unexpectedly, we have observed the aberrant
placental ALP gene expression in the peripheral blood
cells in CML patients (n = 8). The aberrant placental ALP
expression was investigated by real-time RT-PCR with
consequent amplicone-DNA sequencing analysis. We first
found the phenomenon of the phenotypic molecular
switching from normal TNAP to ALPP in the hematopoi-
etic niche induction during leukemogenesis. We have
proposed that aberrant ALPP highlights the leukemic
stem cell niche feature in CML progression. Moreover,
we have firstly elucidated the epigenetic reverse regula-
tion of aberrant ALPP gene expression in CML patients
by histone deacetylase inhibitor (HDACi) sodium butyrate
in culture of blood cells. In addition, the targeted increase
of G-CSFR mRNA expression has been shown. In con-
clusion, we have suggested that aberrant placental ALP
is the leukemogenic-derived alkaline phosphatase that
drives HSC plasticity in leukemic stem cell survival. Based
on the date obtained, we discuss targeting potentially
capable of epigenetic reversion of the leukemic stem
cell phenotype.
AUTOSELECTION PHENOMENON IN THE NORMAL
STEM CELLS: FROM POPULATION
HETEROGENEITY TO CANCER PHENOTYPE
G.I. Solyanik
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
gsolyanik@gmail.com
Introduction. The development and functioning
of a multicellular organism is impossible without regulation
of cell division and cell differentiation processes. It is just
their mutual regulation, on the one hand, causes step-by-
step narrowing of cell potentiality during embryogenesis
and, on the other hand, maintains the renewal of cellular
composition and spatial arrangement of tissues during the
reparation processes in adult organism. Disturbances in the
above regulation are then considered to be responsible for
a number of pathological processes in organism including
carcinogenesis.
At present there is no consensus on the mechanisms
of the regulation of cell division/differentiation processes.
However, none of the existing concepts are capable
of giving a complete solution of the regulation problem
till they do not touch the question of interdependence
between the proliferation and differentiation processes
in the heterogeneous populations of normal stem cells.
Aim. The purpose of the present study is to show
that in the heterogeneous population of stem cells the
mechanism of proliferation — differentiation processes
based on the inhibition of the proliferating stem cells
by differentiation factors (special messengers synthe-
sized by the differentiated cells) can result in the selec-
tion of actively proliferating cells that are insensitive
to the action of differentiation factors.
Methods. The problem of regulation of the prolifera-
tion — differentiation processes is analyzed in the frame-
work of the mathematical model of unipotential stem cell
clone dynamics, assuming two alternative scenarios
of an every new-born cell development, which in the long
Experimental Oncology 39, 234–256, 2017 (September) 253
run define correspondingly the proliferating or differenti-
ated cell status. The scenario choice is then supposed
to be a random event influenced by both the inherent
mitotic activity of stem cell and the differentiation factors.
Results and discussion. Taking into account the
heterogeneity of stem cells it was shown that such
mechanism of the regulation of proliferation — differen-
tiation processes can result in the selection of actively
proliferating and cells that are insensitive to the action
of differentiation factors (that is to say, the cells with
cancer phenotype). We called this phenomenon an au-
toselection. In the framework of the model, the low level
of the intraclonal heterogeneity of the mitotic activity
index of stem cells is necessary but not sufficient for the
emergence of the cells with cancer phenotype. Another
important condition, which is necessary for the appear-
ance of such cells in the clone, is the increase of the
interclonal heterogeneity of stem cells.
Conclusions. The stem cells with cancer phenotype
appears through the series of the permanent nonmuta-
tional changes by autoselection mechanism retaining
for the division and generation of the progeny only that
actively proliferating cells which are less sensitive with
respect to the action of the differentiation factors. The
progression of normal stem cells towards the appear-
ance and increase of the number of the cells with cancer
phenotype can be considered as a possible mechanism
of carcinogenesis.
MODIFYING EFFECT OF EXOGENOUS
LACTOFERRIN ON THE HUMAN BREAST CANCER
CELL LINES wITH PHENOTYPE OF CANCER STEM
CELLS
d.M. Storchai1, n.Y. Lukianova1, I.v. Zalutski2,
v.S. Lukashevich2, v.F. Chekhun1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
2State Scientific Institution “Institute of Physiology”,
National Academy of Sciences of Belarus, Minsk,
Republic of Belarus
chekhun@onconet.kiev.ua
Introduction. Cancer stem cells (CSCs) today are the
subject of intensive research in experimental and clinical
oncology. Their presence in the tumor area is associ-
ated with aggressive course and resistance to therapy.
