International Immunopharmacology 13 (2012) 219–224

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International Immunopharmacology

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Comparison of immunomodulatory effects of placenta mesenchymal stem cells withbone marrow and adipose mesenchymal stem cells

Jung Min Lee a, Jieun Jung b, Hyun-Jung Lee b, Su Jin Jeong c, Kyung Jin Cho d,Seong-Gyu Hwang a, Gi Jin Kim b,⁎a Department of Internal Medicine, CHA Bundang Medical Center, CHA University, 351 Yatap-dong, Bundang-gu, Seongnam 463-712, Republic of Koreab Placenta Research Laboratory, Department of Biomedical Science, CHA University, 606-1 Yeoksam 1-dong, Kangnam-ku, Seoul 135-081, Republic of Koreac Department of Pediatrics, CHA Bundang Medical Center, CHA University, 351 Yatap-dong, Bundang-gu, Seongnam 463–712, Republic of Koread Department of Clinical Laboratory Science, College of Health Science, Korea University, 1 JeongReung-Dong, Sungbuk-Gu, Seoul 136-702, Republic of Korea

Abbreviations: AD-MSCs, adipose-derived MSCs; Abone marrow-derived MSCs; CCl4, carbon tetrachloridderived mesenchymal stem cells; GM-CSF, granustimulating factor; HLA, human leukocyte antigen; IFNhistocompatibility complex; MNCs, mononuclear cells; MPDSCs, placenta-derived stem cells; TNF-α, tumor necrocord blood.⁎ Corresponding author at: Dept. of Biomedical Sc

Yeoksam 1-dong, Kangnam-ku, Seoul 135-081, Republ3687; fax: +82 2 538 4102.

E-mail address: gjkim@cha.ac.kr (G.J. Kim).

1567-5769/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.intimp.2012.03.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 January 2012Received in revised form 16 March 2012Accepted 27 March 2012Available online 6 April 2012

Keywords:Mesenchymal stem cellsImmunomodulatory effectsPlacentaBone marrowAdipose

Mesenchymal stem cells (MSCs) are powerful sources for cell therapy in regenerativemedicine because they canbe isolated from various tissues, expanded, and induced into multiple-lineages. Of note, their immunomodu-latory effects maximize the therapeutic effects of stem cells engrafted on host, making them an especiallyattractive choice. Recently, several varieties of placenta-derived stem cells (PDSCs) including chorionic plate-derivedMSCs (CP-MSCs) have been suggested as alternative sources of stem cells. However, comparative studiesof immunomodulatory effects for CP-MSCs among variousMSCs are largely lacking.We examined and comparedimmunomodulatory function of CP-MSCs with that of BM-MSCs and AD-MSCs using co-culture system withactivated T-cells derived from human umbilical cord blood (UCB) exposed to anti-CD3 and anti-CD28 which areT-cell activating monoclonal antibodies. All MSCs expressed markers of stem cells and three germ layers by RT-PCR. These cells also exhibited comparable immunomodulatory effects when they were co-cultured withactivated T-cells in dose-dependentmanner. However, expression of HLA-ABC and HLA-Gwas highly positive inCP-MSCs compared to otherMSCs, and higher levels of cytokines of IL-2, IL-4, IL-13, andGM-CSFweredetected indose-dependent manner in CP-MSCs. Taken together, the results of the present study suggest that while CP-MSCs, BM-MSCs, and AD-MSCs all have immunomodulatory effects, CP-MSCs may have additional advantageover the other MSCs in terms of immunomodulation. In conjunction with other previous studies, CP-MSCs aresuggested to be a useful stem cell source in cell therapy.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Mesenchymal stem cell (MSC) is defined by adherency to tissueculture plastic and capacity to differentiate into multiple lineages withprofiles of certain cell surfacemarkers [1]. MSCswere originally isolatedfrom bone marrow (BM) with subsequent isolation from other organsincluding adipose tissue, placenta, and umbilical cord blood [2–5].MSCshave gained much attention because they have demonstrated a greatpotential for clinical use with the capacity to differentiate not only into

FP, a-fetoprotein; BM-MSCs,e; CP-MSCs, chorionic plate-locyte macrophage colony--γ, interferon-gamma; MHC,SCs, mesenchymal stem cells;

sis factor-alpha; UCB, umbilical

ience, CHA University, 606-1ic of Korea. Tel.: +82 2 3468

rights reserved.

mesoderm but also endoderm and neuroectoderm [6,7]. Furthermore,the characteristics of differentiation in tissue-specific manner endow agreat promise to the use of these multipotent stem cells in the fields ofregenerative medicine [8,9].

