Original Article the CD200/CD200R Mechanism in Mesenchymal Stem Cells’ Regulation of Dendritic Cells

Am J Transl Res 2021;13(8):9607-9613 www.ajtr.org /ISSN:1943-8141/AJTR0130885

Original Article The CD200/CD200R mechanism in mesenchymal stem cells’ regulation of dendritic cells

Yulei Zhao, Guohong Su, Qing Wang, Ruihuan Wang, Minjuan Zhang

The Second Department of Hematology, Cangzhou Central Hospital, 16 West Xinhua Road, Yunhe, Cangzhou, China Received January 27, 2021; Accepted April 12, 2021; Epub August 15, 2021; Published August 30, 2021

Abstract: Objective: To investigate the CD200/CD200R pathway mechanism in mesenchymal stem cells’ (MSC) regulation of dendritic cells (DC) (MSc). Methods: We collected marrow samples from 40 patients admitted to our hospital from January 2018 to December 2019. The bone marrow MSCs were cultivated, and the peripheral blood mononuclear cells (PBMC) and peripheral blood DC were isolated to establish an in vitro immune response model. The expressions of the CD200 molecule on the surface of MSC were measured. Anti-CD200 blocking antibodies were added to the culture system to observe the effect of the PBMC differentiation and the immature DC (imDC) to mature DC (mDC). Then the impact of the different positive rates of CD200 in the same MSC on imDC maturity was measured. Results: After adding mitogen pHA, the IL-4, IL-10, and TNF-α secretions were increased (all P<0.05), and the OD value of the PBMC+pHA group was higher than it was in the PBMC group. After stimulated by pHA, the CD200 of the MSC group was higher than it was in the MSC+PBMC group (P<0.05). The MSC+PBMC group co- culture inhibited the development of imDC to mDC. Adding anti-CD200 antibodies to the MSC+PBMC co-culture system, MSC could still inhibit the differentiation of PBMC to imDC, and MSC had a significant inhibition effect on imDC to mDC maturation (P=0.006). The addition of MSC reduces the maturation markers on the surface of mDC

(P<0.05). The addition of MSC inhibited the ability of mDC to stimulate PBMC (POD<0.05) and decreased the IL-12

(PIL-12<0.05) levels. The addition of the anti-CD200 antibody increased the proliferation ability of mDC to stimulate

PBMC (POD<0.05), and it also increased the IL-12 levels in mDC (PIL-12<0.05). The expression of the DC mature immune phenotype in the CD200 high expression group was weak (PCD83, CD86<0.05). Conclusion: The mechanism by which MSC inhibits DC may be achieved through the CD200/CD200R pathway, and the CD200/CD200R pathway mainly acts on the process from imDC to mDC.

Keywords: Mesenchymal stem cells, dendritic cells, CD200/CD200R

Introduction nity. It is reported that MSCs enable DCs to express an inhibitory phenotype through inter- Mesenchymal stem cells (MSC) are a kind of cellular interactions, further inhibiting DC matu- non-hematopoietic adult stem cells with a mul- ration and immune tolerance [3]. Yet, the spe- tidirectional differentiation ability. They are cific mechanism MSCs use to regulate DCs has mainly derived from the bone marrow, bones, not been elucidated. Many scholars have paid fat, muscles, and other tissues. They are pre- increased attention to the role of CD200 in dominantly used to treat various blood system immune rejection. CD200 is widely expressed diseases and for the identification of the in DC, thymocytes, B cells, MSCs, the central immune response in vitro due to their unique nervous system nerves, and other hematopoi- immunosuppressive effect [1, 2]. Their immu- etic and non-hematopoietic tissue-derived cells nomodulatory mechanism is mainly achieved [4, 5]. However, CD200 lacks signal motifs and by inhibiting the expansion of B cells and affect- transmembrane anchor sites for binding signal ing the further differentiation and maturation of proteins and can only perform its biological dendritic cells (DCs). As the most powerful anti- functions by binding to other cell surface recep- gen-presenting cells of naive T cells, DCs are tors. CD200R, the CD200 receptor that exists crucial in the process of transplantation immu- on DCs, is not only similar in structure to The CD200/CD200R mechanism in the regulation of dendritic cells

