Immunogenicity of human umbilical cord membrane derived stem cells

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					Immunogenicity of human umbilical cord membrane derived stem cells

                                     Koh J.Y.’
             Division of Bioengineering, National University of Singapore
                    21 Lower Kent Ridge Road, Singapore 119077

ABSTRACT

    This project focuses on the study of immunosuppressive properties of human
umbilical cord lining stem cells by detecting the expression of HLA-G. It was our
hypothesis that the expression of HLA-G in CLEC or CLMC could render the
cord lining favourable for being a stem cell source for transplantation and
engraftment as HLA-G minimizes the chances of immunorejection of foreign
artifacts in the body of the recipient. Investigations were carried out on CLSCs
from different donors and passages in varied media and also on CLSCs that were
induced to differentiate into fat and bone cells. In the differentiated CLSCs, the
cells were harvested after periods of 3, 10 and 20 days to investigate if HLA-G
continues to be expressed after differentiation. Methods of conditioned medium,
protein     quantification,  1D    gel  electrophoresis,   western    blotting and
chemiluminescence (ECL) were adopted. The primary antibody used to bind to
and recognize HLA-G was anti HLA-G and the secondary antibody used was anti-
mouse IgG for the amplification of signals. JEG3, a human choriocarcinoma cell line
which express HLA-G endogenously, was used as experimental control. An
additional experiment was also carried out by transfecting shRNAs into JEG3 to
target different exons in the MHC and find out which shRNAs inhibit the
expression of HLA-G in JEG3. Our reasoning was that these shRNAs when
transfected into HLA-G positive CLSCs would serve in subsequent functional
studies. Methods of conditioned medium, protein quantification, 1D gel
electrophoresis, western blotting and ECL were similarly adopted. Our results
revealed that some CLSC samples showed signs of HLA-G being expressed.
sh319 and sh748 were also shown to be able to partially knock down the
expression of HLA-G.

INTRODUCTION

    Traditionally the umbilical cords of newborn infants have been rendered useless
and discarded after birth. However, it has been recently found that cord lining
rich in stem cells can be used to treat health problems that were conventionally
treated with bone marrow stem cells and PBSCs. Because umbilical cord lining
stem cell have yet to develop features that will allow them to be identified and
attacked, their transplants are less likely to be rejected by the immune system of
the recipient. The absence of well developed immune cells in umbilical cord
lining also renders such transplantations a lower likelihood of being attacked by
the recipient's immune system.
   HLA-G has been suggested to be responsible for the immunological tolerance
of the fetus by the maternal immune system despite its immunological distinction
from the mother. Because of its limited polymorphism, tissues with high levels of
HLA-G expressed are identified as self, preventing auto- or allo-reactive T cells
and natural killer cells activation. In addition, HLA-G also promotes the
development of immune-regulatory lymphocytes that down-regulate alloreactivity.
   In our study we hypothesized that similar expression of HLA-G in CLEC and
CLMC would          provide umbilical cord lining stem cell transplants with
immunosuppressive properties necessary to decrease chances of immunorejection by
the recipient’s body. It was thus our aim to detect and find out if HLA-G is
being expressed in the cord lining and investigate the immunosuppressive
properties of umbilical cord lining stem cells.

MATERIAL AND METHODS

Experiment 1 & 2: CLEC and CLMC Samples
          Table 1. Expt 1 CLEC samples           Table 2. Expt 2 CLMC samples

            CLEC10 P2 DMEM                             CLMC1 P3
            CLEC17 M2BM P2                           CLMC4 DMEM
             CLEC17 MeE P3                          CLMC4 10% FCS
               CLEC22 P3                             CLMC17 M2BM
             CLEC22 P8 GFP                        CLMC17 10%FCS sai1 P1
             CLEC22 P9 Ctrl                           WJ17 sai1 P1
               CLEC33 p3                                CLMC18
               CLEC34 P1                              CLMC22 P2
                JEG3 Ctrl                              JEG3 Ctrl