Currently, the search and development of new therapy
approaches for the treatment of malignant tumors with
CSCs is conducted worldwide. Experimental studies
have shown that higher antitumor activity have natural
antioxidant proteins, including lactoferrin (LF), compared
to synthetic analogs. LF is characterized by a unique set
of biological properties of a different nature. At the same
time, the peculiarities of the LF influence on breast cancer
(BC) cells with the CSC phenotype have not been finally
determined.
Aim. To investigate the cytotoxic and genotoxic ef-
fects of exogenous LF on hormone receptor-positive and
receptor-negative human BC lines with CSC phenotype.
Materials and Methods. The hormone receptor-
positive (MCF-7) and receptor-negative (MDA-MB-231)
human BC cell lines, positive for CSC markers, were
cultured with an exogenous LF at a dose corresponding
to IC30 (10 µg/ml for receptor-positive cells, 5 µg/ml for
receptor-negative cells). Flow cytometry, immunocyto-
chemical, and statistical methods were used.
Results. Cultivation with exogenous LF resulted
in reducing the number of cells with the CSC-like phe-
notype both in hormone-receptor-positive and receptor-
negative BC cell lines. This is evidenced by a reduction
in the number of cells positive for CD44 expression,
in MCF-7 by 55% and the 4.5-fold increase of CD24 ex-
pression in MDA-MB-231cells. We have found that after
incubation with LF, MCF-7 and MDA-MB-231 cells dem-
onstrated increased levels of reactive oxygen species
(2.0–1.7-fold, p < 0.05), inducible NO generation rate
of NO-synthase (1.6–1.9-fold, p < 0.05) and the level of
“free iron” (1.7–2.3-fold, p < 0.05). In addition, the effects
of LF were more pronounced in MDA-MB-231 receptor-
negative cell line. These changes resulted in the increased
expression of proapoptotic protein Bax (by 10–16%,
p < 0.05), reduced expression of the antiapoptotic protein
Bcl-2 (16–21%, p < 0.05) and lowering level of unoxidized
mitochondrial phospholipid cardiolipin (by 1.6–2.4 times,
p < 0.05). This, in turn, caused an increase in the number
of cells killed by apoptosis (14–29.1%, p < 0.05). Cy-
totoxic effects of LF were accompanied by an increase
in the percentage of DNA in “comet tail” (by 1.8–4 times,
p < 0.05) and the blockage of cell cycle at G2/M phase,
especially in receptor-negative cell line.
Conclusions. The obtained data indicate that ex-
ogenous LF shifts pro-/antioxidant balance of hormone
in receptor-positive and receptor-negative human
BC cells with the CSC phenotype by increasing the
level of reactive oxygen species, “free iron” and the rate
of NO generation. These disruptions lead to the blockage
of the cell cycle in the G2/M phase and the death of malig-
nant cells by apoptosis. Further study of the mechanisms
of LF influence will justify the need for its use to modify
the phenotype of cells positive for the expression of CSC
markers.
PROTEOME RESPONSE OF DPSCS
TO TREATMENT wITH EXOGENOUS FGF8
R. Tsikandelova1, P. Mladenov2, Z. Mihaylova3,
v. Mitev1, n. Ishkitiev1
1Department of Medical Chemistry and Biochemistry,
Medical University-Sofia, Bulgaria
2Agrobioinstitute, Bulgarian Academy of Sciences
Sofia, Bulgaria
3Department of Oral and Maxillofacial Surgery, Medical
University-Sofia, Bulgaria
mstsikandelova@gmail.com
Introduction. Human dental pulp is a rich source
of postnatal mesenchymal stem cells which can self-
renew and undergo differentiation into multiple lineages.
FGF8 is a paracrine-acting FGF family ligand, expressed
during the earliest stages of tooth site determination and
indispensable for mediating epithelial-to-mesenchymal
interactions initiating odontogenic differentiation during
embryogenesis.
Aim. To examine differential protein analysis in adult hu-
man dental pulp in response to short- and long- term treat-
ment with FGF8 in order to assess its value as a the rapeutic
agent for the purposes of pulp regeneration.
