Title: Adipose Tissue derived
Mesenchymal Stem Cells’ Influence on
Monocytes’ Differentiation into
Dendritic Cells and Maturation of these
Project period: 25th of April 2012 – 29th
of May 2012
Department of Health Science and Technology
Medicine and Medicine with Industrial
Specialization
Fredrik Bajers Vej 7D2
DK- 9220 Aalborg
Phone 99 40 99 40
Fax 98 15 40 08
Project group: 412
Members:
___________________________________
Anne Frederiksen
___________________________________
Ida Brix
___________________________________
Jesper Lütkemeyer Kjeldsen
___________________________________
Michael Sloth Trabjerg
___________________________________
Mike Sæderup Astorp
___________________________________
Sofie Pagter
Supervisor: Romana Maric
Pages: 26
Appendix: 5 papers
Finished on the: 29th of May 2012
Abstract:
Bone marrow mesenchymal stem cells (BM-MSCs)
have proven to be immunosuppressive. The purpose
of this study is to obtain more knowledge of the
immunosuppressive effect of adipose tissue-derived
mesenchymal stem cells (AD-MSCs) regarding their
effect on differentiation from monocytes into
dendritic cells (DCs) and the maturation of these.
The study constitutes three separate trials
investigating
different
aspects
of
the
immunosuppression under various influences of ADMSCs. Monocytes were isolated from buffycoat and
were stimulated with granulocyte-macrophage
colony stimulating factor (GM-CSF) and interleukin 4
(IL-4) to induce differentiation into immature DCs.
The aspects explored were the effect of conditioned
medium from AD-MSCs investigating the cytokines
secreted, cell-to-cell contact with co-culture of
monocytes and AD-MSCs, and the effect of soluble
factors using transwell inserts. Lipopolysaccharide
(LPS) was added in all three experiments to induce
maturation of the DCs, and furthermore, addition of
antibodies (CD80-PE-Cy5, CD14-PE-Cy7, HLA-DRAPC-Cy7, CD86-PE and CD83-APC) for the use of
flow cytometry.
The data obtained suggest the immunosuppressive
effect of the AD-MSCs to be minimal compared to
earlier studies. The control cells (HEK293) used
seemed to be more immunosuppressive compared
to the AD-MSCs. Further, the transwell inserts used
(Sigma-Aldrich) seemed to have an
immunosuppressive effect on individually cultured
monocytes.
Page 1 of 41
TABLE OF CONTENTS
Introduction ............................................................................................................................... 4
Dendritic Cells ....................................................................................................................... 4
Definition of Dendritic Cells .............................................................................................. 4
Differentiation of Monocytes into Dendritic Cells ............................................................ 4
Function of Dendritic Cells ................................................................................................ 5
Mesenchymal stem cells ....................................................................................................... 5
Description of Mesenchymal Stem Cells .......................................................................... 5
Isolation of Mesenchymal Stem Cells ............................................................................... 5
Immunomodulatory Properties of Mesenchymal Stem Cells........................................... 5
Clinical Applications of Mesenchymal Stem Cells............................................................. 6
Bone Marrow derived Mesenchymal Stem Cells .................................................................. 6
The effect of Bone Marrow derived Mesenchymal Stem Cells on the immune system .. 6
The Effect of Bone Marrow derived Mesenchymal Stem Cells on Dendritic Cells ........... 6
The Effect of Bone Marrow derived Mesenchymal Stem Cells on Dendritic Cells in
Transwell Inserts ............................................................................................................... 7
Comparison of Bone Marrow derived Mesenchymal Stem Cells and Adipose Tissue
derived Mesenchymal Stem Cells ......................................................................................... 7
Description and Purpose of the Experiment ........................................................................... 10
Conditioned Medium Setup ................................................................................................ 10
Cell-to-Cell Contact Setup ................................................................................................... 11
Transwell Inserts Setup ....................................................................................................... 11
Data Analysis ....................................................................................................................... 11
Method .................................................................................................................................... 12
Monocytes .......................................................................................................................... 12
Adipose Tissue Derived Mesenchymal Stem Cells .............................................................. 12
Human Embryonic Kidney Cells .......................................................................................... 12
Control Wells ....................................................................................................................... 13
Flow Cytometry ................................................................................................................... 13
Overview Table ................................................................................................................... 14
Sources of Interference .......................................................................................................... 15
Seeding of the Cells......................................................................................................... 15
Environmental Conditions during Purification of Monocytes ........................................ 15
Centrifugation with Lymphoprep ................................................................................... 15
KM10 Medium as Buffer-System .................................................................................... 15
No Use of Flow Cytometry .............................................................................................. 15
Suggestions for Improvements ....................................................................................... 15
Page 2 of 41
The Second Experiment ...................................................................................................... 15
Buffycoat ......................................................................................................................... 16
Flow cytometry ............................................................................................................... 16
Sterile filtration ............................................................................................................... 16
Results ..................................................................................................................................... 16
Microscope Images ............................................................................................................. 17
Flow Cytometry Data .......................................................................................................... 18
Discussion ................................................................................................................................ 23
Adipose tissue derived mesenchymal stem cells ................................................................ 23
Human Embryonic Kidney cells ........................................................................................... 23
Dendritic Cell Controls ........................................................................................................ 23
A Critical View on the Results ............................................................................................. 24
Immunosuppression in the Conditioned Medium Trial.................................................. 24
Impossible to distinguish AD-MSC and HEK-cells ........................................................... 24
Unspecific Bindings of Isotypes ...................................................................................... 24
pH Change ....................................................................................................................... 24
Transwells’ Supression of Dendritic Cell Control ............................................................ 25
Subpopulations ............................................................................................................... 25
Ratio ................................................................................................................................ 25
Summary ............................................................................................................................. 25
Future Perspectives ................................................................................................................. 25
References ............................................................................................................................... 26
Appendix.................................................................................................................................. 29
Page 3 of 41
INTRODUCTION
Mesenchymal stem cells (MSCs) are of
high interest in the scientific environment
today, as they are able to perform selfrenewal and multi-directional
differentiation when injected into an
allogen host. The research within MSCs
seems to be going in four directions:
Tissue regeneration, a delivery method
for genetic therapy, enhancement of
hematopoietic stem cell engraftment and
treatments of immune diseases (Yi &
Song, 2012). However, there are currently
several difficulties connected to working
with MSCs in vitro in regard to isolation
and proliferation. Adipose derived MSCs
(AD-MSCs) have shown to be easier
attainable as well as easier to work with in
vitro compared to bone marrow derived
MSCs (BM-MSCs). The AD-MSCs can be
hard to distinguish from the BM-MSCs as
their phenotypes are very similar
(Puissant et al., 2005). Even so, there
seems to be a great variance in their
functionality concerning their ability to
differentiate into different kinds of tissue
(Al-Nbaheen et al., 2012).
This Study tries to clarify the role of ADMSCs in differentiation of monocytes into
dendritic cells (DCs). This has previously
been tested with BM-MSCs, where it
seemed that these cells had an inhibitory
effect on this differentiation process (Yi &
Song, 2012). It is investigated whether
this is the case for AD-MSCs as well.
DENDRITIC CELLS
DEFINITION OF DENDRITIC CELLS
DCs are a part of the immune system, and
they are the most potent antigenpresenting cells, in other words the
principle initiator of adaptive immune
responses both in vivo and in vitro assays
(O’Neill, 2007).
All DCs originate from hematopoietic
stem cell. There have been found
different kinds of DCs from both myeloid,
lymphoid as well as a third progenitor cell
type, that does not have any myeloid or
lymfoid potential. One type of DC is the
DC2, which originates from the
plasmacytoid cells of the blood. Another
kind is the interstitial DC1 developed from
monocytes. These are found in the
connective tissue in many kinds of organs
(Agger, 2007; O’Neill, 2007). This study
maintains a focus upon the DC1 type.
A monocyte is a progenitor cell which can
differentiate into either a DC or a
macrophage (Randolph, 1998). A
monocyte stimulated by granulocytemacrophage colony stimulating factor
(GM-CSF) and interleukin-4 (IL-4) for
several days will differentiate into an
immature DC with the phenotype CD14and CD83-, meaning that they do not
express CD14 and CD83 (O’Neill, 2007).
Because there has not been found an
unambiguous marker specific for DCs,
they are often defined by their lack of
markers, for example CD14 expressed on
the surface of monocytes and
macrophages but not on DCs (Agger,
2007).
DIFFERENTIATION OF MONOCYTES
INTO DENDRITIC CELLS
Two steps of differentiation characterize
DCs. Recently developed unstimulated
DCs are called immature DCs and express
relatively high numbers of MHC I- and to a
lesser extend MHC II-molecules on their
surface (Agger, 2007). In this stage they
are able to actively gather antigens in
their environment (O’Neill, 2007). If they
are subjected to an appropriate stimulus,
for example lipopolysaccharides (LPS)
from a gram-negative bacteria or by
proinflammatory cytokines, such as tumor
necrosis factor α (TNF-α), they will
differentiate into mature dendritic cells
Page 4 of 41
expressing a greater amount of MHC Imolecules, a much greater number of
MHC II-molecules and furthermore CD80,
CD83 and CD86 (Agger, 2007; O’Neill,
2007).