CD200, it can also transmit inhibitory immune ject the cells to an adherent culture, the culture regulatory signals with CD200, thereby inhibit- medium was discarded after being incubated ing the DC immune response. Hence, the explo- for 3 h, and then we washed the solution three ration of the CD200/CD200R pathway mecha- times with a PBS buffer. The PBMCs were sus- nism in MSCs’ regulation of DC is of vital signifi- pended in an RPMI 1640 medium (containing cance and can provide a theoretical basis 10% FBS) and inoculated into inactivated for clinical treatment involving allogeneic MSCs. transplantation. Phytohemagglutinin was added to accelerate Materials and methods the transformation of the mononuclear cells. The PHA stimulated mononuclear cells without Material collection MSC were defined as the positive control group, and those without PHA were defined as the We collected bone marrow samples from 40 negative control group, and the two groups of patients admitted to our hospital from January cells were incubated. The optical densities (OD) 2018 to December 2019, and we collected 10 of the negative control group (PBMC), the posi- ml of bone marrow from each patient’s posteri- tive control group (PBMC+PHA), and the ex- or superior iliac spine using heparin anticoagu- perimental group (MSC+PBMC, PBMC+MSC+ lation tubes. All the samples were examined PHA) were measured at λ=492 nm using ELISA, using bone marrow cytology to determine the and the measurement was repeated three disease type. And all patients voluntarily signed times to get the average. an informed consent form, and the Ethics Committee of Cangzhou Central Hospital After inoculating the MSCs into a 6-well plate approved the protocol of the experiments, and and co-culturing them with the PBMCs, PHA the approval No. is 2017-252-01 (Z). was added. And the MSC+PBMC and the MSC+PHA were set as the control group. After Experimental methods 72 hours of culturing, the adherent cell MSCs were collected and resuspended in PBS, and Cultured bone marrow MSCs and isolated the CD200 expression changes were measured human peripheral blood mononuclear cells using FACS. (PBMC): After aseptically harvesting the bone marrow, a bone marrow cell suspension was Culturing and measuring the human peripheral prepared, bone marrow mononuclear cells DC: We took 50 ml of peripheral blood from were separated using density gradient cen- healthy adults and isolated the PBMCs using trifugation, and bone marrow-derived MSCs Ficoll density gradient centrifugation. Then, the were obtained through adherent cultures, inoc- cells were washed twice with PBS, inoculated 6 ulated in a petri dish (medium: 10% fetal bo- at 2×10 /well on a 6-well culture plate (10% vine serum+2 mM glutamine low-sugar DMEM) fetal calf serum RMPI+1640 medium was used at a density of 5×105/cm2, and cultured in a to resuspend the cells), and then the cells were placed in an incubator (37°C, 5% 37°C, 5% CO2 incubator, and the medium was changed after 48 h. The bone marrow MSCs CO2) for two hours. Next, the non-adherent were digested and passaged when the adher- cells were discarded, and the solution was ent cells’ confluence reached over 90%. washed with a RMPI-1640 medium once more. 2 ml of 10% fetal bovine serum RMPI-1640 We collected 20 ml of peripheral blood from medium (containing IL-4+GM-GSF) was add- healthy volunteers, and we separated the ed, cultured in an incubator (37°C, 50% CO2) PBMCs using density gradient centrifugation. until day 3 and day 5 to half medium change, and the immature DCs (imDC) could be Establishing an in vitro immune response observed on day 5. Subsequently, the antigen model to determine the expressions of the differentiation cluster was fluorescently labeled CD200 molecules on the MSCs’ surfaces: The and its fluorescence intensity was measured. digested and washed MSCs were counted, the Lipopolysaccharide (LPS) was added to pro- cell concentration was adjusted, and then we mote the maturation of some cells, and mature inoculated 1×104 cells per well in a 96-well DC (mDC) was obtained on day 7. The fluores- plate. 20 μg/ml mitomycin was added to sub- cence intensity was measured as above.