Besides the regenerative function, another important potential ofMSCs is immune-related property [10]. In a number of in vitro studies,immunosuppressive effects of MSCs and their mechanism have beenwell described in which suppression of T-cell proliferation andinhibition of dendritic cell differentiation have been suggested asthe key events. Immunosuppressive effects have been furtherconfirmed in in vivo studies and are being evaluated in clinical trialsin diseases such as refractory graft-versus-host disease and Crohn'sdisease. In addition, immune privilege and hypoimmunogenicity ofMSCs are other aspects of immune-related property. Althoughcontroversy exists, there are evidences that MSC might be immune-privileged to freshly isolated NK cells preventing them from lysis andthat low expression of human leukocyte antigen (HLA) majorhistocompatibility complex (MHC) class I with no expression of co-stimulatory molecules may play an important role. These propertiesare anticipated to give advantage to survival and engraftment of MSCsin transplanted setting [11,12].

Table 1Sequence of primers and length of fragments used for RT-PCR.

Genes Sequence Tm (°C) Size (bp)

Oct4 F: 5′-ACA CTC GGA CCA CGT CTT TC-3′ 54 300R: 5′-CGT TCT CTT TGG AAA GGT GTT C-3′

Nanog F: 5′-TTC TTG ACT GGG ACC TTG TC-3′ 54 300R: 5′-GCT TGC CTT GCT TTG AAG CA-3′

Sox2 F: 5′-GGG CAG CGT GTA CTT ATC CT-3′ 52 200R: 5′-AGA ACC CCA AGA TGC ACA AC-3′

NF-68 F: 5′-GAG TGA AAT GGC ACG ATA CCT A-3′ 58 500R: 5′-TTT CCT CTC CTT CTT CTT CAC CTT C-3′

Cardiac F: 5′-GGA GTT ATG GTG GGT ATG GGT C-3′ 58 500R: 5-AGT GGT GAC AAA GGA GTA GCC A-3′

AFP F: 5′-AGC TTG GTG GAT GAA AC-3′ 50 200R: 5′-TCC AAC AGG CCT GAG AAA TC-3′

HLA-G F: 5′-GCG GCT ACT ACA ACC AGA GC-3′ 58 900R: 5′-GCA CAT GGC ACG TGT ATC TC-3′

TERT F: 5′-GAG CTG ACG TGG AAG ATG AG-3′ 55 300R: 5′-CTT CAA GTG CTG TCT GAT TCC AAT G-3′

β-actin F: 5′-TCC TTC TGC ATC CTG TCA GCA-3′ 58 300R: 5′-CAG GAG ATG GCC ACT GCC GCA-3′

220 J.M. Lee et al. / International Immunopharmacology 13 (2012) 219–224

MSCs from diverse origins are currently available, and each typehas its own strength and shortcomings. Recently, placenta-derivedMSCs (PDSCs) have been suggested as an alternative source of stemcells. Similar to other MSCs derived from bone marrow and adipose,PDSCs were shown to differentiate into 3 germ layers and haveimmunomodulatory properties [13,14]. The merits of using PDSCs liein that they are free from ethical concern in procurement and thatrelatively large amount of PDSCs can be readily obtained. Moreover,from a developmental point of view, placenta is an organ thatoriginates during the early period of embryological stages and isfundamental for maintaining fetomaternal tolerance, which possiblyindicate that PDSCs might harbor similar traces as well [15,16]. Ourgroup has recently shown that chorionic plate-derived mesenchymalstem cells (CP-MSCs), a subset of PDSCs, can be used in in vitroscreening system for hepatotoxicity, in which CP-MSCs were betterthan the bone marrow-derived mesenchymal stem cells (BM-MSCs).We also performed in vivo study to document the feasibility of CP-MSCs in ameliorating liver damage in CCl4-induced cirrhotic ratmodel [17,18]. However, no study has been conducted yet to directlycompare immune-related aspects of CP-MSCs with other MSCs.