Materials and Methods. Intact human premolar
teeth were obtained from healthy patients between the
ages of 18 and 40 following the acquisition of written
consent. Dental pulps were exposed using diamond burs
and extracted using sterile barbed broaches. Tissue ex-
plants were enzymatically digested with collagenase type
I (3 mg/ml) and disease (4 mg/ml) for 1 h at 37 °C and
254 Experimental Oncology 39, 234–256, 2017 (September)
cultured in DMEM with added 20% heat-inactivated fetal
bovine serum (FBS). Hetero genous cell culture was main-
tained at standard culture conditions with supplemented
10% FBS. Upon reaching the third passage, samples run
in triplicates were subjected to short- (24 h) and long-term
(10 days) treatment with 10 ng/ml human recombinant
FGF8. 2D DIGE analysis was conducted using 24-cm strips
in the narrow 4–7 pH range for the first dimension and
SDS-PAGE (8–16%) for the second. Protein spots were
subsequently identified by MALDI-TOF/TOF-MS.
Results and discussion. 20 protein spots identified
by MALDI-TOF/TOF-MS with high confidence were found
to be differentially expressed among treatment groups
as compared to untreated controls. These belonged
to clusters of gluconeogenesis (tri osephosphate isomer-
ase), cellular structure and motility, inflammation, protein
biosynthesis, trafficking and degradation, cell cycle and
DNA repair, osteogenic differentiation, cytokinesis and
vesicle trafficking.
Conclusions. In summary, the proteomic ap-
proach has yielded novel information about the effect
of FGF8 on cells of adult dental pulp with identified proteins
being linked to regulation of actin cytoskeletal rearrange-
ments, migration, osteogenic differentiation, neuronal cell
function, consistent with what has been reported about the
role of FGF8 in the context of embryonic tooth development
and the neural crest origin of dental pulp cells. FGF8 is likely
to be able to instigate pulp cell migration and odontogenic
differentiation in response to pulp injury in an appropriate
model organism, however further studies need to be con-
ducted to evaluate its therapeutic potential.
ADULT NEURAL CREST-DERIVED MULTIPOTENT
STEM CELLS FROM HAIR FOLLICLE: PHENOTYPE,
FUNCTIONAL PROPERTIES AND PROSPECTS
FOR USE IN REGENERATIVE MEDICINE
R.G. vasyliev1, 2, v.F. Grytsyk2,
A.e. Rodnichenko1, 2, О.S. Gubar2, 3,
A.v. Zlatska1, 2, I.M. Gordiienko2, 4, d.О. Zubov1, 2
1State Institute of Genetic and Regenerative Medicine,
National Academy of Medical Sciences of Ukraine,
Kyiv, Ukraine
2Biotechnology Laboratory ilaya Regeneration, Kyiv,
Ukraine
3Institute of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine, Kyiv, Ukraine
4R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
rvasiliev@ukr.net
Introduction. Neural crest is a transient structure
during embryonic development of vertebrates which
gives rise to peripheral nervous system; melanocytes;
cartilage, bone and connective tissue in head; some
endocrine cells, etc. Several tissues and organs of adult
mammals contain adult neural crest-derived multipotent
stem cells (NC-MSCs) and they have great potential
to use in regenerative medicine.
Aim. To obtain the NC-MSCs from human hair fol-
licle, characterize their morphological and functional
properties; based on our positive results in animal models
to conduct pilot human studies for treatment of spinal
cord injuries (SCI), degeneration of intervertebral disks
(IVD) and calvarial bone defects.
Materials and Methods. All procedures were per-
formed with the voluntary informed consent of patients and
in accordance with the current legislation of Ukraine. NC-
MSCs were obtained from hair follicle by explant method
and expanded by our original protocol. Cultured NC-MSCs
were characterized by qPCR, immunocytochemistry, flow
cytometry, cytogenetic and functional assays. Cytokines
and growth factors produced by NC-MSCs were determined
by ELISA and Bio-Rad multiplex assay. Patients subjected
to non-effective standard drug therapies, surgery approaches
or neuro-rehabilitation have received the cell therapy treat-
ment or transplantation tissue-engineered (TE) bone equiva-
lent. Three patients with contusion SCI received single com-
bined paravertebral and intrathecal injections (20•106 cells
per injection/patient). Ten patients with IVD degeneration,
herniation and pain syndrome received only paravertebral
cell injection (10•106 cells per patient). Three patients with
critical sized calvarial bone defects had the transplantation
of TE bone equivalent. Minimal follow-up period was 1 year.
