Haematological malignancies represent approximately 7 of all malignant [601205]

Abstract
Background
Haematological malignancies represent approximately 7% of all malignant
diseases [1]. Acute myeloid leukaemia (AML) is an aggressive and fatal disease.
AML treatment basically remained unimproved in the last 20 years and consists
of induction of cytotoxic chemotherapy. An average less than 30% of AML
patients survive for long-term. Mesenchymal stem cells (MSCs) are currently
being investigated for an ever-expanding number of clinical indications based on
their tissue-regenerative, immunomodulatory and anti-inflammatory effects.
Aim
The aim of the present work is to detect the immunomodulatory effect of MSCs
on the AML.
Materials and methods
The samples of the MSCs were separated from human umbilical cord blood
(HUCB), and co-cultured with samples of recently-diagnosed adult AML prior to
chemotherapy. Cells identification was done using flow cytometric analyses.
Leukaemia inhibitory factor gene (LIF) and interleukin-10 (IL-10) were
measured using real -time polymerase chain reaction (RT-PCR) and ELISA
techniques, respectively before and after the co-culture; aiming to evaluate the
immunomodulatory effect of the MSCs on AML.
Results
In the present study, the group of AML cells showed a significant increase in
the level of relative expression of LIF after co-culture with MSCs. As regards
IL-10 concentrations, there was a significant decrease in the IL-10 concentration
in the cultured medium for the AML group after co-culture with MSCs compared
with that of the control group.
Conclusion
Co-culture of AML with MSCs represents a simple approach to inhibit
leukaemia cells in vitro. This effect may be through the immunomodulatory and
anti-inflamatory effects of MSCs.
Key words: MSCs, AML, LIF, PCR, ELISA
Introduction
AML is the most frequent haematological malignancy in adults. Less than
50% of adult AML patients have a 5-year overall survival rate, and in the
elderly, only 20% of AML patients survive for 2 years [2].
The MSCs are undeveloped cells capable of proliferation, self-renewal,
conversion to differentiated cells and regenerating tissues. The MSCs have
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unique immunomodulatory properties. Stem cells may be potential candidat: [anonimizat] "MS-5" can block apoptosis [4].
LIF is a pluripotent cytokine with pleiotropic activities. The LIF is
characterized by its ability to stimulate the differentiation of the murine
myeloblastic leukaemia cell line "M1" [3].
Materials and methods
The present study included four groups (10 each) of cultured cells; peripheral
blood samples derived from healthy subjects (group A), peripheral blood
samples derived from patients who recently diagnosed with AML (group B),
human umbilical-cord blood samples (group C) and co-cultured cells of both
groups B and C (group D). Flow cytometric analyses were done to identify the
stem cells. LIF and IL-10 were measured using RT-PCR and ELISA
techniques, respectively before and after the co-culture; aiming to evaluate the
immunomodulatory effect of MSCs on AML.
1. Mesenchymal stem cells isolation, culture, expansion and identification
2 ml human cord blood (UCB) was diluted with 4 ml phosphate buffer saline
(PBS) with a 2:1 ratio, respectively. Mononuclear cells (MNCs) were isolated
by density gradient centrifugation at 1800xg for 30 minutes at room
temperature and re-suspended in complete culture medium (5 ml DMEM, 10%
foetal bovine serum FBS, supplemented with 100 U/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine (GIBCO/BRL). The cells were incubated
at 37° C in 5% humidified CO 2 for 12-14 days as primary culture or upon
formation of large colonies. Identification of MSCs was done by CD34 and
CD105 surface markers as human MSCs are CD34 negative and CD105
positive, as was proven by using flow cytometry analysis.
2. leukaemic samples
2 ml Peripheral blood samples were collected from each of ten patients
recently diagnosed with AML. The peripheral blood samples were diluted with
equal volume of 2% dextran solution and incubated at room temperature for 45
minutes. The supernatant was collected and centrifuged at 1.600 rpm for 10
minutes. Pellet was re-suspended in 4 ml PBS with 0.1 % EDTA. Mixture was
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transferred into 20 ml tube containing Ficoll-Paque with a 2:1 ratio,
respectively and centrifuged at 2,200 xg for 20 minutes. MNCs (buffy coat)
were thawed in human cell complete culture medium (5 ml DMEM, 10% FBS,
supplemented with 100 U penicillin, 1000 U streptomycin, and 2 mM L-
glutamine (GIBCO/BRL) [5].
3. Isolation of leukaemic cells and co-culture with MSCs
The cells from leukaemic samples were characterized by their adhesiveness
and fusiform shape. Besides, these cells are CD34 negative and CD13 positive
as was proven by using flow cytometry analysis. These cells were incubated at
37° C in 5% humidified CO 2 for 12-14 days as primary culture or upon
formation of large colonies. When large colonies developed (80-90%
confluence), the cultures were washed twice with PBS and the cells were
treated with 0.25 % trypsin in 1mM EDTA (GIBCO/BRL) for 5 minutes at 37°
C. After centrifugation (at 2400 rpm for 20 minutes), 2.5× 105 cells were co-
cultured with an equal number of irradiated MSCs and re-suspended with
serum-supplemented complete culture medium (5 ml DMEM, 10% FBS,
supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-
glutamine) and incubated in 50 cm2 culture flask (Falcon) [6].
4. Enzyme-linked immunosorbent assay (ELISA) technique.
To demonstrate IL-10 secretion by the adherent layers, 24 hours before