FUNCTION OF DENDRITIC CELLS
One of the important functions of the DCs
is, as already mentioned, their ability as
antigen-presenting cells. They are able to
initiate the immune response through
activation of naive T-cells (Agger, 2007).
More specific, antigen-presenting DCs
activate naive T-cells. The response is
dependent on the antigen dose and the
state of maturation of the DCs (O’Neill,
2007). The DC is capable of activating
several types of T-cells. The most
important in relation to this experiment is
however the differentiation of naïve Tcells into T-helper cells. This is the
fundamental reaction in the adaptive
immune response, because T-helper cells
are necessary for other immune cells to
expand and differentiate. An example of
this is activation of B-lymphocytes by help
of Th2 cells. This gives the B-cells the
ability to produce antibodies against
foreign molecules (Agger, 2007).
Since the DCs are the principal initiator of
the adaptive immune response, it is of
high interest to clarify the influence of
MSC on them. In the following section
MSC is described in a general perspective.
MESENCHYMAL STEM CELLS
DESCRIPTION OF MESENCHYMAL
STEM CELLS
MSCs are adult stem cells from various
types of tissues (Yi & Song, 2012).
According to the Mesenchymal and Tissue
Stem Cell Committee of the International
Society for Cellular Therapy, a MSC must
conform to the following criteria:

When maintained in standard
culture conditions, a MSC must be
plastic-adherent.
A MSC must express CD105, CD73
and CD90.
A MSC must lack expression of
CD45, CD34, CD14 or CD11b,
CD79a or CD19 and HLA-DR
surface molecules.
A MSC must be able to
differentiate into osteoblasts,
adipocytes and chondroblasts in
vitro. (Dominici et al., 2006)
ISOLATION OF MESENCHYMAL STEM
CELLS
Isolation of MSC is not an easy task. One
of the key problems in this isolation
process is that no unique MSC surface
markers have yet been identified.
Furthermore, it is difficult to distinguish
subpopulations of MSCs (Yi & Song, 2012).
In the isolating process, mononuclear cells
are fractionated from the remaining
tissue by a density gradient (Yi & Song,
2012). Despite the massive effort to gain a
pure sample of MSC, this has not yet been
achieved. The isolation process gives rise
to the probability, that true MSCs will get
lost in the specter of mononuclear cells
living up to the criteria for MSCs (Yi &
Song, 2012).
IMMUNOMODULATORY PROPERTIES
OF MESENCHYMAL STEM CELLS
MSCs have a general immunosuppressive
effect, which functions via paracrine
activity by cytokines, and via cell-to-cell
contact (Yi & Song, 2012). This effect is a
result of the ability of MSCs to stop the
differentiation and possibly also the
maturation of DCs. (Jiang et al., 2005) This
means that the activation of T-cells, which
are required for activation of the adaptive
immune response, will not occur. Thereby
the immunosuppressive effect can be
acquired by using MSCs in vivo.
Page 5 of 41
CLINICAL APPLICATIONS OF
MESENCHYMAL STEM CELLS
Regarding the clinical applications, MSCs
have been shown to have a tissue
regenerative effect, as well as the ability
to differentiate into bone-, cartilage-,
muscle-, tendon- and neural cells. There is
however, still doubt about the exact
mechanism involved. Furthermore, MSCs
have been shown to enhance the
hematopoietic stem cell engraftment, and
show promise in gene therapy.
Moreover, studies of the effect of MSCs
on autoimmune diseases such as multiple
sclerosis and type II diabetes have been
investigated (Yi & Song, 2012).
BONE MARROW DERIVED
MESENCHYMAL STEM CELLS
In the 1970ies, Friedenstein first identified
BM-MSC as an adherent fibroblast-like
population, which was able to
differentiate into bone. Later studies
show that these cells fulfill stem cell
criteria, which are self-renewal and
multilineage differentiation capacity.
Since MSCs from bone marrow were the
first to be discovered, BM-MSCs have
been object of a range of studies,
including investigations of their
immunomodulatory effect (Nauta, Fibbe,
& Dc, 2012a).
THE EFFECT OF BONE MARROW
DERIVED MESENCHYMAL STEM CELLS
ON THE IMMUNE SYSTEM
BM-MSCs have a suppressive effect on
the immune system, and are likely to be
involved in the induction of immune
tolerance (Aggarwal & Pittenger, 2005).
The immunomodulatory properties of
BM-MSCs include suppression of T-cell
proliferation, suppression of B-cell
proliferation and their terminal
differentiation, effect on the maturation
and function of DCs, and immune
modulation of other immune cells such as
the natural killer (NK) cells (Yi & Song,
2012). When BM-MSCs are co-cultured
with purified subcultures of immune cells,
experiments have shown that they change
the cytokine secretion profile of DCs, NKcells and T-cells, both the effector and
naive T-cells. This change induces a more
anti-inflammatory or tolerant phenotype.
The BM-MSCs cause the mature DC1s to
decrease their secretion of tumor necrosis
factor- (TNF-), a proinflammatory
cytokine, and they cause DC2s to increase
the secretion of the anti-inflammatory
cytokine interleukin-10 (IL-10). Levels of
prostaglandin E2 (PGE2) are elevated, and
inhibitors of PGE2 production weaken the
BM-MSC-mediated immune modulation
(Aggarwal & Pittenger, 2005). However,
Puissant found that PGE2 is not a major
soluble inhibitory factor released by BMMSC (Puissant et al., 2005; Agger, 2007;
Bortesi et al., 2009).
THE EFFECT OF BONE MARROW
DERIVED MESENCHYMAL STEM CELLS
ON DENDRITIC CELLS
As mentioned, the BM-MSCs have an
effect on the differentiation, maturation
and function of the DCs. When coculturing MSC and monocytes with GMCSF and IL-4, the differentiation of
monocytes into DCs was strongly
inhibited. Monocytes cultured with BMMSCs developed macrophage
morphology. The cells retained the same
amount of CD14 without acquisition of
CD1a or up-regulation of CD80, CD83 and
CD86, in contrast to monocytes cultured
without BM-MSCs (Jiang et al., 2005). The
monocytes were blocked from entering
the G1 phase of the cell cycle, resulting in
an accumulation of cells in the G0 phase
with an impaired antigen-presenting
ability (Ramasamy et al., 2007). An altered
cytokine production pattern of the MSCinfluenced DCs was observed, showing a
Page 6 of 41
decreased production of TNF-α , INF- and
IL-12, and an increased production of the
anti-inflammatory cytokine IL-10 (Nauta,
Fibbe, & Dc, 2012b). It shows that the
ratio between MSCs and monocytes have
an influence on the inhibitory effect
observed (Jiang et al., 2005). At a BMMSC/monocyte ratio higher than 1:10, the
suppressive effect was observed even
without intercellular contact. With
intercellular contact, the blockage of the
differentiation of monocytes into DCs first
became minor when the BMMSC/monocyte ratio was as low as 1:200.
This indicates a very strong BM-MSCmediated inhibition of the differentiation
of monocytes into DCs (Jiang et al., 2005)
When co-culturing mature DCs with BMMSC, the expression of CD83, MHC II,
CD80 and CD86 were reduced, and the
secretion of IL-12 was down regulated
(Jiang et al., 2005).
THE EFFECT OF BONE MARROW
DERIVED MESENCHYMAL STEM CELLS
ON DENDRITIC CELLS IN TRANSWELL
INSERTS
In the same study with co-culture of BMMSC with monocytes, a transwell insert
was used to determine whether
intercellular contact between monocytes
and BM-MSCs was necessary for the
inhibitory effect of monocytes into DCs
(Jiang et al., 2005). At a higher BMMCS/monocyte ratio of 1:10, the
differentiation of monocytes into DCs was
completely blocked. At a ratio of 1:20 and
1:50, the monocytes were able to
differentiate into immature DCs when
cultured with GM-CSF and IL-4. These
immature cells had down-regulated CD14,
but also an up-regulated CD1a expression.
When stimulated with LPS, the immature
DCs underwent maturation even though
the BM-MSCs were present. These results
pointed toward that BM-MSCs are
capable of suppressing the generation of
DCs through secretion of cytokines at a
higher BM-MSC/monocyte ratio. At lower
ratios however, the suppression occurs
mainly through intercellular contact (Jiang
et al., 2005).
To determine whether the suppressive
factors were constitutively secreted from
the MSCs, supernatant from these cell
cultures were added to monocytes. This
showed no inhibitory effect, unless the
MSCs had been co-cultured with
lymphocytes. This indicated, that the
MSCs required a dynamic cross talk with
T-lymphocytes to secrete inhibitory
factors (Nauta, Fibbe, & Dc, 2012a).
In 2001 Zuk et al. found adipose tissue to
be an alternatively source of MSCs with
great advantages, since the cells were
obtainable in larger quantities than in the
bone marrow. Furthermore, the medical
procedure of cell acquisition could be
done under local anesthesia with minimal
discomfort for the patient (Zuk et al.,
2001).