Results and discussion. Adult NC-MSCs were
successfully isolated and expanded from all patients.
NC-MSCs have stable karyotype, Sox2+ Sox10+ Nestin+
CD73+ CD90+ CD105+ CD140a+ CD140b+ CD146+ CD166+
CD271+ CD349+ CD34- CD45- CD56- HLA-DR- phenotype
and were able to self-renewal and directed differentia-
tion in vitro into neurons, Schwann cells, melanocytes,
osteoblasts, chondrocytes and adipocytes. NC-MSCs
secreted the following proteins: NGF, BDNF, NT-3, NT-
4/5, IL-1ra, IL-10, bFGF, VEGF, and GM-CSF. Two patients
with lumbar SCI caused by shrapnel improved from ASIA
C to ASIA D after cell therapy. One patient with thoracic
SCI caused by bullet improved from ASIA B to ASIA C af-
ter treatment. Treated patients showed the restoration
of sensitivity as well as the emergence of active move-
ments and strength grown in paretic limbs. Ten patients
with IVD degeneration showed a significant improvement
in pain syndrome and reduction in size of a protrusion/
extrusion. In three patients, the calvarial bone defects
were successfully restored with TE bone equivalent.
Conclusion. NC-MSCs can be efficiently obtained
and expanded from adult human hair follicle. Our pre-
liminary results have shown significant therapeutic and
regenerative potential of this cell type.
TISSUE-ENGINEERED BONE FOR TREATMENT
OF COMBAT RELATED LIMB INjURIES
R.G. vasyliev1, 2, v.M. oksymets2,
A.e. Rodnichenko1, 2, A.v. Zlatska1, 2, О.S. Gubar2, 3,
d.О. Zubov1, 2
1State Institute of Genetic and Regenerative
Medicine, National Academy of Medical Sciences
of Ukraine, Kyiv, Ukraine
2Medical Company Ilaya®, Kyiv, Ukraine
3Institute of Molecular Biology and Genetics,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
zoubov77@yahoo.com
Introduction. Combat-related bone defects are
manifested in 10–12% of casualties, and 30% of these
defects are complicated with osteomyelitis. The greatest
difficulties arise in the treatment of diaphyseal bone de-
fects more than 5 cm and metaepiphyseal defects that ex-
ceed 15–20 cm3. Treatment of critical sized bone defects
is an actual clinical challenge. The “gold standard” in this
case is autologous bone grafting. But disadvantage of this
method is associated with limited donor bone resources.
Aim. Based on our preliminary positive clinical results
with use of cultured bone marrow-derived multipotent
mesenchymal stem/stromal cells (BM-MSCs) in trau-
Experimental Oncology 39, 234–256, 2017 (September) 255
matology, our aim was to develop 3D tissue-engineered
bone equivalent (3D-TEBE) transplantation technology
for restoration of critical sized bone defects.
Materials and Methods. To fabricate 3D-TEBE
we used devitalized allogeneic bone (blocks and chips)
seeded with cultured autologous cells: BM-MSCs in mix
with periosteum progenitor cells (PPCs) and endothelial
progenitor cells (EPCs). Quality/identity of cell cultures was
assured by flow cytometry (cell phenotype), cytogenetic
analysis (GTG-banding), donor and cell cultures infection
screening (IFA, PCR), functional analysis (cell kinetics, CFU
analysis, multilineage differentiation assay, cell senescence
assay). 3D-TEBE transplantation was performed in 47 com-
bat-injured with 49 bone defects. New bone formation
was assessed by the radiographic examination. Treatment
was based on local clinical protocol approved by Ministry
of Health of Ukraine.
Results and discussion. Patients were included
in a treatment program 8–19 months after injury, provid-
ed the ineffectiveness of conventional surgery methods.
All cell cultures had a normal karyotype and phenotype,
differentiation potential and functional properties, 30%
CFU frequency and hadn’t any signs of cell senescence.
FDA/PI staining, MTT-assay and histological analysis
of 3D-TEBE samples showed their regular seeding with
viable cells. Histological analysis of 3D-TEBE biopsies
3 months after transplantation taken within adoptive
resection surgery showed immature bone tissue forma-
tion. Restoration of all bone defects was observed after
5–6 months (by radiographic examination).
Conclusions. The developed biotechnology of 3D-
TEBE transplantation allows restoring the bone integrity,
forming new bone tissue in a site of bone defect, and
significantly reducing the rehabilitation period of a patient.