harvesting the confluent adherent layers were refed with the standard culture
medium and 24 hours later, the conditioned medium was pooled and stored for
later analysis. The level of IL-10 in cell culture supernatant was measured
using ‘Orgenium Laboratories IL-10 ELISA kit’. 50 µl of sample and 50 µl of
each diluted standard (starting from 125pg/ml) were added into appropriate
wells. 50 µl of Green coloured Biotinylated antibody were added to all wells
containing standards and samples (total reaction volume is 100 µl). 100 µl of
prepared streptavidin-HRP solution were added to each well and incubated for
30 minutes at room temperature. 50 µl of TMB were added to each well, and
incubated in the dark for 20 minutes at room temperature. 25 µl of stop
solution were added to each well, and the plate was read at 450 nm within 15
minutes. The results of the unknown samples were calculated against the
standard.
5.Polymerase chain reaction (PCR)
A) RNA extraction and cDNA synthesis:
Total RNA was extracted from cultured cells using extraction kits (Promega,
Madison, WI, USA) SV Total RNA Isolation System. The amount of RNA was
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measured spectrophotometrically. The RNA integrity was tested on the
Nanodrop. All samples had an OD 260/280 nm ratio >1.5, indicating high
purity. The extracted RNA was reverse transcribed into cDNA using real-time
PCR kit (Stratagene, USA). 3 μl of random primers were added to the 10 μl of
RNA which was denatured for 5 minutes at 65 °C in the thermal cycler.
B) Quantitative real-time PCR (qPCR):
cDNA specific LIF Taqman primer and probe sets were developed using
primer express software. The forward primer of LIF gene (5'
-GTCAACTGGCTCAACTCAACG -3' ), the reverse primer (5 ’-
TACGCGACCATCCGATACAGC -3’) commercially available primers and
probe for reference β-actin gene were used for normalization (Applied
Biosystems). All PCR reactions were performed on the ABI Prism 7700
Sequence Detection System (Applied Biosystems) using the fluorescent
Taqman methodology. The PCR cycle at which the fluorescence arises above
the background signal is called the Cycle threshold (Ct) and it is inversely
proportional to the log of the initial copy number. In total 10 µl of the reverse
transcription volume was used for each PCR reaction in a total volume of 50
µl. A real time PCR reaction was carried out. One primer pair amplified the
target gene and the other amplified the endogenous reference gene. Primer and
probe concentrations for the target gene were optimized according to the
manufacturer’s procedure. For LIF mRNA; the gene-specific forward and
reverse primers pair was normalized. Each primer (forward and reverse)
concentration in the mixture was 5pmol/µl and 10 pmol for the probe. The
thermal cycling conditions comprised 10 min. at 50°C, 10 min. at 95°C, 15
seconds at 95°C (45 cycles of 15 seconds denaturation at 95°C) and 30 seconds
annealing at 72° C.
Statistical analysis
Statistical analysis was performed with statistical package for social sciences
(SPSS, 10.01, SPSS Inc., Delaware, US). Data analysis was performed using
χ2-test for tables with frequencies, percentages, range mean, and SD. Values
were considered statistically significant when p-value less than 0.05.
Results:
Cell culture
The present study recorded the fibroblast-like cultured MSCs, and thier
adherence capabilities to the plastic surface in the culture and the
differentiation capabilities into connective tissue before and after co-culture
with AML cells ([Figure 1], [Figure 2], [Figure 3], [Figure 4]) .
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Immunophenotyping
The current study showed that MSCS were CD34 negative and CD105
positive ([Figure 5], [Figure 6]), while AML cells were CD34 negative and CD
13 positive as was proven by immunophenotyping ([Figure7], [Figure 8]).
LIF gene expression
The gene-specific forward and reverse primers pair was normalized. Each
primer (forward and reverse) concentration in the mixture was 5 pmol/µl. At
the end of a RT-PCR running with SYBR Green chemistry, the relative
quantification was used. This frequently-used method presents expression
levels in number of folds as compared to expression level of calibrator which is
usually the biological control sample. Quantification is calculated by Livak
method [7]. The actual operation of this quantification method was performed
by qPCR software. To quantify the relative expression of LIF, the Ct
(threshold cycle) values were normalized for housekeeping gene ( ß-actin).
The results obtained using the 2-ΔΔCT method showed that the mean & SD of
relative expression of LIF increased significantly from 1.00±0.00 in the control
group (group A) to 1.911±0.932 in the AML group (group B) (p<0.05).
Following MSCs co-culture, the mean & SD of relative expression of LIF
increased significantly reaching 4.247±0.983 compared to the AML group
(group B) ([Table 1], [Figure 9]).
IL-10 concentration
The level of IL-10 in cell culture supernatant was measured using
‘Orgenium Laboratories IL-10 ELISA kit’. The mean & SD of IL-10
concentration increased significantly from 1.13±0.43 in the control group
(group A) to 4.28±0.73 in the AML group (group B). Following MSCs co-
culture, the mean & SD of IL-10 concentration decreased significantly
reaching 1.33±0.49 ([Table 1], [Figure 10]).
Discussion:
The present study population included 10 newly-diagnosed AML patients
and 10 healthy controls. LIF and IL-10 were measured using RT-PCR and
ELISA techniques, respectively before and after the co-culture; aiming to
evaluate the immunomodulatory effect of MSCs on AML.
In the present study, the AML cells group showed a significant increase in