COMPARISON OF BONE MARROW
DERIVED MESENCHYMAL STEM
CELLS AND ADIPOSE TISSUE
DERIVED MESENCHYMAL STEM
CELLS
Earlier studies with focus on BM-MSCs
have provided a good amount of
knowledge regarding their abilities
(Puissant et al., 2005). In recent years,
there has been an increased focus on the
extraction and use of AD-MSC instead of
BM-MSC, based on similarities and easier
attainability of these (Al-Nbaheen et al.,
2012). This section will make a
comparison between the BM-MSCs and
AD-MSCs.
Interestingly, the phenotypes of BM-MSCs
and AD-MSCs are nearly similar according
to Puissant et al. However, studies show
discrepancy between the expression of
Page 7 of 41
CD34 (Al-Nbaheen et al., 2012; Puissant et
al., 2005). Puissant and colleagues
concluded that AD-MSC expressed CD34,
whereas Al-Nbaheen et al. did not find
any expression of CD34 on AD-MSCs (AlNbaheen et al., 2012; Puissant et al.,
2005). Both sources agreed that BM-MSC
did not express CD34 (Al-Nbaheen et al.,
2012; Puissant et al., 2005). Though, their
phenotype seems nearly identically, it
seems that their abilities vary from one
another.
Larger amounts of MSCs are possible to
extract from adipose tissue compared to
bone marrow (Puissant et al., 2005). In
vitro experiments reveal that AD-MSCs
have a greater proliferation potential than
that of BM-MSCs. This makes them very
suitable for culturing (Puissant et al.,
2005). A study performed by Al-Nbaheen
and colleagues indicates a difference in
the MSCs’ ability to differentiate into
specific tissues. BM-MSCs seem to evolve
more easily into osteoblast, while ADMSCs evolve more easily into adipocytes
(Al-Nbaheen et al., 2012). Also, it seems
that the AD-MSCs have a higher ability to
induce angiogenesis than the BM-MSCs
(Petit JY, 2012).
A recent study concludes there to be a
significant difference between BM-MSCs
and AD-MSCs, in their ability to suppress
differentiation from monocytes to DCs
(Ivanova-Todorova et al., 2009). The study
showed that AD-MSCs are more potent
suppressors than BM-MSCs. This was
expressed by an experiment carried out
by Ivanova-Todorova and colleagues were
this effect was tested. Monocytes were
isolated and cultured. The description of
their experiment was as follows:
“Peripheral blood mononuclear cells
(PBMC) were passed through a column
with magnetic beads coated with antiCD14 anti-body and the CD14+ cells were
isolated and cultured independently or
co-cultured with different types of human
MSCs in the presence of cytokines (IL-4,
GM-CSF) to induce their differentiation
into dendritic cells and their further
maturation induction by LPS added on day
6th of culture.” (Ivanova-Todorova et al.,
2009, page 39).
They tested the cells for the following CD
markers: CD14, CD80, CD83, CD86 and
HLA-DR. The experiment showed that of
the independently cultured CD14 positive
cells, only 1.3% of them remained CD14
positive. Compared to those co-cultured
with BM-MSCs, 53.2% remained CD14
positive, and for AD-MSCs, 69.8%
remained CD14 positive.
The same was tested with CD83, where
76% of the independently cultured cells
showed to be positive. Compared to those
co-cultured with BM-MSCs, 23.6% were
positive for CD83, and for AD-MSCs,
21.5% showed a positive result. These
results indicate that AD-MSCs have a
more powerful ability to suppress the
differentiation of monocytes into
dendritic cells compared to BM-MSC.
Furthermore, the study indicated that ADMSCs were more potent in stimulating
the secretion of IL-10 than the BM-MSCs
(Ivanova-Todorova et al., 2009).
Although, co-culture studies have already
been conducted with AD-MSCs and
monocytes there are yet undefined
elements in this field of study. These
earlier studies have shown that AD-MSCs
inhibited differentiation of monocytes
into DCs. However, these earlier studies
have investigated the role of cell-to-cell
contact in the AD-MSC induced inhibition
of monocytes differentiation. For future
perspectives it is of great relevance to
investigate how the AD-MSCs will act
when not in direct contact with the
monocytes. This is to find out if it is their
secreted product, which induce the
immunosuppressive effect or not. Thus, in
this study it is investigated whether direct
cell-cell contact is necessary for AD-MSC
Page 8 of 41
induced inhibition of monocyte
differentiation, or whether potential
soluble factors released into the
surroundings by AD-MSCs are involved in
this inhibitory effect.
Page 9 of 41
DESCRIPTION AND PURPOSE
OF THE EXPERIMENT
Based on the results and research
presented in the introduction (by IvanovaTodorova et al., 2009 & Jiang et al., 2005)
this experiment has been designed to gain
a broader knowledge on the area of ADMSCs’ immune-modulatory effects on
DCs. It is expected to verify the results
gained by Ivanova-Todorova and
colleagues. These results showed an
immune-suppressive effect of AD-MSCs
on the differentiation of monocytes into
DCs.
In this experiment, monocytes, AD-MSCs
and control cells (human embryonic
kidney cells (HEK293-cells)) will be
cultured with different setups. It is divided
into three parts each investigating
different aspects of cell differentiation of
monocytes into DCs under various
influences of AD-MSCs. One setup will be
exploring the effect of conditioned
medium from AD-MSCs on the
differentiation. Another will explore the
influence on differentiation when cell-tocell contact between AD-MSCs and
monocytes is present. A third will explore
the effect of AD-MSCs on the
differentiation through transwell inserts,
thus without physical cell-cell contact.
These three setups will make it possible to
determine if a potential reaction will be
caused by cytokines (conditioned medium
setup), physical contact between the cells
(cell-to-cell contact setup) or short lived
cytokines (transwell inserts setup).
Additionally it will be investigated if the
same methods of interaction between
non-MSCs (HEK-cells) and monocytes will
have a similar effect as the AD-MSCs.
CONDITIONED MEDIUM SETUP
This trial will explore if conditioned
medium from AD-MSCs and HEK-cells
introduced to monocytes will have an
immunoregulatory effect. Conditioned
medium is defined by being the medium,
in which the cells have lived for a period
likely to contain cytokines produced by
the AD-MSCs and HEK-cells. If a
suppression of differentiation within the
monocyte population is observed, these
cytokines are likely to be the cause. See
figure 1.
of time. The conditioned medium will be
Page 10 of 41
CELL-TO-CELL CONTACT SETUP
In this trial AD-MSCs and HEK-cells are cocultured with monocytes with cell-to-cell
contact. This will make it possible to
determine if direct contact between the
AD-MSCs and monocytes will influence
the differentiation of the monocytes into
DCs. The same will be investigated for
HEK-cells. It is possible that cell-to-cell
contact is necessary for the AD-MSCs to
influence the dif-ferentiation. This kind of
requirement for cell-to-cell contact, to
initiate internal cell processes, is known
from immunology; exemplified in the
necessity for cell-to-cell contact in T-cell
activation by an antigen-presenting cell.
See figure 2.
TRANSWELL INSERTS SETUP
The third trial will explore the influence of
AD-MSCs and HEK-cells influence the
differentiation process of monocytes
when co-cultured, but physically
separated by a transwell inserts. When
the AD-MSCs and monocytes are cultured
together, but denied cell-to-cell contact, it
is possible to determine if short-lived
cytokines may be able to induce an
immunosuppressive effect on the
differentiation process. See figure 3.
DATA ANALYSIS
Cells will be analyzed by flow cytometry
which will provide information about the
specific phenotypes. Based on the data
obtained, it will be possible to conclude if
the AD-MSCs and HEK-cells have had an
immunomodulatory effect on the
monocytes.
Page 11 of 41
METHOD
This study was conducted by three
separate project groups each investigating
one aspect of the above mentioned. This
study will only describe the protocol for
the transwell insert trial.
MONOCYTES
Peripheral blood mononuclear cells
(PBMCs) are isolated by density gradient
centrifugation from buffycoat, donated by
the Blood Bank, Aalborg Sygehus Nord
with Lymphoprep (Axel-Shield).
Monocytes are isolated from PBMCs by
adherence to bottom of 6-well plates
according to the protocol (see appendix 2)
Buffycoat is diluted 1:4 in 0.9% NaCl,
added Lymphoprep and centrifuged
(380xg 20min) to isolate PBMCs, which
are found in the interphase after density
gradient centrifugation. PBMCs have a
density of 1.077g/ml as Lymphoprep and
cannot migrate through the medium,
whereas erythrocytes and granulocytes
have larger densities, and fall to the
bottom. PBMCs are washed 3 times with
PBS+1mM EDTA by centrifugation 2x
300xg and 1x 200xg to wash away any
thrombocytes. It is important that
washing is performed at 4oC as monocytes
stick to plastic surfaces at room
temperature. PBMCs are counted with
methyl violet supplemented with acetic
acid. Methyl violet stains leucocytes and
acetic acid kills erythrocytes. Cells are
sown in 6-well cell culture plates (Costar;
Corning Incorporated) at a concentration
of 3.4x106 cell/mL in each well. After 3
hours at 37oC in a CO2-incubator, cells are
washed with warm KM10 medium
consisting of RPMI 1640 medium
supplemented with L-Glutamine,
Penicillin/Streptomycin and 10% fetal calf
serum (FCS). During the 3 hour
incubation, it is expected that monocytes,
which make up approximately 15% of
PBMCs, will adhere to the bottom of the
wells, while the rest of the cells will not
adhere and therefore will be washed
away. The cells are grown in KM10 media
supplemented with 100ng/ml GM-CSF
and 20ng/ml IL-4 (Perprotech) for 6 days.