   The samples were isolated by Dr. Phan Toan Thang from the umbilical cords
of healthy donors. Each sample was tagged with a donor number and the passage
number from which it was extracted. CLMC17 10%FCS sai1 P1 and CLMC4
10%FCS were both bathed in 10% fetal calf serum cell culture medium. WJ17
sai1 P1 was a sample that was extracted from the Wharton jelly while CLEC22
P8 GFP was transfected with GFP. JEG3 was used as the positive control in this
experiment to ensure that all procedures were carried out correctly. The JEG3 cell
line was purchased by Dr. Gan Shu Uin from the American Type Culture Collection.
   CLEC10 P2 DMEM and CLMC4 DMEM were bathed in Dulbecco's Modified
Eagle's Medium (DMEM). 4ml of the samples in conditioned medium was loaded
in millipore amicon and centrifuged for 15min at 3700rpm. The samples were then
again centrifuged with distilled water to wash away saline medium that the cells
were previously kept in, and two times further for 15min to reduce the sample
volume to around 100ul. The sample was finally removed from the filter unit
sample reservoir.
   Protein quantification was then carried out to these samples with BSA standards.
Each sample was diluted 10x with distilled water and aliquot into a well. 200ul
of dye (1 part dye: 4 parts ddH2O) was added to each of the sample and
incubated for 5min. O.D. was then measured at 595nm wavelength. The results
were compared to BSA standards.
   All of the samples have been aliquoted into 100ug aliquots when 1D gel
electrophoresis was carried out and kept in eppendorf tubes under conditions of
minus 20 degree Celsius. 1D gel electrophoresis was carried out to separate
protein according to molecular weight. 6x loading dye was loaded into each
sample. The dye served as an indicator and also denatured the proteins. The
samples were water bathed with distilled water at 100 degree Celsius for 5min for
further denaturation and later centrifuged for 10s at 10,000rpm to wash down
droplets of protein along the sides of the tubes. The experiment was carried out in
running buffer where 50V was applied to run the stacking gel and 100V for the
resolving gel. The procedure was immediately stopped after the dye run down.
Contents along each lane in the gel was finally transferred to a membrane in a
sandwich. In the sandwich, the gel and a piece of membrane were clasped
between 2 pieces of filter paper, and this was further sandwiched between 2
layers of sponge. The sandwich was dipped in transfer buffer with the gel aligned
to the negative terminal. Voltage was set at 29V for 18h.
  The membrane was washed in 1x TBS after 18h for 5min. Ponceau was then run
at 90rpm and the image scanned. The membrane was later washed using TBS
tween twice for 5min.
   Western blotting was carried out at 90rpm. The membrane was incubated with
5ml 1:1000 primary antibody anti HLA-G, purchased from BD pharmingen, for 1h
and 1:5000 Mouse IgG as secondary antibody for another 1h. Anti HLA-G was
applied to specifically recognize by binding to HLA-G and Mouse IgG served to
further amplify the signals. 1ml luminolreagent and 1ml oxidizing reagent were
applied to the membrane for ECL with 10-minute exposure time. The presence of
HLA-G was identified as bands on a film and the positions of the bands were
compared to Precision Plus Protein Standard. The molecular weight of HLA-G is
37kDa.

Experiment 3: JEG3 ShRNA Experiment
                  Table 3. Sample list of JEG3 ShRNA experiment

                                       JEG3 Ctrl
                                      JEG3 shAll
                                    JEG3 scramble
                                      JEG3 sh319
                                      JEG3 sh512
                                      JEG3 sh621
                                      JEG3 sh748
                                     JEG3 sh1046



  In this experiment, ShRNA designed against HLA-G was purchased
commercially by Dr. Gan Shu Uin and transfected into JEG3. The JEG3 cell line
was purchased also by Dr. Gan Shu Uin from the American Type Culture Collection.
Each shRNAs was labeled according to the position of the corresponding sequence
in the target exon. ShAll referred to a mixture of Sh319, Sh512, Sh621, Sh748
and Sh1046 that were applied to the JEG3 cells while Scramble was a random
selection of shRNA acting as technical control. A positive control was used
without inhibition by shRNA to ensure that each step of the experiment was
carried out accurately.
   Concentration of conditioned medium, protein quantification, 1D gel
electrophoresis and western blotting methods have been described previously.

Experiment 4: CLMC29 p2 Fat and Bone Differentiation
                        Table 4. Sample list CLMC29 p2

                                       ctrl d3
                                       fat d3
                                      bone d3
                                      ctrl d10
                                      fat d10
                                     bone d10
                                      ctrl d20
                                      fat d20
                                     bone d20
                                     JEG3 ctrl



    Cord lining mesenchymal cells from donor number 29 were isolated by Dr.
Phan Toan Thang. The cells from passage 2 were harvested for this experiment.
Some of the cells which were used as control in this experiment were allowed to
remain in their native states while others were induced to become either
adipocytes and osteocytes. The CLMCs were immersed in DMEM/ 10% FBS and
supplements were added for induction. 0.5mM IBMX, 1 microM dexamethasone,
10 uM insulin, 200 uM indomethacin were supplemented to some of the CLMCs
for adipogenic induction. 0.01 uM 1,25-dihydroxyvitamin D3, 50 uM ascorbate-2-
phosphate, 10mM beta-glycerophosphate were supplemented to other CLMCs for
osteogenic induction. 1% antibiotic/ antimycotic was added to both to prevent
bacterial and fungal growth. After periods of 3, 10 and 20 days, the cells were
harvested . JEG3 was used as a positive control in this experiment to ensure that
all procedures were carried out correctly.
       Concentration of conditioned medium, protein quantification, 1D gel
electrophoresis and western blotting methods have been described previously.
RESULTS