the level of relative expression of LIF gene after co-culture with MSCs
separated from HUCB. As regards IL-10 concentrations in the present study,
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there was a significant decrease in the IL-10 concentrations in the cultured
media for the AML group after co-culture with MSCs compared with that of the
control group.
In agreement with the data previously reported [8], the present study recorded
the fibroblast-like cultured MSCs, and thier adherence capabilities to the plastic
surface in the culture and the differentiation capabilities into connective tissue.
The current study showed that MSCS are CD34 negative and CD105 positive, as
were proven by immunophenotyping. This is in accordance with the data
previously reported [9, 10], that MSCs must express CD105, CD90 and CD73;
and that MSCs lack expression of CD45, CD34 and CD14.
The current study also showed that AML cells were CD34 negative as was
proven by flow cytometry and this was coincided by researchers [11] who
showed that CD34 expression in AML is highly variable; they classified their
patients into 3 groups based on the extent of CD34 expression.
The present study recorded that the co-cultivation of MSCs with AML cells
tends to increase the LIF expression and then LIF protein induces the
differentiation of several myeloid leukaemia cells and inhibits their growth. The
present study showed that there was statistically-significant difference between
LIF expressions in AML samples before and after co-culture with MSCs.
Following MSCs co-culture, the mean and SD of relative expression of LIF
increased significantly, reaching 4.247
compared to the AML group.
The high relative expression of LIF in the MSCs samples is in accordance to
the results previously reported by [12] who reported that LIF maintains the
pluripotency of embryonic stem cells, while induces the differentiation of
several myeloid leukaemia cells and inhibits their growth. In agreement with the
previously reported data [13], the protein LIF maintains the stem state of MSCs
and other stem cells [14]. The present study recorded that the mean and standard
deviation of relative expression of LIF in the MSCs group (MSCs) (group D)
was 1.974
0.781.
The present study recorded high relative expression of LIF in the AML
samples; this is in agreement with the data previously reported by [15, 16], These
authors reported that adherent layers derived from patients with chronic myeloid
leukaemia (CML), AML, myeloid dysplastic syndrome (MDS), or hairy cell
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leukaemia (HCL) secrete significantly higher levels of the LIF into the
conditioned media as compared to adherent layers derived from normal controls.
The present study showed that following MSCs co-culture with AML cells,
the mean and SD of relative expression of LIF increased significantly, reaching
4.247
compared to the AML group ( p<0.05). The high relative
expression of LIF in AML samples that were co-cultured with MSCs makes
MSCs a promising candidate for AML cell-based therapy; this is in accordance
with the results previously reported [17, 18].
The present study also showed that there was statistically-significant
difference between IL-10 concentration in the AML medium before and after
co-culture with MSCs. The mean and SD of IL-10 concentration increased
significantly from 1.13
pg/ml in the control group to 4.28
pg/ml in
the AML group.
Following MSCs co-culture, the mean and SD of IL-10 concentration
decreased significantly, reaching 1.33
pg/ml, this is in accordance with
the results previously reported [19]. The mean and SD of IL-10 concentration in
the MSCs group (group D) was 13.4
5pg/ml. These results are in agreement
with the results previously reported [20], these authors showed that the MSCs
interactions with macrophages can change macrophages pro-inflammatory
activity into anti-inflammatory via release of prostaglandin E 2 (PGE2) from
MSCs, which, in turn, binds to prostaglandin E 2 receptor 2 (EP2) and
prostaglandin E2 receptor 4 (EP4) on macrophages to induce high-level IL-10
production.
The high IL-10 concentration in MSCs cultured medium makes MSCs a
perfect candidate for AML cell-based therapy; this is in accordance with the
results previously reported [21], These authors pointed out that IL-10 has the
ability to inhibit tumour metastases; they also showed that IL-10 is an efficient
inhibitor of tumour metastasis in vivo at doses that do not have a direct effect on
normal cells.
The current study also pinpointed the immunomodulatory properties of the
MSCs; this is in agreement with the findings of [22]. The mean and SD of IL-10
concentration decreased significantly in the medium following MSCs co-culture.
The statistically-significant decrease in concentration levels of IL-10 in media
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of AML samples after co-culture with MSCs is in accordance with the
previously reported data [23, 24].
The human MSCs are an attractive target for advanced researches in a wide
range of therapeutic applications due to their paracrine effects, multi-lineage
differentiation potential and, most importantly, their immunomodulatory
properties.
Conclusion
The current study showed that Co-culture of AML with MSCs represents a
simple approach to inhibit leukaemia cells in vitro. This effect may be through
the immunomodulatory and anti-inflamatory effects of MSCs. The MSCs
represent the optimal candidate for cell therapy because they can be easily
obtained from BM or HUCB and expanded on a large scale before
autotransplantation, raising no ethical problems.