These cytokine are used to drive the
differentiation of monocytes into
immature DCs. On day 6, 10ng/ml LPS is
added to induce maturation of DCs. LPS
are large molecules consisting of lipids
and polysaccharides and are found in the
outer membrane of gram-negative
bacteria. On day 7 cells are analyzed and
photographed in a reverse phase contrast
microscope. Furthermore the
differentiation and maturation state is
analyzed by flow cytometry.
ADIPOSE TISSUE DERIVED
MESENCHYMAL STEM CELLS
AD-MSCs are isolated by lipectomy. Cells
are initially grown in alfa-MEM, but have
been adjusted stepwise to RPMI 1640
(Lifetechnologies) media before use. This
is because AD-MSCs are more robust than
monocytes, and are easier adjusted to
new media than monocytes (Trine Fink,
associate professor) AD-MSCs are
harvested at day one by trypsinization
using trypsin/EDTA which breaks the
integrins and cadherins between the ADMSCs and the wells. Cells are sown into
two transwell inserts (Sigma-Aldrich) at a
density of 25000 cell per well. This
number was calculated to get a ratio of
1:20 (AD-MSC:monocytes).
HUMAN EMBRYONIC KIDNEY
CELLS
HEK293-cells originate from a human
embryonic kidney and were obtained in
the 1970ies. As AD-MSCs, HEK-cells are
initially grown in alfa-MEM, and slowly
adjusted to RPMI 1640 (Lifetech-nologies)
media before use. Cells are harvested at
Page 12 of 41
day 1 by trypsinization using
trypsin/EDTA. Cells are sown in two
transwell inserts (Sigma-Aldrich) at a
density of 25000 cell per well.
CONTROL WELLS
The last two wells function as control for
normal monocyte differentiation. This
means, that no cells were added in the
transwell inserts, only KM10 media.
(Sigma-Aldrich).
FLOW CYTOMETRY
Flow cytometry is a method used to
analyze a range of parameters of
individually cells in a heterogenic
population. It is for instance applied for
immunophenotyping and cell counting,
which both can be illustrated graphically
and analyzed statistically (Flowcytometry,
n.d.).
cells from the bottom of all the wells are
collected in RPMI 1640, centrifuged
(300xg for 10 min.) and resuspended in
PBS supplemented with 0.5% BSA and
0.01% sodium azid at a pH of 7.4. BSA is a
stabilizer and deliver nutrients and binds
free radicals. Furthermore, it prevents the
targeted cells from adhering to the tubes.
Sodium azid prevents bacterial growth of
gram-negative bacteria. Cells are counted
with trypan blue (Sifma) in a microscope,
which dyes the dead cells blue. Cells were
incubated with anti-CD80-PE-Cy5, antiHLA-DR, anti-CD86-PE, anti-CD83-APC
(Beckton Dickinson) and anti-CD14-PE-Cy7
(BD Pharmingen) and their corresponding
isotypes for 30 minutes at 4oC in darkness.
Exposure to light will compromise the
activity of the antibodies. After washing
the cells twice with PBS supplemented
with 0.5% BSA and 0.01% sodium azid by
centrifugation (300xg for 5 min.), the cells
are resuspended in 300μl PBS with 1%
formaldehyde for preservation. The
samples are analyzed on a FACSCanto and
thereafter by FACSDiva. Subsequently the
results are edited with FlowJo.
In this study flow cytometry is used to
analyze the differentiation and
maturation state of the DCs. On day 7,
Page 13 of 41
OVERVIEW TABLE
This table is produced to give a further overview of the experiment
Monocytes
Day -5
Day -3
Day 0
Day 1
The cells are purified
from buffycoat and
seeded into the 6 wells
KM10, GM-CSF and IL-4
are added to the
medium in the wells
The cells are washed
and added new KM10,
GM-CSF and IL-4
Day 3
Other
The new medium contains 50% alpha-mem and
50% RPMI 1640
The medium is changed to
contain 25% alpha-mem
and 75% RPMI 1640
The medium is again
changed. It does now
consist of 100% RPMI
1640
The cells are seeded into
transwell systems in four
wells, two of each cell
type
The transwells are put
into all 6 wells
All wells are added fresh
KM10, GM-CSF and IL-4
All wells are added fresh
KM10, GM-CSF and IL-4
LPS is added to all wells
and the cells will
afterwards be incubated
at 37o C
Day 5
Day 6
Day 7
AD-MSCs and HEK-cells
The cells are separately
counted and seeded in
new containers with new
medium
The monocytes are
harvested and counted
with trypan blue
The monocytes are
prepared for flow
cytometry
The monocytes were
analyzed using flow
cytometry.
Page 14 of 41
SOURCES OF INTERFERENCE
The first experiment failed to show any
results, why the purpose of this chapter is
to clarify the possible errors. Before the
cells were harvested on day 7, they were
observed using a microscope. Also the
control wells, without any other cells than
DCs, contained very few cells. The
possible errors are listed below.
SEEDING OF THE CELLS
When seeding the cells in a specific
concentration, it is of great importance
that the resuspention of cells is carefully
mixed before a portion is seeded into a
well. If not, it is possible that the cells
precipitate. This would make the
concentration in the resuspention too low
to provide any useful data. This could
explain the small amount of cells
observed in each well.
ENVIRONMENTAL CONDITIONS
DURING PURIFICATION OF
MONOCYTES
When the monocytes were purified from
the buffycoat, they were outside the 37°C
CO2-incubator for many hours. These
conditions could cause cell death or make
them adhere to the plastic during
preparation, and thus fewer cells were
seeded out in the wells. Cells should be
kept at 4oC, in between handling.
CENTRIFUGATION WITH LYMPHOPREP
During purification of monocytes from the
buffycoat, Lymphoprep was used as
density gradient to isolate the PBMCs. It
was discovered that the centrifugation
was conducted at too great a force, which
possibly would have interfered with the
results by possibly causing cell death.
KM10 MEDIUM AS BUFFER-SYSTEM
At the end of the experiment the KM10
medium changed colour, indicating a
change in pH value, thereby providing a
disadvantageous environment for the
cells. This could inactivate the cytokines,
why the monocytes possibly were not
stimulated to develop into DCs. Whether
or not the change in pH had an effect on
the cytokines or the cells depends on how
acidic or alkaline the medium had
become. This was not investigated
further.
NO USE OF FLOW CYTOMETRY
By using flow cytometry it would have
been possible to determine the
phenotype of the cell cultures. This was
not done and therefore it was not
possible to detect if the cells had
differentiated as well as matured
successfully.
SUGGESTIONS FOR IMPROVEMENTS
It would be useful to observe the cells
regularly using a microscope to determine
potential errors in the process as they
occurred. The KM10 medium contained a
bicarbonate-buffer system, which is a
major physiologic buffer maintaining the
pH value at 7.0-7.4. It would be beneficial
to add HEPES-buffer to provide a pH value
of 7.2-7.6. This addition would give the
medium a better buffer capacity when
working long periods outside the CO2incubator (“Life Technologies Co.,” n.d.).
THE SECOND EXPERIMENT
A second identical experiment was
conducted to avoid previously suspected
interference sources in an attempt to
achieve useful results. This trial was
conducted by the supervising PhD.
student Romana Maric, and laboratory
technician Brita Holst Jensen. Hence it
Page 15 of 41
became possible to minimize some
sources of interference. Previously, the
lack of the practitioners’ laboratory
experience was suspect to the errors
experienced. The second experiment
obtained the same low amount of cells as
the previous one. From here it can be
concluded that the method was
conducted in a correct manner. The focus
must be applied elsewhere.
BUFFYCOAT
The attention could be directed towards
the buffycoat (Ralf Agger, associate
professor). Buffycoat is isolated from a
blood sample that has been kept
overnight. Pre-buffycoat is isolated from a
fresh blood sample. Buffycoat was used in
this experiment. Hence both trials failed
to achieve optimal results, even in the
hands of skilled professionals, it seems
rational to suspect the buffycoat. If a new
trial was to be conducted, it would be of
great interest to apply pre-buffycoat or
full blood instead.
cytometry. They were indistin-guishable
resulting in inaccurate data. The AD-MSCs
could have been added antibodies giving
them the same green emission as that of
the HEK-cells. This makes it possible to
discard any data collected on cells having
this specific emission. Thereby the DCs or
monocytes could have been isolated in
the data obtained. A flow cytometry
testing the five antibodies on the ADMSCs and HEK-cells should have been
conducted. This would clarify if the ADMSCs and HEK-cells have any surface
markers in common with monocytes or
DCs.
10.000 events were preferred when
performing the flow cytometry test. The
material available for the test was though
insufficient, which resulted in an
acceptance of 2000 – 10.000 events
instead. In addition, a flow cytometry test
of the HEK-cells without antibodies and
isotypes would clear the suspicion if the
cells’ autofluorescence could have had an
impact on the data collected.