Therefore, in this study, we investigated differences in immuno-modulatory function between CP-MSC and other MSCs including BM-MSCs and adipose-derived MSCs (AD-MSCs).

2. Materials and methods

2.1. Culturing of CP-MSCs, BM-MSCs, and AD-MSCs

Full term normal human placentas (gestation≥37 weeks) withoutmedical, obstetrical, or surgical complications were collected afterinformed consent. Sample collection and utilization for this researchwere approved by the Institutional Review Board of CHA GeneralHospital, Seoul, Korea. To isolate CP-MSCs, the chorioamnioticmembrane was peeled off and separated from the chorionic plate ofplacenta. They were chopped into small pieces, then washed withphosphate-buffered saline (PBS), and initially digested with 0.5%collagenase IV in PBS at 37.8 °C for 40 min, followed by vigorousshaking for 15 min at 15-min intervals over 90 min in 0.25%collagenase IV at 37.8 °C. An equal volume of Dulbecco's modifiedEagle's medium (DMEM; Gibco-Invitrogen, Grand Island, NY) con-taining 10% fetal bovine serum (FBS; Gibco-Invitrogen) was addedand centrifuged at 1000 g for 5 min to get the cell pellet. Theharvested cells were resuspended in the culture medium containedDMEM/F12 (Gibco-BRL) supplemented with penicillin (100 U/ml),streptomycin (100 mg/ml), 25 ng/ml FGF4 (Peprotech, Inc., NJ), 1 mg/mlheparin (Sigma), 50 mg/ml gentamicin (Gibco-BRL), and 10% FBS(Gibco-BRL) and then incubated at 37.8 °C in an incubator with 5%CO2. BM-MSCs (Cambrex Bioscience Walkersville, East Rutherford, NJ),AD-MSCs (provided by Dr. JH Sung, CHA University, Korea), and normalfibroblast cell line WI-38 (ATCC; Manassas, VA) were cultured using aculture medium containing alpha-MEM (Gibco-BRL-Invitrogen), sup-plemented with penicillin (100 U/ml), streptomycin (100 mg/ml),1 mM sodium pyruvate (Gibco-BRL), and 10% FBS (Gibco-BRL).

2.2. RT-PCR

For RT-PCR analysis, CP-MSCs, BM-MSCs, AD-MSC, and WI38 werehomogenized and lysed in 1 ml of TRIzol (Invitrogen, Carlsbad, CA).Total RNA was extracted with 200 ml of chloroform and precipitatedwith 500 ml of 80% (v/v) isopropanol. After the supernatant wasremoved, the RNA pellet was washed with 75% (v/v) ethanol, air-dried, and dissolved in 0.1% (v/v) diethyl pyrocarbonate-treatedwater. The RNA concentration was determined by measuringabsorbance at 260 nm using a spectrophotometer. A reverse tran-scription reaction was performed with 1 mg of total RNA andSuperScriptTM III reverse transcriptase (Invitrogen). The cDNA was

amplified using h-Taq DNA polymerase (Solgent, Seoul, Korea),according to the manufacturer's instructions. First-strand cDNAswere amplified in a final volume of 20 ml containing 0.5 U Taq DNApolymerase (Solgent) and 20 pmol of each human-specific targetprimers. The PCR primers and the size of the amplified products areshown in Table 1. Amplification reactions were performed on thefollowing conditions: denaturation at 95.8 °C for 15 min followed by35–40 cycles of denaturation at 95.8 °C for 30 s, annealing at50–60.8 °C for 40 s, and elongation at 72.8 °C for 5 min. The PCRproducts were visualized and photographed following electrophore-sis on a 1% (w/v) agarose gel containing 0.5 mg/ml ethidium bromide.cDNA samples were adjusted to yield equal actin amplifications.