Experiment 1: CLEC Samples
                                17MeEP3                    22P3 22P8GFP JEG3

 37kDa
 HLA-G


                             Figure 1. CLEC first result


                         17MeEP3       22P3 22P8GFP
               37kDa
               HLA-G

                           Figure 2. CLEC second result


    According to ECL results, positive bands at 37 kDa were observed for the last
3 lanes: CLEC22 P3, CLEC22 P8 GFP and JEG3 Ctrl. A faint band was also
observed for CLEC17 MeE P3. The positive band that came up for our positive
control indicated that experimental procedures were carried out correctly. To
confirm the results and that presence of HLA-G in CLEC22 P3 and CLEC22 P8
GFP were not due to spillage of JEG3 control in the last lane when the samples
were loaded into the wells during gel electrophoresis, the experiment was repeated
a second time for CLEC22 P3, CLEC22 P8 GFP and CLEC17 MeE P3. The
results turned out negative for all of these samples.
   From the results of our experiments, we concluded that only in CLEC17 MeE
P3 was HLA-G expressed. Due to the nature of the bands showed up in our first
film, we came to the conclusion that the faint band which showed up for
CLEC17 MeE P3 was remote from our JEG3 sample and unlikely to have been
derived from spillage. The absence of band showing up in the second film could
thus most likely be due to experimental fault. The bands which showed up for
CLEC22 P3 and CLEC22 P8 GFP, on the other hand, appeared to have come
from spillage of JEG3 samples during loading when 1D gel electrophoresis was
carried. They all turned out negative when relocated in lanes further from Jeg3 in
our second experiment.
    CLEC22 P8 GFP was done transfecting GFP into CLEC22 P8 to investigate if
the transfection will affect expression of HLAG. CLEC22 P9 Ctrl acted as the
control. Results showed that no positive band turned up for either sample. HLA-G
was not expressed CLEC22 in the first place.
    Further experiments can be carried out to investigate the effect of passage
number and media type on the expression of HLA-G in CLECs. In these
experiments, it is desirable to keep all conditions invariable except one we are
testing for.

Experiment 2: CLMC Samples
                                                              18   22P2 JEG3


 37kDa
 HLA-G

                             Figure 3. CLMC first result


                                 18           22P2
                    37kDa
                    HLA-G


                           Figure 4. CLMC second result


   According to ECL results, positive bands at 37 kDa were observed for the last
3 lanes: CLMC18, CLMC22 P2 and JEG3 Ctrl. A positive band that came up for
our positive control indicated that experimental procedures were carried out
correctly. However to confirm that the results were not due to spillage of JEG3
control in the last lane when the samples were loaded into the wells during gel
electrophoresis, the experiment was repeated a second time with CLMC18 and
CLMC22 P2.
   Our conclusion was that HLA-G was expressed in both CLMC18 and CLMC22
P2. The results shown in our second film confirmed positive results. The nature
of the bands in our second film appeared unlikely to have been caused by
spillage.
   Further experiments can be carried out to investigate the effect of passage
number and media type on the expression of HLA-G in CLMCs. In these
experiments, it is desirable to keep all conditions invariable except one which we
are testing for.

Experiment 3: JEG3 ShRNA Experiment
            Ctrl    shall scramble sh319        sh512      sh621   sh748   sh1046
37kDa
HLA-G


                            Figure 4. JEG3 shRNA result
   According to ECL results, positive bands at 37 kDa were observed for all
lanes. Fainter bands were observed for sh319, sh748 and shAll. Because shAll
contained each of Sh319, Sh512, Sh621, Sh748 and Sh1046 to serve as a
experimental confirmation, it will produce a positive band if any of Sh319, Sh512,
Sh621, Sh748 and Sh1046 does. This is validated from the results of this
experiment. Shall also validated that each of the shRNA will continue to function
when transfected together. The positive band that came up for our positive control
indicated that the experimental procedures were carried out correctly. Scramble
also showed positive results that indicated that no other functions were inhibited
in the process which would interfere with HLA-G expression.
   In this experiment, we want to find out the shRNA that can decrease the
expression of HLA-G when transfected into JEG3. This will serve as a negative
control to apply to other HLA-G positive stem cells. From the results of our
experiment, sh319 and sh748 seem possible candidates. By forming a complement
with the selected sequence on the exon, sh319 and sh748 inhibit the expression of
the phenotype corresponding to the exon responsible for the expression of HLA-G.
However, whether an ShRNA works depend on factors more than just the exon it
targets. It is currently also an uncertainty if these shRNA functioning in JEG3
correlates to the fact that they might similarly inhibit HLA-G expression in CLSC.