FLOW CYTOMETRY
STERILE FILTRATION
When analyzing the results gained from
flow cytometry, it was in this case not
possible to distinguish the cell populations
from each other. The AD-MSCs and the
HEK-cells could have been dyed with the
same fluorescent colour, whereby these
populations could have been ignored, and
the data obtained would only represent
the potential DCs and monocytes.
In the conditioned medium trial, there
were AD-MSCs and HEK-cells present in
the medium transferred to the wells. This
could have been avoided if the
conditioned medium was sterile filtrated
during the transfer.
Further, CD14-PE-Cy7 should have been
tested on the monocytes to establish a
point of reference to compare the
obtained data from the experiment.
IL-4 and GM-CSF were added to
differentiate monocytes into DCs. LPS was
added to induce the maturation process.
The AD-MSCs and HEK-cells were cultured
with monocytes in different trials to
investigate their influence on the
differentiation and maturation.
In the experiment HEK-cells and AD-MSCs
influenced the obtained data from flow
RESULTS
Page 16 of 41
MICROSCOPE IMAGES
Before harvesting the cells, pictures were taken to estimate the state of the cells. All photos
were taken at 20x optics.
Photo 1: DC in conditioned
medium extracted from ADMSCs.
Photo 2: DC from AD-MSC
co-culture.
Photo 3: DC from well with
transwell insert, containing
AD-MSCs.
Photo 4: DCs and HEK-cells
in conditioned medium
from HEK-cells.
Photo 5: DCs and HEKcells in co-culture.
Photo 6: DCs from well
with transwell insert
containing HEK-cells.
Photo 7: DCs in control
well.
Photo 8: DCs in control
well.
Photo 9: DCs in control
well with transwell insert.
Since DCs were observed in all the trials, it
was relevant to do a flow cytometry test.
Fluorescent antibodies were added to the
three different cell suspensions to
distinguish between different surface
markers. In each trial three FACS tubes,
each containing a single cell suspension,
were added following antibodies; CD80PE-Cy5, HLA-DR-APC-Cy7, CD86-PE, CD83APC. Furthermore, in each trial, their
corresponding isotypes were added to
three different FACS tubes, each
containing one of the single cell
suspensions.
Page 17 of 41
FLOW CYTOMETRY DATA
Data 1 - Data from flow cytometry of cells in conditioned medium from AD-MSCs.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Page 18 of 41
Data 2 - Data from flow cytometry of cells co-cultured with AD-MSCs.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Data 3 - Data from flow cytometry of cells co-cultured with AD-MSC separated by transwell
insert.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Page 19 of 41
Data 4 - Data from flow cytometry of cells in conditioned medium from HEK-cells.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Data 5 - Data from flow cytometry of cells co-cultured with HEK-cells.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Page 20 of 41
Data 6 - Data from flow cytometry of cells co-cultured with HEK-cells separated by
transwell insert.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Data 7 - Data from flow cytometry of control cells in KM10 medium from the setup with
conditioned medium.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Page 21 of 41
Data 8 - Data from flow cytometry of control cells in KM10 medium from the setup with coculturing.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Data 9 - Data from flow cytometry of control cells in KM10 medium from the setup with
transwell inserts.
A) 2D scatter plot of the cell population showing the morphologic gating.
B) Histogram showing antibody CD80-PE-Cy7 and isotype CD80-PE-Cy7.
C) Histogram showing antibody CD83-APC and isotype CD83-APC.
D) Histogram showing antibody CD86-PE and isotype CD86-PE.
E) Histogram showing antibody CD14-PE-Cy7 and isotype CD14-PE-Cy7.
F) Histogram showing antibody HLA-DR-APC-Cy7 and isotype HLA-DR-APC-Cy7.
Page 22 of 41
DISCUSSION
Whether AD-MSCs have had an effect on
the differentiation of the monocytes will
now be discussed. To detect if the
observed cells are DCs, this study will use
the expression of CD80 and CD86 as an
indicator of this. If the potentially
observed DCs are mature or immature
DCs will be decided by the amount of
HLA-DR and CD83 expressed. Also the
expression of CD14 among the cell
populations is used to say if the
potentially non-DCs are monocytes or
macrophages.
ADIPOSE TISSUE DERIVED
MESENCHYMAL STEM CELLS
The results obtained in this study indicate
that the AD-MSCs have had an
immunosuppressive effect. This effect is
however doubtful as none of the data
collected was as conclusive as that of
Ivanova-Todorova (Ivanova-Todorova et
al., 2009). The largest suppression was
observed for the AD-MSCs in the cell-tocell contact trial. A subpopulation here
was suspected to be suppressed
monocytes. Despite this potential
suppression, many DCs were still
observed. The second inhibitoriest effect
of the AD-MSCs was detected in the trial
using conditioned medium. A smaller
subpopulation suspected to be
suppressed monocytes was observed in
this trial as well. Also many DCs were
detected. The least, and seemingly noninhibited monocytes, grown with ADMSCs, were observed in the data from the
transwell insert trial. Here no cell
differentiation seemed to have been
inhibited.
The subpopulation for the cell-to-cell
contact trial seems to be more
suppressed than those of the conditioned
medium trial, whereas the monocytes
from the transwell insert trial seems to
have all differentiated into mature DCs.
However this study will not conclude
whether an actual immunosuppressive
effect, has been present.
HUMAN EMBRYONIC KIDNEY
CELLS
The results obtained from the HEK-cells
indicated that these cells had a more
immunosuppressive effect than the one
observed for the AD-MSCs. The HEK-cells
had a strong suppressive effect on the
monocytes in both the conditioned
medium trial as well as the cell-to-cell
contact trial. In each, a smaller
subpopulation was observed and
suspected to be DCs. The primary
population in those two trials was
believed to be the HEK-cells themselves,
since the population was overall negative
in all tests. The largest amount of DCs
seemed to be present in the transwell
insert trial, although only a small amount
was detected here.
The HEK-cells seems to be most
immunosuppressive when there is cell-tocell contact compared to the conditioned
medium trial. The ones cultured in
transwell inserts do not seem to be as
suppressive as in the other trials. Also, it
seems that the HEK-cells have a stronger
immunosuppressive ability than that of
the AD-MSCs.
DENDRITIC CELL CONTROLS
The data from the conditioned medium
trial and the cell-to-cell contact trial
showed almost the same result. This was
expected as they were cultured using the
same protocol. The transwell insert trial
gave results indicating suppression. This is
remarkable as the only difference from
the other two trials was the presence of
Page 23 of 41
the transwell insert. This could indicate
something interesting, which will be
discussed later.
A CRITICAL VIEW ON THE RESULTS
The results obtained in this study must
however be viewed in a critical context.
IMMUNOSUPPRESSION IN THE
CONDITIONED MEDIUM TRIAL
The conditioned medium contained HEKcells and AD-MSCs, which could have
interfered with the differentiation of
monocytes into DCs and the maturation
of these. There could be several reasons
for this interference. The HEK-cells and
AD-MSCs could have emptied the KM10
medium for nutrients, possibly causing
cell death. Furthermore, no studies have
been conducted regarding whether HEKcells have the ability to suppress the
differentiation of monocytes into DCs.
Also the ratio between the conditioned
and non-conditioned medium (KM10)
could be of great importance. In the coculture with HEK-cells and DCs, the
suppression observed could also be
caused by the HEK-cells potentially being
immunosuppressive.
IMPOSSIBLE TO DISTINGUISH AD-MSC
AND HEK-CELLS
It is complex to distinguish the presence
of HEK-cells and AD-MSCs from the
monocytes and DCs, due to the lack of
specific markers targeting HEK-cells and
AD-MSCs in this experiment. Additionally,
it is unknown if the cells expressing HLADR could be HEK-cells, since it is known
that AD-MSCs do not express this surface
marker.
UNSPECIFIC BINDINGS OF ISOTYPES
In the different trials the CD14-PE-Cy7
isotype marker binds as strongly as the
CD14-PE-Cy7 antibody marker. The CD14PE-Cy7 isotype test showed that it bound
unspecific, even in the control containing
only monocytes and DCs. An explanation
of this could be that the isotype is able to
bind to the monocytes or DCs themselves.
The CD14-PE-Cy7 isotype therefore
proved to be a poor isotype to use.
Because of this, the data obtained for
CD14 was analyzed with a skeptical view.
In the trials using HEK-cells, both in the
conditioned medium trial and the cell-tocell contact trial, the CD86-PE isotype
bound unspecific. This was not the case in
the transwell insert trial. This indicates
that CD86-PE possibly binds unspecific to
HEK-cells, since these were present in
both the cell suspension from the coculture setup and the setup with
conditioned medium and not in the
transwell trial.
For the HLA-DR-APC-Cy7 isotype marker
the same tendency occurred as with the
CD14-PE-Cy7 isotype marker. Though the
antibody marker showed to have a higher
affinity for HLA-DR than the isotype’s
affinity for its unknown receptors,
possibly being Fc-receptors. This makes it
possible to use the results in the analysis.
