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Immunogenicity of human umbilical cord membrane derived stem cells
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
"Immunogenicity of human umbilical cord membrane derived stem cells"