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immunological features, and potential for homing. Stem Cells
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Primary
culture
of
MSCs
on
day
7.
The
Figure
shows
colony
forming
unit-fibroblast
(CFU-F)
with
a
rapidly
proliferating
early
passage
mesenchymal
stromal
cells
(MSCs).
Optical
lens
20X.
Figure-1
kamj_7_17I577.jpg
Comparison
between
the
mean
&
SD
of
IL-10
concentration
(pg/ml)
in
the
cultured
media
in
the
four
studied
groups
Figure-10

kamj_7_17I574.jpg
First-passage
culture
on
day
5,
showing
large
colonies
and
their
adhesiveness
into
50
cm2
culture
flasks.
Optical
lens
40X.
Figure-2

kamj_7_17I578.jpg
Primary
culture
of
leukaemic
blast
stem
cells
on
day
7
in
complete
culture
medium.
Notice
that
the
size
of
the
initial
colony
is
very
large
with
a
relatively
equal
distribution
and
elongated
cells.
Optical
lens
10X.
Figure-3
kamj_7_17I579.jpg
Co-culture
of
mesenchymal
stem
cells
(MSCs)
with
acute
myeloid
leukaemia
cells
(AML)
on
day
8
in
complete
culture
medium.
Representative
images
for
the
co-cultured
cells
indicating
preserved
morphology
of
the
leuakemic
blasts
cultured
for
8
days
in
the
presence
of
MSCs.
The
total
number
of
viable
leukaemic
cells
was
diminished
after
co-culture
with
MSCs;
while
MSCs
tend
to
make
colony
forming
unit-fibroblast
(CFU-F).
Optical
lens
20X.
Figure-4

kamj_7_17I580.jpg
Flow
cytometric
characterization
analyses
of
human
umbilical
cord-derived
MSCs
showing
gated
histogram.
Each
coloured-dot
represtens
one
single
event.
96.1%
of
cells
are
CD34
negative
and
represented
in
region
[B3]
Figure-5

kamj_7_17I575.jpg
Flow
cytometric
characterization
analyses
of
human
umbilical
cord-derived
MSCs
showing
gated
histogram.
Each
coloured-dot
represtens
one
single
event.
86
%
of
cells
are
CD105
positive
and
represented
in
region
[l2]
Figure-6

kamj_7_17I732DC81B.jpg
Flow
cytometric
characterization
analyses
of
human
myeloid
leukaemia
cells
showing
gated
histogram.
72.4
%
of
cells
gated
in
region
are
CD13
positive
and
represented
in
regions
[l2]
and
[l4]
Figure-7

kamj_7_17I571.jpg
Flow
cytometric
characterization
analyses
of
human
myeloid
leukaemia
cells
showing
gated
histogram.
83.6
%
of
cells
are
CD34
negative
and
represented
in
regions
[l3].
Figure-8
kamj_7_17I572.jpg
Comparison
between
the
mean
and
standard
deviation
of
relative
expression
of
LIF
in
the
four
studied
groups
Figure-9

kamj_7_17I573.jpg
The
mean
of
relative
expression
of
ILF
and
IL-10
concentration
in
the
four
studied
groups
Table-1
kamj_7_17I576.docx