In an experiment, executed by Beavis and
Pennline, investigating this tandem dye
combined of APC and Cy7, as used in this
experiment, it was shown that monocytes
are able to bind APC-Cy7 non-specifically
(Beavis & Pennline, 1996). Though this
was minimal, it might still have influenced
the data obtained.
PH CHANGE
It was observed that the KM10 medium
had changed pH. The wells containing
HEK-cells became alkaline (orange),
whereas the wells containing AD-MSCs
and the wells only containing DCs became
acidic (pink). The changes in pH could
Page 24 of 41
have interfered with the differentiation of
monocytes into DCs.
TRANSWELLS’ SUPRESSION OF
DENDRITIC CELL CONTROL
The transwell inserts seemed to have a
suppressive effect on the differentiation
of monocytes compared to the other DC
controls. This could indicate that the
transwell inserts may contain the
cytokines, which would suppress the
differentiation. Furthermore, it is possible
that the transwell inserts consist of some
substances inhibiting the differentiation
of monocytes into DCs.
SUBPOPULATIONS
Subpopulations were detected in most of
the data gained from the experiment. An
explanation could be that some of the
cells were not expressing the surface
markers of interest. This indicates that the
subpopulations observed were not DCs.
The data obtained for CD14 was
inconclusive, and therefore it was not
possible to distinguish these cell types
from each other. Therefore, in the trials
containing both AD-MSCs and HEK-cells,
the subpopulations could represent either
one of those, monocytes or macrophages.
The reason for this problem, in relation to
the conditioned medium trials, was
caused by the fact that this medium
contained cells.
RATIO
In the experiment AD-MSCs and
monocytes were cultured in a 1:20 ratio.
Since no clear immunosuppression was
observed and monocytes had
differentiated into DCs, it could indicate
that the ratio used might have been too
low. This is seen on the graphs for the
conditioned medium trial and cell-to-cell
contact trial (Data 1, Data 2). A
suppression seems to be present in both,
but stronger in the one with the highest
ratio.
An earlier study from Jiang et al. show an
immunosuppressive effect when BMMSCs and monocytes are co-cultured
using transwell inserts with a ratio of
1:10. As the ratio was lowered to 1:20 and
even 1:50, the monocytes became able to
differentiate into DCs (Jiang et al., 2005).
It would therefore be relevant to consider
an alteration of the ratio in the
experiment to find a more suitable ratio.
SUMMARY
The results of this experiment indicate
that AD-MSCs have a decreased inhibitory
effect on the differentiation of DCs
compared to the results obtained by
Ivanova-Todorova and colleagues.
Based on the results obtained in this
experiment, it is not possible to draw any
conclusions. However, indications have
been observed and noted. The results
indicate AD-MSCs to be
immunosuppressive, although not in a
degree formerly observed in other
studies. Also, the HEK-cells used seem to
be more suppressive than AD-MSCs, and
finally, the data indicates the transwell
inserts themselves to have an inhibitory
effect on the monocytes differentiation
into DCs.
FUTURE PERSPECTIVES
Whether or not the AD-MSCs have an
immunosuppressive ability was not
properly clarified. There are still aspects
that need to be investigated further.
It would be ideal to correct the issues
discussed in the source of interference
chapter. Thereby the experiment might
result in useful data, which potentially
could show the immunosuppressive effect
of the AD-MSCs, previously indicated by
Page 25 of 41
Ivanova-Todorova et al. An essential
correction to the experiment could be the
ratio between monocytes and AD-MSCs.
In the experiment by Jiang et al., they
used a BM-MSC/monocyte ratio of 1:10
compared to the AD-MSC/monocyte ratio
of 1:20 in this study. It could be of interest
to conduct further studies, investigating
the influence of different ratios. This also
applies for ratio of conditioned medium in
the trials regarding this.
Another consideration for future
perspectives is whether to use HEK-cells
as control cells. Because of the lack of
knowledge regarding HEK-cells’ effect on
monocytes, it could be beneficial to try
with cancer cells. Cancer cells have
previously shown a tendency of
immunosuppression (Pinzon-Charry,
Maxwell, & López, 2005). In other words,
the use of cancer cells would make it
possible to compare their known
immunosuppressive effect versus the
presumable effect of the AD-MSCs.
Remarkably the HEK-cells showed an
immunosuppression. This event could be
worth making a closer investigation
through an experiment, where the HEKcells are co-cultured with monocytes or
other types of immune cells.
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mesenchymal stromal cells, 3499-3506. doi:10.1182/blood-2007-02-069716
O’Neill, D. (2007). Exploiting dendritic cells for active immunotherapy of cancer and chronic
infections. Molecular biotechnology, 131-141. doi:10.1007/s12033-007-0020-6
Petit JY, B. F. M.-P. I. G. G. M. P. M. P. C. A. C. C. P. G. M. M. L. V. R. M. (2012). The white
adipose tissue used in lipotransfer procedures is a rich reservoir of CD34+ progenitors
able to promote cancer progression. (We only had acces to the articles review and can
therefor not comment on its deeper content). Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/22052460
Pinzon-Charry, A., Maxwell, T., & López, J. A. (2005). Dendritic cell dysfunction in cancer: a
mechanism for immunosuppression. Immunology and cell biology, 83(5), 451-61.
doi:10.1111/j.1440-1711.2005.01371.x
Puissant, B., Barreau, C., Bourin, P., Clavel, C., Corre, J., Bousquet, C., Taureau, C., et al.
(2005). Immunomodulatory effect of human adipose tissue-derived adult stem cells:
comparison with bone marrow mesenchymal stem cells. British journal of
haematology, 129(1), 118-29. doi:10.1111/j.1365-2141.2005.05409.x
Ramasamy, R., Fazekasova, H., Lam, E. W.-F., Soeiro, I., Lombardi, G., & Dazzi, F. (2007).
Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing
entry into the cell cycle. Transplantation, 83(1), 71-6.
doi:10.1097/01.tp.0000244572.24780.54
Randolph, G. J. (1998). Differentiation of Monocytes into Dendritic Cells in a Model of
Transendothelial Trafficking. Science, 282(5388), 480-483.
doi:10.1126/science.282.5388.480
Yi, T., & Song, S. U. (2012). Immunomodulatory Properties of Mesenchymal Stem Cells and
Their Therapeutic Applications, 35(2), 213-221. doi:10.1007/s12272-012-0202-z
Zuk, P. a, Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, a J., Benhaim, P., et al. (2001).
Multilineage cells from human adipose tissue: implications for cell-based therapies.
Tissue engineering, 7(2), 211-28. doi:10.1089/107632701300062859
Page 27 of 41
Title: Neurogenic Induction of Human Adipose Der
Project period: 26th of April 2011 – 1st of June 2011
Project group: 402
Members:
Cecilie Weiersøe Skovholm
Danny Klindt Josephsen
Heidi Kasten
Malene Cording Christensen
Marie Gerstrøm
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
APPENDIX
Title: Neurogenic Induction of Human Adipose Der
List of Contents:
Project period: 26th of April 2011 – 1st of June 2011
Project group: 402
1 – CD-List
Members:
2 – Protocol
Cecilie Weiersøe Skovholm
3 – Catalog and Materials
Danny Klindt Josephsen
4 – Cell Surface Markers
5 – APV
Heidi Kasten
Malene Cording Christensen
Marie Gerstrøm
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
APPENDIX 1: CD LIST
Title: Neurogenic Induction of Human Adipose Der
Cluster of Differentiation
CD1a
CD11b
CD14
CD19
CD34
CD45
CD73
CD80
CD83
CD86
CD90
CD105
Others
IL-4
IL-10
IL-12
TNF-α
TNF-γ
Project period: 26th of April 2011 – 1st of June 2011
Projectbygroup:
402
These surface markers are especially expressed
cells that
are
specialized for antigen presentation.
Expressed on myoloide and NK-cells. It is known mediate leukocyte
Members:
adhesion.
This CD is mainly expressed by macrophages and monocytes and to lesser
extent dendritic cells. It is also able to recognize pathogen-associated
molecular patterns.
Cecilie Weiersøe Skovholm
B-lymphocyte antigen - this CD can only be located B-lymphocytes.
This CD can be found on the surface of hematopoietic cells. CD34 is
equally important for adhesion and T-cell migration into lymph notes.
Klindt Josephsen
This CD is specific for mesenchymal stem Danny
cells compared
to hem poetic
stem cells which do not possess CD45.
Is found on T- and B-cell subsets.
Heidi
Kastencells and to a lesser
Is especially found on the surface of mature
dendritic
extent on the B-cell and monocytes. CD80 is also important in regards to
T-cell activation.
Is found on the mature dendritic cell. ButMalene
also on B-cells
and
langerhans
Cording
Christensen
cells.
Is found on the mature dendrictic cell, monocytes and active b-cells.
Marie Gerstrøm
Supervisor: Meg Duroux
Printactivation
run: 7 by IL-4, Th2 cells
Is a cytokine that activates naive t-cells. Upon
subsequently produce additional IL-4. ThePages:
cell that
42initially produces IL-4
has not been lidentified. It has many biological roles, including the
Appendices: 4
stimulation of activated B-cell and T-cell proliferation, and the
differentiation of CD4+ T-cells into Th2 cells.