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Table 1. Changes in the cytokine IL-4, IL-10, and TNF-α cules with different positive rates of the same MSC (positive rate <25% or levels in PBMC before_ and after adding mitogen PHA into the culture system ( x±s) >80%) were obtained, and we inocu- 5 IL-4 (ng/L) IL-10 (ng/L) TNF-α (ng/L) lated them into 6-well plates at 2×10 / Before adding 15.72±5.03 48.79±4.35 21.36±6.28 well. Next, imDC was added to the After adding 287.34±96.85 226.41±86.30 136.78±36.53 adherent MSC6 well plates as a simple imDC group, catalyzed, and then t 8.857 6.500 9.847 we obtained the mDC. The immuno- P <0.001 <0.001 <0.001 phenotype of the mDC was deter- mined. The effect of DC on the prolif- The effect of anti-CD200 blocking antibodies eration of PBMC was observed using ELISA, on PBMC differentiation: MSCs with CD200 and the IL-12 level in the supernatant was positive rate >50% after digestion were measured. obtained, washed, and resuspended, and anti- CD200 blocking antibody was added to the Statistical analysis MSC resuspension, incubated in an incubator (37°C) for three hours, then inoculated 2×105/ SPSS 23.0 software was used to conduct the well in a 6-well plate, and the adherent was data analysis and GraphPad Prism 7.0 to plot cultured for 8 hours. The PBMCs were ex- the graphics. The measurement data were tracted and added to the adherent MSC6 well expressed as (χ±s), and analyzed using t tests. The count data were expressed as n (%), and plates and divided into two culture groups: 2 PBMC and MSC+PBMC. We referred to the DC analyzed using χ tests. P<0.05 indicated sta- culture procedure to obtain imDC and mDC, tistical significance. and we measured their immunophenotypes. Results The effect of the anti-CD200 blocking antibod- The expressions of the CD200 molecules on ies on the tendency of imDC to mDC: The the surface of MSC in the immune response PBMCs were isolated, cultivated, and then environment imDC was obtained. 8 hours before the culturing, the MSC expressing CD200>50% was In the in vitro immune response model of the digested, washed, and resuspended. Then the PBMCs, it was found that all groups secreted a anti-CD200 blocking antibody was added, incu- small amount of cytokines such as IL-4, IL-10, bated in an incubator (37°C) for 3 hours, inocu- and TNF-α, and the IL-4, IL-10, and TNF-α secre- 5 lated at the rate 2×10 /well in a 6-well plate. tions in each group after adding the pHA Then ImDC was added to the adherent MSC increased (P<0.05, Table 1). The proliferation 6-well plate, divided into two culture groups, in each group was measured using the ELISA imDC and MSC+imDC, and LPS was added to method, and the measurements showed that obtain mDC. The FACS method was used to the OD value of the PBMC+pHA group was determine the immunophenotypes of imDC significantly higher compared to the PBMC and mDC. The mDC was cultured adherently group [(0.85±0.07) vs (0.38±0.06)] (t=16.12, and inactivated by adding mitomycin, wash- P<0.05), as shown in Figure 1. At the same ed 3 times with PBS, and cultured with PBMC time, after the PBMC was stimulated by the from different sources. And it was divided into PHA, the expressions of the CD200 molecules the simple PBMC group and the simple MSC on the surface of the MSC group increas- group, and cultured for 68 h. And then the ed compared with the MSC+PBMC group CCK8 was added, and the culture was contin- (51.73±9.68 vs 36.79±8.63, t=3.643, P= ued for 6 hours under the original conditions. 0.002). The OD at λ=492 nm was measured using ELISA. The supernatant was collected and the DC general characteristics IL-12 level was measured using ELISA. The cell phenotype results demonstrated that The effect of the different CD200 positive rates PBMC showed a high expression of CD14 and a of the same MSC on the maturation of imDC: low expression of CD1a. After we induced the The PBMCs were isolated to obtain imDC. 8 differentiation of the PBMCs, the imDC showed hours before culturing the imDC, CD200 mole- low expressions of CD83 and CD86, and no

9609 Am J Transl Res 2021;13(8):9607-9613 The CD200/CD200R mechanism in the regulation of dendritic cells

ated into the imDC phenotype. In addition, it could inhibit the development of imDC to mDC. See Table 2.

The effect of the anti-CD200 blocking antibodies on the DC immune phenotypes

After adding the anti-CD200 blocking antibody to the MSC+PBMC co-culture system, MSC can still inhibit the differentiation of PBMC towards imDC, and there was no significant difference in the changes in the surface immune molecules of imDC (P>0.05), but MSC had a significant inhibitory effect on the maturation of imDC to mDC (P=0.006).

MSC was added to treat the maturation pro- Figure 1. This OD bar graph of each group of cells cess of imDC to mDC, and it was found that the at λ=492 nm was created using ELISA measurement expression levels of the maturation markers on values. *** indicated P<0.001. the surface of mDC decreased (P<0.05). After the intervention with the anti-CD200 blocking antibodies, the expressions of the mature immune phenotypes of mDC were significantly higher than the expressions of the MSC sup- pression group (P<0.05). See Table 3.