2.3. T-cell proliferation analysis using ELISA

To assess the ability of MSCs to suppress T-cell proliferation, MSCswere first treated with mitomycin C of 50 μg/ml for 50 min toinactivate the proliferation of MSCs themselves. Next, humanumbilical cord blood (UCB) mononuclear cells (MNCs) were preparedby centrifugation on a Ficoll Hypaque density gradient, and 2×105

cells of UCB MNCs were co-cultured with 2×103, 4×103, 1×104, and2×104 cells of each MSC per well in 96-well culture plate with orwithout 1 μg/ml anti-CD3 and anti-CD28 T-cell activating mAbs(eBioscience, Inc., San Diego, CA, USA) for 72 h. To analyze thesuppression of proliferative response of T-cell clonal expansion,clustering of T-cells was examined in bright field and BrdU ELISA(Roche) was performed according to the manufacturer's protocol at72 h cultivation.

2.4. Flow cytometry analysis

For flow cytometry analysis, CP-MSCs, BM-MSCs, AD-MSC, andWI-38 (5×105 cells) were dissociated with cell dissociation buffer(Gibco-Invitrogen) and washed with PBS containing 2% (v/v) FBS.They were incubated with isotype control IgG or antigen-specificantibodies with various fluorescence-conjugated anti-human IgGantibodies (diluted 1:200; BD Biosciences, San Diego) for 30 minand propidium iodide (PI, 5 ng/ml; Sigma-Aldrich) was used toidentify nonviable cells. Flow cytometry analysis was performedusing a vantage Flow Cytometer (BD Biosciences, San Jose, CA).

2.5. Multiplex supernatant cytokine assay (Luminex)

The supernatant from co-culture of MSCs and T-cells washarvested with or without 1 μg/ml anti-CD3 and anti-CD28 activatingmAbs after 72 h cultivation, and 50 μl of supernatant was combined

221J.M. Lee et al. / International Immunopharmacology 13 (2012) 219–224

with coated beads in MILLIPLEXTMMAP kit (Millipore Corp., Billerica,MA, USA). Commercial kits were run in individual plates with buffersand standards according to the previous reports [19]. Incubations andwashes were done in 1.2-μm filter membrane 96-well microtiterplates (Millipore Corp.). After the final wash, beads from the 96-wellmicrotiter plates were resuspended in a 125 μl cuvette of a Luminexinstrument. An acquisition gate was set between 7500 and 13,500 fora doublet discriminator, sample volume was 75 μl, and 100 events/region were acquired. Raw data (mean fluorescence intensity) fromall the bead combinations tested were analyzed with Master PlexQT3.0 quantification software (MiraiBio Inc., Alameda, CA, USA) inorder to obtain concentration values. All samples were assayed induplicate and analyzed with a Luminex 200 Labmap system (Luminex,Austin, TX, USA). Data analyseswere performedusing Bio-PlexManagersoftware version 4.1.1 (Bio-Rad Laboratories). Cytokine/chemokineconcentrations were interpolated from an appropriate standardcurve.

2.6. Statistical analysis

Results are presented as the means±SD. Statistical significancewas tested using t-test with a significance level of Pb0.05.

3. Results

3.1. Expression of stemness-related markers of CP-MSCs, BM-MSCs, andAD-MSCs

Fig. 1A shows the typical spindle shape of MSCs from chorionicplate of placenta, bone marrow, and adipose. To analyze theexpression for stemness markers, RT-PCR for Oct-4, Nanog, Sox-2,NF-68, cardiac muscle, AFP, and HLA-G were investigated among CP-MSCs, BM-MSCs, and AD-MSC (Fig. 1B). Oct-4, a specific marker ofembryonic stem cells was strongly detected in CP-MSCs and BM-MSCs, whereas it was weakly observed in AD-MSCs. Nanog, Sox2, andTERT, which are markers of stem cells, were also detected. In addition,NF68, cardiac muscle, and α-fetoprotein (AFP), which are markers ofthree germ layers, and HLA-G, an immunomodulatory gene were allexpressed in CP-MSCs, BM-MSCs, and AD-MSCs. These results indicatethat these MSCs express genes compatible with the ones expressed byMSCs able to self-renew and differentiate in multiple lineages.

Fig. 1. Morphology and marker expression in MSCs derived from placenta, bonemarrow, and adipose. (A) The morphology of CP-MSCs, BM-MSCs, and AD-MSCs allshow typical round-spindle shape of MSCs. (B) RT-PCR analysis for markers of stemcells and three germ layers in MSCs derived from placenta, bone marrow, and adipose.MSCs: mesenchymal stem cells, CP-MSCs: chorionic plate-derived MSCs, BM-MSCs:bone marrow-derived MSCs, AD-MSCs: adipose-derived MSCs. Scale bars: 50 μm.