It is a key
regulator
Finished
on the
1st of in
June 2011
humoral and adaptive immunity. IL-4 decreases the production of Th1
cells, macrophages, IFN-gamma, and dendritic cell IL-12. Further more IL-4
stimulates monocytes to differentiate into immature dendritic cells under
the influence of GM-CSF.
IL-10 inhibits the synthesis of proinflammatory cytokines such as TNF-alfa
The contents of this report is freely accessible, but re
and INF-gamma. There by inhibiting APC.
be done in agreement with the authors
IL-12 is involved in the maturation of a naive T-cell into a Th1/Th2-cell.
This cytokin is primarily produced by macrophages, NK- and T-cells. It is
proinflamatoric and induce apoptose.
1
TNF-γ is the most important mediator of acute inflammation in response
to Gram-negative bacteria and other infectious microbes. It mediates the
PGE-2
MHC-1
recruitment of polymorphonuclear leukocytes and monocytes to the site
of infection.
It’s been proposed for a longer time that it is involved in the
Title: Neurogenic Induction of Human Adipose Der
immunosupression during example given burns and other serverer
Project period: 26th of April 2011 – 1st of June 2011
injuries.
Their function is to display fragments of proteins
within
Project from
group:
402the cell to T
cells; healthy cells will be ignored, while cells containing foreign proteins
will be attacked by the immune system.
Members:
Class I MHC molecules bind peptides generated mainly from degradation
of cytosolic proteins by the proteasome.
MHC-2 (HLA-DR
Cecilie
Weiersøe
Skovholm
Major Histocompability Complex (Called HLA
in humans)
is used
to
present antigens to the imunosystem.
TCR
Stands for T-celle-receptor. It is for important
in Klindt
the process
of T-cell
Danny
Josephsen
activition.
GM-CSF is a cytokine that functions as a white blood cell growth factor.
GM-CSF stimulates Monocytes whereupon
theyKasten
mature
Heidi
into macrophages and dendritic cells.
GM-CSF
Malene Cording Christensen
Marie Gerstrøm
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
Title: Neurogenic Induction of Human Adipose Der
Project period: 26th of April 2011 – 1st of June 2011
APPENDIX 2: PROTOCOL
Project group: 402
DYRKNING AF HUMANE DC VED ADHÆRENS
Members:
(GRUPPE III: CO-KULTUR I TRANSWELL
INSERTS)
Cecilie Weiersøe Skovholm
KORT OVERSIGT OVER FORSØGETS OPSÆTNING:
Dag 0
Oprensning af PBMC fra buffycoat
Danny Klindt Josephsen
Monocyt oprensning ved adhærens
Afvask af lymfocytter
Heidi Kasten
Tilsætning af KM10 medium + cytokiner
Dag 1
Afvask af lymfocytter
Malene Cording Christensen
Tilsætning af KM10 medium + cytokiner
Marie Gerstrøm
Tilsætning af stamceller og HEK celler i Transwell inserts
Dag 3
Tilsætning af KM10 medium + cytokiner
Dag 5
Tilsætning af KM10 medium + cytokiner
Dag 6
Modning
Dag 7
Høst af celler + flowcytometri
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
MATERIALER:
Title: Neurogenic Induction of Human Adipose Der
Project period: 26th of April 2011 – 1st of June 2011
Kemikalier
Plast
Buffycoat
group: 402
50ml TPP Project
rør
0.9% NaCl (sterilt)
15ml TPP rør
Lymfoprep (sterilt)
Sterile engangs-plast pipetter
PBS pH 7.4 + 1mM EDTA
Celledyrknings 6-brøndsplader
Members:
FACS rør Cecilie Weiersøe Skovholm
Methylviolet-eddikesyre
Sterilfiltre
Trypan blåt 0,4 %
Sprøjte Danny Klindt Josephsen
RPMI 1640 (- glutamin)
Lang kanyle
L-Glutamin , stock 200mM
Heidi Kasten
Penicillin/Streptomycin, stock
Føtalt kalveserum (varmeinaktiveret ved
56°C)
GM-CSF, stock: 100μg/ml (50μl aliquot)
Malene Cording Christensen
Marie Gerstrøm
IL-4, stock: 100μg/ml (10/20μl aliquot)
LPS, stock: 10μg/ml
Supervisor: Meg Duroux
PBS + 0,5% BSA + 0,01% natriumazid
Print run: 7
PBS + 1% formaldehyd
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
FØLGENDE LAVES INDEN FORSØG PÅBEGYNDES
Title: Neurogenic Induction of Human Adipose Der
KM10 medium med 10% føtalt kalveserum (FCS)
Project period: 26th of April 2011 – 1st of June 2011
Fremstilles af
Project group: 402
RPMI 1640
Members:
2 mM Glutamin (tilsættes ud fra stockopløsning på 200mM)
1% Penicillin/Streptomycin (af en stockopløsning på P:10000U/ml og S:10000µg/ml)
10% FCS (varmeinaktiveret ved 56°C)
Cecilie Weiersøe Skovholm
- Mediet steril-filtreres inden brug og holdes sterilt. Hældes på 50ml rør, idet det holder sig
bedst i disse rør (O2-mætningen)
Danny Klindt Josephsen
PBS + 1mM EDTA
-Steril-filtreres inden brug og holdes sterilt
Heidi Kasten
NaCl 0.9%
-Steril-filtreres inden brug og holdes sterilt
Malene Cording Christensen
PBS + 0,5% BSA + 0,01% sodium azid
-Steril-filtreres inden brug (pga. flowcytometer kørsel)
Marie Gerstrøm
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
DAG 0
Oprensing af PBMC fra buffycoat
Title: Neurogenic Induction of Human Adipose Der
1. Fortynd buffycoat 1:4 i 0.9% NaCl.
Project period: 26th of April 2011 – 1st of June 2011
- Der bruges ca. ___20___ml buffycoat
Project group: 402
2. Fordel det fortyndede buffycoat på 50ml TPP rør (ca.15-20ml pr rør) og lejr
FORSIGTIGT Lymfoprep under buffycoated med en lang kanyle.
3. Centrifuger cellerne i 20min, 180xg, 18-20oC, bremse
0.
Members:
4. Sug FORSIGTIGT ca. 7.8 ml supernat fra med en lang pipette.
5. Centrifuger cellerne i 20min, 380xg, 18-20oC. bremse 0.
6. Høst interfasen med en engang plast-pipette, og saml to
Cecilie
interfaser i et 50ml TPP rør indeholdende 10-15ml
kold Weiersøe Skovholm
PBS+1mM EDTA. Fyld op med kold PBS+1mM EDTA til 25ml.
Danny
Klindt Josephsen
7. Vask cellerne 3 gange i kold PBS+1mM EDTA- efter
vask to
pooles alle cellerne i ét 15ml TPP rør. Vask:
1. 300xg, 4oC, 10min, bremse 0
2. 300xg, 4oC, 10min, bremse
2 Kasten
Heidi
o
3. 200xg, 4 C, 10 min, bremse 2
8. Efter 3. vask resuspender cellerne i KM10 medium.
- Der resuspenderes i ____3____ml Malene Cording Christensen
9. Tæl cellerne i methylviolet (90μl methylviolet + 10μl celler) (Dette gøres ved at
overføre 10μl celler fortyndet i methylviolet på et tællekammer, og dernæst tælles
celler indtil der er talt min. 100 celler (se figur) og cellekoncentrationen udregnes)
Marie Gerstrøm
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
Talt antal celler= _____350______
Celletal (C)= antal talte celler * fortyndingen af celler*104 / antal kamre talt
Title: Neurogenic Induction of Human Adipose Der
350 celler ⦁10 mL ⦁ 10^4
𝐶=
1
↔
Project period: 26th of April 2011 – 1st of June 2011
Project group: 402
𝐶 = 35⦁106 𝑐𝑒𝑙𝑙𝑒𝑟/𝑚𝐿
Indstil cellerne på 3,4x106 celler/ml i KM10 medium
Members:
Oprensning af monocytter ved adhærens
1. Udså i hver brønd i en 6-brøndsbakke 3,4x106 celler, dvs. 1 ml cellesuspension.
2. Tilsæt yderligere 2 ml KM10 medie pr. brønd. Cecilie Weiersøe Skovholm
3. Sæt bakken i CO2-inkubator i 3 timer, hvorved monocytterne vil adhærere til bunden
af brøndene.
4. Efter 3 timer sug supernatanten fra (indeholdende non-adhærente celler), og skyl
Danny Klindt Josephsen
efterfølgende hver brønd 2 gange med ca. 1ml varmt cytokinfrit KM10 medium. Gør
dette ved at holde bakken skrå, så mediet blot løber ned over bundende af
brøndene (gentages 2-3 gange med samme medium). Gentag proceduren fra den
Heidi Kasten
modsatte af bakken med nyt skyllemedium (gentages 2-3 gange med samme
medium)
5. PBMC’er indeholder ca. 15% monocytter, resten adhærerer ikke og skylles derfor
Malene Cording Christensen
fra. Tilsæt KM10 medium med cytokiner (se nedenfor)
Tilsætning af KM10 medium og cytokiner til adhærente celler
Marie Gerstrøm
6. Optø og slyng cytokinerne (GM-CSF + IL-4). Cytokinerne
antages for friske i op til 5
o
dage ved 4 C efter optøning.