The effect of the anti-CD200 blocking antibody on the tendency of imDC to mDC

After adding the MSCs to treat the maturation of mDC, they inhibited the ability of mDC to

stimulate the PBMCs (POD value<0.05), and reduced the mDC’s inflammatory factor IL-12

level (PIL-12<0.05) After adding the anti-CD200 blocking antibody, compared with the MSC Figure 2. The expressions of CD200 and CD200R on inhibition group, the PBMC proliferation ability PBMC and imDC. *** indicated P<0.001. stimulated by mDC increased (POD value<0.05), and the mDC’s inflammatory factor IL-12 level related CD14 expression, but the mDC showed also increased (PIL-12<0.05). See Table 4. high expressions of CD83 and CD86. After adding the PBMCs, it can promote its activation The effect of the CD200 MSCs on the different and proliferation, showing the effect of the anti- positive rates on imDC gen presentation. In the comparison between the CD200 low The expressions of CD200 and CD200R on expression group and the high expression PBMC and imDC group, the CD200 high expression group showed a weaker DC mature immunopheno-

A total of five samples of PBMC and imDC were type expression (PCD83, CD86<0.05). The prolifera- tested, and their CD200 and CD200R expres- tion inhibition rate and the secretion ability of sions are displayed in Figure 2. IL-12 in the high expression group on PBMCs

were weakened by ELISA (POD, IL-12<0.05). See The effect of MSC on the immune phenotype Table 5. of DC Discussion The PBMC group and the MSC+PBMC group were co-cultured for five days. No cell surface In recent years, evidence supporting the role of molecules in the MSC+PBMC group differenti- MSCs in immune regulation has increased.

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Table 2. The inhibitory effect of MSC on the DC immu- the MSCs, suggesting that the PBMCs nophenotype have produced an immune response PBMC MSC+PBMC t P under the action of the immune stimulat- CD14 (%) 33.76±9.27 6.43±1.28 9.235 <0.001 ing factors. In addition, in the model of adding PHA to stimulate the immune CD1a (%) 12.48±3.13 76.35±8.51 22.270 <0.001 response of MSC+PBMC co-culture, the CD83 (%) 60.83±15.74 91.42±12.16 4.863 0.001 PBMCs activated the proliferation, and CD86 (%) 67.28±11.23 85.49±9.53 4.658 0.001 the expressions of CD200 on the surfaces of the MSCs was up-regulated. MSC MSCs can regulate immunity by inhibiting the alone has no significant immunosuppressive expression of DC, affecting the activity of T effect on T lymphocytes. But after activating cells, and the immune response of transplanta- the immune response, it can increase the IL-4, tion, and they have achieved certain results in IL-10, and TNF-α levels, plays a certain role in the treatment of graft-versus-host disease [6, inhibiting the proliferation of immune effector 7]. Many scholars have stated that soluble fac- cells, reduces the inflammatory factor levels, tors play a key role in mediating the inhibition of and regulates the tendency of T lymphocytes MSC, and the isolation of MSC and lympho- subsets to Th2 [13, 14]. Therefore, the immune cytes in a co-culture system has no impact on regulation mechanism of MSC may guide the the inhibition of MSCs [8, 9]. However, there immune response to stabilize the internal envi- are studies suggesting that the method of con- ronment [15]. tact between cells may also play an important part in mediating the MSC inhibitory pathway Some foreign scholars have pointed out that [10]. The CD200/CD200R signaling pathway MSC can inhibit the differentiation and ma- has a significant inhibitory effect on immune turation of PBMC-derived DC in vitro [16, 17]. regulation. By the comprehensive analysis of Zhang et al. [18] found that MSCs can inhibit the immune function of MSC and the mole- the differentiation stage of PBMC-derived DC cular characteristics of CD200, it is speculated (the expression of CD1a, CD86, human leuko- that the molecular expression of CD200 may cyte DR antigens, etc.), and they also have an be related to the immune mechanism of MSC. inhibitory effect on the later mature stage of Ho et al. [11] pointed out that the immunomod- DC (the CD83 and CD86 expressions). MSC ulatory effect of the route of exposure of MSC changes the cytokine secretions and gradually and the effector cells may be related to the directs DC expressions towards immature CD200/CD200R pathway. Yang et al. [12] immunophenotypes by inhibiting the expres- revealed that for transplanted bone marrow sion of CD83. MSC inhibits the maturation of MSCs in a rat model of ischemic stroke after DC and induces immune tolerance through the injection of CD200R antibodies, the MSCs intercellular contact [19, 20]. We thus exam- can inactivate the microglia around the infarct- ined its specific regulatory mechanism in this ed tissue through ionized calconectin antigens study. In evaluating the effect of CD200 mole- to exert neuroimmunological regulation, sug- cules on the maturation and characteristics of gesting that the transplanted MSC may partici- DCs in MSCs, anti-CD200 blocking antibodies pate in the inactivation of microglia around the were added to the PBMC+MSC co-culture sys- infarction through the CD200/CD200R path- tem. It was observed that the MSCs had high way, exerting an immunosuppressive effect. CD1a expressions and low CD14 expressions, but they had no significant immunosuppressive In our study, we explored the changes in the effect on the immunophenotype of imDC. In CD200 molecules on the MSC surfaces by addition, the CD200R molecule on the surface establishing an immune response environ- of PBMC was lowly expressed, and it is specu- ment. The results showed that the PBMCs pro- lated that there is no CD200/CD200R signal liferated significantly after PHA was added to pathway between MSC and PBMC to achieve the system in vitro, and the proliferation of an immunosuppressive regulation. Moreover, PBMC was significantly inhibited after adding we found that the CD200R molecule on the sur- MSC to the system. If immune stimulating fac- face of imDC was highly expressed, and the tors are excluded, the growth of the PBMCs in addition of the anti-CD200 blocking antibody to the in vitro environment will not be affected by the imDC+MSC co-culture system can improve