RESULTS OF MONITORING GENETIC STABILITY
OF MESENCHYMAL STEM CELLS IN THE
SMARTCELL BIOTECHNOLOGY COMPANY
e.n. Yaremenko
The SmartCell Biotechnology Company, Odessa,
Ukraine
work.en@ukr.net
Introduction. Due to the active development of cellular
technologies, the study of the genetic stability of human
stem cells (SCs) is receiving increasing attention. Since the
amount of SCs isolated from the body is too small to be used
for medicinal purposes, they should be expanded in culture.
In the course of culturing, de novo-induced chromosomal
abnormalities are possible, giving rise to abnormal cell lines.
Accumulation of such abnormalities can lead to malignant
transformation of cells.
Aim. To assess the chromosomal variability of mesen-
chymal stem cells (MSCs), depending on the source of cell
production and the duration of their cultivation.
Materials and Methods. MSCs were isolated from
bone marrow and adipose tissue. Their genetic stability was
evaluated by the method of karyotyping using specialized
software. G-staining in metaphase plates was provided for
cytogenetic analysis.
Results. We conducted a cytogenetic analysis
of a number of MSC cultures originating from bone mar-
row and adipose tissue starting from the 2nd and ending
with the 10th passage. In all samples under study, neither
metaphase plates with an altered chromosome number
nor structural changes in the chromosomes were detected
upon cell culture.
Conclusions. The results of our studies showed that the
methods used for MSC isolation, as well as the conditions
for their cultivation, preserve the normal karyotype of the
cells at the early passages.
MOLECULAR PROFILE FEATURES OF PROSTATE
CANCER CELLS wITH THE CD44+/CD24–
PHENOTYPE
T.v. Zadvornyi, n.Yu. Lukianova, v.F. Chekhun
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv, Ukraine
tito132007@ukr.net
Introduction. The clinical significance of the molecular
heterogeneity of prostate cancer (PC) is actively discussed
in recent publications due to the important role of cancer
stem cells (CSC) in the disease course. The presence
of these cells is a determining factor for the growth and
progression of the tumor, its metastatic activity, and also
sensitivity to therapy. In this regard, the study of the mo-
lecular profile of cells with the CSC phenotype is an urgent
issue, the solution of which will help to deepen knowledge
about the biology of tumor growth.
Aim. To investigate the features of the molecular pro-
file of PC cell lines, depending on the expression of CSC
markers (СD24 and СD44).
Materials and Methods. The study was conducted
on human PC cell lines (DU-145 and LNCaP). Expression
of CSC markers (CD24 and CD44), steroid hormones re-
ceptors (testosterone (AR), estrogen α (ERα), progesterone
(PR) and epidermal growth factor — (HER2/neu)), E-cad-
herin and Kі-67 was determined by the immunocytochemi-
cal method. The evaluation of the results was performed
using optical microscopy by H-Score method. The marker
expression levels were considered to be high at the values
from 201 to 300 points of the H-Score, average — from
101 to 200 points of the H-Score, and low at the range from
0 to 100 points of the H-Score. The invasive activity of the
cells was examined using a standard invasive test accord-
ing to the manufacturer’s instructions (BD Biociences).
STATISTICA 6.0 software was used to process the results.
Results. The study of CD24 and CD44 expression
demonstrated that the PC cell lines differ in expression
of CSC markers. DU-145 cells were positive for the CSC
phenotype (CD44+/CD24–), while LNCaP cells had low
levels of CD44 (65 ± 12.3 points of the H-Score) and did
not express CD24. We established that DU-145 cells with
CSC phenotype exhibit a high level of testosterone recep-
tor (250 ± 14.6 points of H-Score), low level of HER2/neu
(60.3 ± 14.6 points of H-Score), and have lack of ER, PR,
and E-cadherin expression.
In the cells of LNCaP line, negative for the CSC pheno-
type, we observed a high level of testosterone expression
(272.6 ± 10.3 points of the H-Score), the average PR ex-
pression (145.7 ± 11.0 points of the H-Score), the absence
of HER2/neu, and the average level of E-cadherin (187.7 ±
16.0 points of the H-Score).
Cells with the CD44+/CD24– phenotype of DU-145 cell
line were characterized by a high proliferative (Ki-67 le-
vels considered to be 265.0 ± 20.5 points of the H-Score)
and invasive activity (0.37/1000 cells) compared to the
LNCaP cells (Ki-67 level amounted 174.0 ± 24.6 points
for H-Score, the invasive activity was 0.076/1000 cells,
respectively).