7. Tilsæt 3 ml KM10 medium med cytokiner til hver brønd. Tilsæt følgende mængde
Supervisor: Meg Duroux
cytokiner til mediet:
- GM-CSF: slut-koncentration 100ng/ml Print run: 7
- IL-4: slut-koncentration 20ng/ml
Pages: 42
KM10: ____3_____ml
Appendices: 4
GM-CSF: ____3_____μl
Finished on the 1st of June 2011
IL-4: ____0,6______μl
8. Inkuber cellerne i 37oC CO2-inkubator 16-18 timer.
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
DAG 1
Afvask af lymfocyter + tilsætning af stamceller og HEK celler i Transwell inserts + cytokiner
9. Efter ca. 16-18 timers inkubation, sug supernatanten fra brøndende og skyl
Title: medium
Neurogenic
Inductioni of Human Adipose Der
brøndene 2 gange med ca. 1ml varmt cytokinfrit KM10
som beskrevet
punkt 4.
Project period: 26th of April 2011 – 1st of June 2011
10. Der tilsættes 1,5 ml KM10 medium pr. brønd med cytokiner til alle 6 brønde (NB db.
Project group: 402
konc. i forhold til nedenstående koncentrationer). Indsæt Transwell inserts med
stamceller i 1,5 ml KM10 (ca. 5x105celler) til brønde 1 + 2, og transwell inserts med
HEK celler i 1,5 ml KM10 (ca. 5x105celler) til brønde 3 + 4. Til brønd 5 + 6 tilsættes
Members:
Transwells med 1,5 ml KM10 medium. Slutvolumen i alle brønde er så 3 ml,
slutkonc. af cytokiner i alle brønde er følgende:
- GM-CSF: 100ng/ml
Cecilie Weiersøe Skovholm
- IL-4: 20ng/ml
KM10 indeholdende celler: ___1,5______ml
Danny Klindt Josephsen
GM-CSF: ____3_____μl
IL-4: ____0,6_____μl
Heidi Kasten
Malene Cording Christensen
Marie Gerstrøm
Supervisor: Meg Duroux
o
11. Stil cellerne i 37 C CO2-inkubator.
Print run: 7
DAG 3
Tilsætning af medium + cytokiner
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
12. Tilsæt 0.5ml frisk cytokin-indeholdigt KM10 medium til hver brønd.
- GM-CSF: slut-koncentration 100ng/ml
- IL-4: slut-koncentration 20ng/ml
KM10: _____0,5____ml
GM-CSF: ____0,5_____μl
The contents of this report is freely accessible, but re
be done in agreement with the authors
IL-4: ____0,1_____μl
13. Stil cellerne i 37oC CO2-inkubator
1
DAG 5
Tilsætning af medium + cytokiner
Title: Neurogenic Induction of Human Adipose Der
14. Tilsæt 1 ml cytokin-holdigt KM10 medium til hver brønd,
Project period: 26th of April 2011 – 1st of June 2011
-
GM-CSF: slut-koncentration 100ng/ml
IL-4: slut-koncentration 20ng/ml
KM10: _____1____ml
Project group: 402
Members:
GM-CSF: ____1_____μl
IL-4: ____0,2_____μl
Cecilie Weiersøe Skovholm
o
15. Stil cellerne i 37 C CO2-inkubator.
DAG 6
Danny Klindt Josephsen
Modning
Heidi Kasten 10ng/ml, kl 09.00
16. Til alle brønde tilsæt LPS (modningstimulus) i slut-koncentration
Medium: _____4,5____ml/brønd
LPS stock: _____4,5____µl/brønd
Malene Cording Christensen
17. Stil cellerne i 37oC CO2-inkubator
DAG 7
Marie Gerstrøm
Høst af celler + flowcytometri
Supervisor: Meg Duroux
18. Kig på cellerne i mikroskopet, noter hvad der observeres, og tag billeder
Print run: 7
19. Høst alle celler ved at suge væsken fra med pipette. For at høste de ”semiPages: 42
adhærente” celler, spul hver brønd flere gange med koldt RPMI 1640. (Dette kan
Appendices:
tage op til 5min pr brønd). Der tages billeder af hver
brønd efter4høst, for at se hvor
meget der er høstet.
Finished on the 1st of June 2011
20. Centrifuger cellerne i 10min, 300xg, 22oC, bremse 2.
21. Resuspender AD-MSC cellerne i _270 __μl PBS + 0.5% BSA + 0,01% sodium azid.
22. Resuspender HEK cellerne i ___370____ μl PBS + 0.5% BSA + 0,01% sodium azid.
The contents of this report is freely accessible, but re
be done
in agreement
with the authors
23. Resuspender medieceller i _310 _ μl PBS + 0.5% BSA
+ 0,01%
sodium azid.
24. Tæl cellerne i trypanblåt (fortyndes 1:2)
1
AD-MSC Celletal (1 + 2): __6,1 *105celler/mL _____
HEK Celletal (3 + 4): ____6,75 *105celler/mL___
Medie Celletal (5 + 6): __6,1 *105celler/mL _____
Title: Neurogenic Induction of Human Adipose Der
6
25. Indstil cellerne på 2-10 x10 celler/ml i PBS +0.1% BSA. Hvis der er for få
celler, indstil
Project period: 26th6 of April 2011 – 1st of June 2011
så det passer med det antal rør der skal analyseres, dog mindst 0.1x10 celler pr
Project group: 402
FACS rør.
26. Gør 6 FACS rør klar med følgende antistoffer samt isotype kontroller for de
Members:
forskellige markører (se skema):
Rør 1
Rør 2
Cecilie Weiersøe Skovholm
CD80-PE-Cy5
20μl
CD80-PE-Cy5
isotype
HLA-DR-APCCy7
5μl
HLA-DR5μl
Danny Klindt
APC-Cy7
isotype
CD86-PE
20μl
CD86-PE
20μl
Heidi Kasten
isotype
CD83-APC
20μl
CD83-APC
20μl
isotype Malene Cording Christensen
CD14-PE-Cy7
5μl
CD14-PE-Cy7
isotype
20μl
Josephsen
5μl
Marie Gerstrøm
Supervisor: Meg Duroux
1. Overfør 100μl cellesuspension fra hver brønd til ét FACS rør indeholdende
Print run: 7
antistoffer og til ét rør indeholdende isotype kontroller.
2. Inkuber rørene ½ time ved 4oC under sølvpapir
Pages: 42
Appendices: 4
3. Tilsæt 2 ml PBS + 0,5% BSA + 0,01% sodium azid pr. rør
Finished on the 1st of June 2011
4. Centrifuger rørene i 5min, 300xg, 20oC, bremse 2
5. Hæld supernatanten fra og resuspender pellet i 2 ml PBS + 0,5% BSA + 0,01% sodium
azid pr. rør
6. Centrifuger rørene i 5min, 300xg, 20oC, bremse 2The contents of this report is freely accessible, but re
be done in agreement with the authors
7. Hæld supernatanten fra og resuspender pellet i300μl PBS + 1%formaldehyd og
vortex (NB! Formaldehyd er kræftfremkaldende, stå i stinkskab under tilførslen, og
luk FACS rør med prop). Hvis cellerne skal analyseres samme dage, kan pellet
resuspenderes i300μl PBS.
1
8. Analysér på flowcytometer eller gem ved 4oC indtil analyse (inden 7 dage)
Title: Neurogenic Induction of Human Adipose Der
Project period: 26th of April 2011 – 1st of June 2011
APPENDIX 3: CATALOG ANDProject
MATERIALS
group: 402
Produkt
RPMI 1640
Lymfoprep
PBS
EDTA
L-Glutamin
Penicillin/Streptomycin
GM-CSF
IL-4
LPS
BSA
Transwell
Trypan Blå
CD80-PE-Cy5
HLA-DR-APC-Cy7
CD86-PE
CD83-APC
CD14-PE-Cy7
NaCl
Firma
Lifetechnologies
Medinor/Axis-Shield
Lifetechnologies
Sigma-Aldrich
Perprotech
Sigma-Alrich
Sigma-Aldrich
Sifma
Beckton Dickinson
Beckton Dickinson
Beckton Dickinson
Beckton Dickinson
Beckton Dickinson
Sigma
Members:
Katalognummer
31870-074
1114545
Cecilie70011-051
Weiersøe Skovholm
Danny P4333
Klindt Josephsen
200-04
Heidi Kasten
A2153
CLS3412-24EA
93595
Malene559370
Cording Christensen
335831
555658
551073
Marie Gerstrøm
557742
S9888
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1
APPENDIX 4: SURFACE MARKERS
Title: Neurogenic Induction of Human Adipose Der
Project period: 26th of April 2011 – 1st of June 2011
Project group: 402
Members:
Cecilie Weiersøe Skovholm
Danny Klindt Josephsen
Heidi Kasten
Malene Cording Christensen
Marie Gerstrøm
Supervisor: Meg Duroux
Print run: 7
Pages: 42
Appendices: 4
Finished on the 1st of June 2011
The contents of this report is freely accessible, but re
be done in agreement with the authors
1