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Table 3. The effect of the anti-cd200 blocking antibody on the DC immunophenotype PBMC MSC+PBMC t P Anti CD200 blocking antibody CDl4 43.19±11.27 40.36±12.36 0.535 0.599 CDla 11.34±2.45 10.89±2.31 0.423 0.678 CD86 81.05±10.23 67.86±8.64 3.115 0.006 Added MSC CD83 74.57±6.81 51.74±7.36 7.200 <0.001 CD86 92.36±8.49 78.63±7.43 3.848 0.001 Anti CD200 blocking antibody was added CD83 42.54±11.87 71.23±12.36 5.294 <0.001

Table 4. A comparison of the OD values on the effect of anti-cd200 blocking antibodies on the tendency of imDC to MDC imDC imDC+MSC t P Added MSC OD 0.95±0.02 0.67±0.02 31.300 <0.001 IL-12 (pg/ml) 327.59±12.38 207.36±11.75 22.280 <0.001 Anti CD200 blocking antibody was added OD 0.73±0.02 0.86±0.01 18.380 <0.001 IL-12 (pg/ml) 233.41±12.43 289.36±12.57 10.010 <0.001

Table 5. A comparison of the effects of the CD200 MSCs pathway, and the CD200/CD200R with different positive rates on imDC pathway mainly acts on the process CD200 low CD200 high of imDC tending to mDC. Effective t P expression group expression group interventions on this signaling pathway are of a certain significance in CD83 (%) 71.26±9.74 57.79±8.54 3.288 0.004 the clinical treatment of graft-ver- CD86 (%) 82.36±8.77 69.54±9.31 3.170 0.005 sus-host disease and autoimmune OD 0.69±0.14 0.85±0.17 2.297 0.034 diseases. IL-12 (ng/L) 266.52±11.26 216.48±15.37 8.305 <0.001 Acknowledgements

DC mature immune phenotype expression, the This study was supported by the Science and antigen presentation effect and the inflamma- Technology Program of Cangzhou, China tory factor IL-12 level. The above results con- (Program Number: 162302182). firmed that the CD200/CD200R pathway is involved in the process of MSC inhibiting DC Disclosure of conflict of interest maturation, and plays an important role in the None. stage of imDC trending to mDC. ImDC was selected to co-culture with MSCs with different Address correspondence to: Yulei Zhao, The Sec- CD200 positive rates, and the results showed ond Department of Hematology, Cangzhou Central that the immune phenotypic expression of Hospital, 16 West Xinhua Road, Yunhe, Cangzhou, mDC in the CD200 high expression group weak- China. Tel: +86-15720306668; E-mail: zhaoyu- ened, the inflammatory factor IL-12 level was [email protected] reduced, and the antigen presentation function was impaired. It further verified the regulatory References role of the CD200/CD200R pathway in the process of MSC inhibiting DC [21]. However, this [1] Uccelli A, Moretta L and Pistoia V. Mesenchy- study was an exploratory study, which has the mal stem cells in health anddisease. Nat Rev Immunol 2008; 8: 726-736. defect of an insufficient amount of supportive [2] Li N and Hua J. Interactions between mesen- research literature. This study only examined chymal stem cells and the immune system. specific hypotheses, and it lacked clinical Cell Mol Life Sci 2017; 74: 2345-2360. evidence. [3] Ding DC, Chang YH, Shyu WC and Lin SZ. Hu- man umbilical cord mesenchymal stem cells: a To sum up, the MSC inhibiting DC mechanism new era for stem cell therapy. Cell Transplant may be achieved through the CD200/CD200R 2015; 24: 339-347.

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