Conclusions. The association of the presence of CSC
markers with a high proliferative and invasive activity
256 Experimental Oncology 39, 234–256, 2017 (September)
and a decrease in the adhesion properties of PC cells
has been established. The findings point to the need for
a more in-depth study of the role of CSC in the biology
of cell growth.
ENDOMETRIAL STROMAL CELLS:
ISOLATION, EXPANSION, MORPHOLOGICAL
AND FUNCTIONAL PROPERTIES
A.v. Zlatska1, 2, A.e. Rodnichenko1, 2, О.S.Gubar2, 3,
d.О. Zubov1, 2, S.n. novikova1, R.G. vasyliev1, 2
1State Institute of Genetic and Regenerative Medicine,
National Academy of Medical Sciences of Ukraine,
Kyiv, Ukraine
2Biotechnology Laboratory ilaya Regeneration, Kyiv,
Ukraine
3Institute of Molecular Biology and Genetics,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
alenazlatcka@gmail.com
Introduction. The endometrium is a unique struc-
ture that is able to complete self-renewal over a month
cycle, and undergoes these changes over 400 times
during woman reproductive age. A significant regenera-
tive potential is due to the presence of stem cells in the
endometrium, such as mesenchymal, epithelial and
endothelial progenitor cells. That is why endometrium
is a promising object for MSCs isolation and in vitro
short-term expansion for their further exploration and use
in assisted reproductive technologies, reproductology
and regenerative medicine.
Aim. To study the biological properties of human
endometrial stromal cells in vitro.
Materials and Methods. The endometrium samples
(n = 10) were obtained by biopsy at the first phase of the
menstrual cycle from women with endometrial hypopla-
sia. In all cases, a voluntary written informed consent was
obtained from the patients. Endometrial fragments were
dissociated by enzymatic treatment for 1 h in 0.05% col-
lagenase IA and 0.05% pronase and 2% FBS. The cells
were cultured in DMEM:F12 supplemented with 10% FBS,
2 мМ L-glutamine and 1 ng/ml FGF-2 in a multi-gas incu-
bator at 5% CO2 and 5% O2. For the phenotype determi-
nation, the ability to directed adipogenic, osteogenic and
chondrogenic differentiation assays, CFU-test, karyotype
stability, the P3-cells have been used. The cell secretome
was assessed by BioRad Multiplex immunoassay kit.
Results and discussion. Primary population of en-
dometrial cells was heterogeneous and contained cells
with fibroblast-like and epithelial-like morphology, but
at P3 the majority of cell population had fibroblast-like
morphology. The cells possessed MSCs typical pheno-
type CD90+CD105+CD73+CD34-CD45-HLA-DR- assessed
by flow cytometry. Positive expression of CD146+CD166
+CD49f+CD140a+CD140b+nestin+ was determined. Cell
doubling time is 29.6 ± 1.3 h. They were capable of di-
rect osteogenic and adipogenic differentiation. The cells
showed 35.7% colony forming efficiency and a tendency
to 3D spheroid formation. The GTG-banding assay con-
firmed the stability of eMSC karyotype during long-term
culturing (up to P8). After 48 h incubation period in serum-
free medium eMSC secreted anti-inflammatory IL-1ra
(74.6 ± 9.5 pg/ml), as well as IL-6 (29.8 ± 8.3 pg/ml),
IL-8 (138.5 ± 33.3 pg/ml) and IFNγ (55.9 ± 3.8 pg/ml),
angiogenic factors VEGF (92.2 ± 19.8 pg/ml), GM-CSF
(133.2 ± 5.1 pg/ml) and FGF-2 (17.8 ± 4.3 pg/ml), che-
mokines IP-10 (39.9 ± 3.3 pg/ml) and MCP-1 (41.1 ±
6.7 pg/ml).
Conclusion. Thus, obtained endometrial stromal
cells meet minimal ISCT criteria for MSCs, such as adher-
ence to plastic in standard culture conditions, expression
of typical phenotype markers and ability for the directed
differentiation in vitro. eMSCs are stable during long-
term culturing. They also produce a range of cytokines,
chemokines and growth factors. Proliferative potential,
karyotype stability and secretome profile make them
a perspective object for the use in the regenerative
medicine.
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