Experiment 4: CLMC29 p2 Fat and Bone Differentiation
                                                                             JEG3
   37kDa
   HLA-G
               Figure 5. CLMC29 p2 fat and bone differentiation result


     According to ECL results, a positive band at 37 kDa was observed only in
the last lane which contained our experimental control JEG3. This indicated that
  37kDa
experimental procedures were carried out correctly. However none of the samples
  HLA-G
showed positive bands. This indicated that HLA-G was not expressed in CLMC29
p2.
    The experiment was carried out find out if HLA-G continues being expressed
in CLSCs after differentiation. In a previous experiment done with undifferentiated
CLSCs to investigate HLA-G’s correlation with immunosuppression, it has been
found that the CLSCs suppress immune reaction in vitro. However a conclusion
has yet been reached with regards to whether HLA-G has a direct relationship with
immunosuppression. The study is still ongoing.
    Improvement could be made to experiment 4 fat and bone differentiation by
instead working with HLA-G positive CLSCs.

DISCUSSION

   In our study, we found that some of our CLEC/ CLMC samples showed signs
of HLA-G being expressed. The results, however, are insufficient to reach any
conclusion about the nature of HLA-G’s expression in CLSCs. Further experiments
can be carried out to investigate the influence of passage number and media type.
Sh319 and sh748 were found to be able to partially inhibit the expression of
HLA-G when transfected in JEG3. It was our guess Sh319 and sh748 would
similarly be able to partially/ inhibit the expression of HLA-G in CLSCs to serve
in subsequent functional studies. To study HLA-G’s expression in CLSCs after
differentiation, future experiments can be done on HLA-G positive CLSCs to
investigate the difference. If proven that HLA-G continues to be expressed after
differentiation, it represents that CLSC transplants might possibly experience a
lower chance of immunorejection; if proven also HLA-G’s correlation with
immunosuppression.
    Because of the difficulty in obtaining CLSC sources, our experiments were
limited to the few CLSC samples we had. Further studies can be done with a
larger pool of CLSC samples. Possible investigations include the effect of passage
number and media type on HLA-G expression. CLMC29 P2 fat and bone
experiment was done on a CLSC which did not express HLA-G. Future
investigations should be however done on HLA-G positive stem cells and the
influence of media type on HLA-G’s expression during differentiation. It is
desirable also to conduct the study with a larger pool of HLA-G positive CLSCs
and their HLA-G expression when induced to differentiate into other cell types
besides fat and bone. As cell culture environment is usually two dimensional
which differs from three dimensional in vivo conditions, methods of molecular
crowding can be adopted. Elements added should only take up space and have no
other effects on the cells. Also to facilitate future studies it is suggested that cell
culture conditions should be optimized for control of HLA-G expression.
    It was our hypothesis that HLA-G should be expressed in CLSCs considering
that it is expressed in the placenta. While there have been ongoing studies to
investigate the expression of HLA-G in other stem cells like the hematopoietic
stem cell, we have pioneers where it comes to the study of HLA-G expression in
CLSCs. CLSCs will prove to become a valuable potential source of stem cells
because of their almost infinite supply, high yield in primary cultures, minimal
contamination of blood and the lack of ethical implications, a property which is
absent in embryonic stem cells.

REFERENCES

Crisa, L. (2007). Retrieved May 20, 2007, from:
   https://www.scripps.edu/mem/eht/torbett/hp_crisa.html
Genetic Science Learning Center University of Utah. (2007). Stem cell therapy today.
   Retrieved May 20, 2007, from:
   http://learn.genetics.utah.edu/units/stemcells/sctoday/
Human Leukocyte Antigen. (2007). Wikipedia. Retrieved May 20, 2007, from:
   http://www.answers.com/topic/human-leukocyte-antigen
Koc, S., Kather, A., Markert, U.R., Durst, M., Schneider, A., & Kaufmann, A.M. (2007).
  Enhancement of immunogenicity of JEG3 cells by ectopic expression of HLA-
  A*.0201 and CD80. [Abstract]. INIST-CNRS. Retrieved May 20, 2007, from:
  http://cat.inist.fr/?aModele=afficheN&cpsidt=15027153

				
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Description: Immunogenicity of human umbilical cord membrane derived stem cells