3.2. Analysis of T-cell inhibition using co-culture with MSCs

When we cultured mononuclear cells (MNCs) isolated fromhuman umbilical cord blood (UCB) with anti-CD3 and anti-CD28 T-cell activating mAbs for 3 days, clustering of activated T-cells wasincreased. We determined whether MSCs had immunomodulatoryeffects in co-culture system with UCB MNCs and thus inhibitedproliferation and aggregation of activated T-cells. As shown in Fig. 2A,regardless of number of cells, fibroblast WI38 as a control did notcause any change in aggregation of UCB MNCs after activation withanti-CD3/CD28. However, starting with cell number of 4×103, CP-MSCs, BM-MSCs, and AD-MSCs all showed suppressive effects onproliferation and aggregation of UCBMNCs, which were more evidentwith increasing number of co-cultured MSCs. In comparing amongMSCs, AD-MSCs seemed to have slightly superior inhibitory effects onactivated T-cells than CP-MSCs or BM-MSCs. These results indicatethat all MSCs co-cultured with activated UCBMNCs have an inhibitoryeffect in dose-dependent manner.

3.3. T-cell proliferation using BrdU ELISA assay

To quantify and compare the immunomodulatory effects of MSCson activated T-cells, we performed BrdU ELISA assay in T-cells afterco-culture with MSCs. Ratio of with and without anti-CD3/CD28activation was calculated and used as a measure to indicate theeffects. As shown in Fig. 2B, there was no change in ratio with varyingnumber of cells in experiments with fibroblast WI38 as a control.However, this was significantly contrasted with experiments withMSCs, which showed decreasing ratio with increasing number ofcells, indicating that proliferation of T-cells was suppressed with co-culture of MSCs (Pb0.05). Notably, CP-MSCs showed a clear reductionof the ratio in dose-dependent manner with increasing cell number.These findings confirm that MSCs have an inhibitory effect onactivated T-cells.

3.4. Expression analysis of HLA phenotypes in MSCs using flow cytometryanalysis

It is well known that the patterns of HLA expression are importantin immunomodulation. To confirm the expression of surface pheno-types of HLA, MSCs were analyzed with flow cytometry using mouseanti-human antibodies. The expression of HLA-DR was negative in allMSCs. On the other hand, the expression of HLA-ABC was detected toa varying degree according to cell type, in which the expression washigher in CP-MSCs (96.7%) than in BM-MSCs (87.2%), and AD-MSCs(86.4%) (Fig. 3). Notably, the expression of HLA-G, which plays a rolein immune tolerance, was highly positive in CP-MSCs reaching 84.2%,compared to 58.7% in BM-MSCs and 64.6% in AD-MSCs. These dataindicate that there is a notable difference in expression pattern ofHLA-ABC and HLA-G among MSCs suggesting a superior immunesuppressive function of CP-MSCs.

3.5. Comparison of cytokine expression of MSCs by multiplex supernatantcytokine assay

To analyze the profiles of cytokines related to immunomodulationin MSCs, we performed multiplex human cytokine assay thatincluded IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFN-γ, TNF-α, and GM-CSF (Fig. 4). Compared to normal fibroblast cell line, WI-38, lowerconcentration of IFN-γ and IL-10 was observed in supernatant ofMSCs. Of note were the cytokines of IL-2, IL-4, IL-13, and GM-CSF,which were detected in higher concentrations in dose-dependentmanner in CP-MSCs compared with BM-MSCs and AD-MSCs. Inaddition, RANTES was assayed. The concentration was much higher inCP-MSCs than in other MSCs, starting with cell number of 4×103.These results indicate that the secretion of various cytokines by MSCs

Fig. 3. Expression of surface markers for immunomodulation in MSCs derived from placenta, bone marrow, and adipose using flow cytometry analysis. 5x105 cells were incubatedwith isotype control IgG or antigen-specific antibodies with various fluorescence-conjugated anti-human IgG antibodies and propidium iodide (PI) used to identify nonviable cells.The expression of HLA-ABC was detected to a varying degree according to cell type. Notably, the expression of HLA-G, which plays a role in immune tolerance, was highly positive inCP-MSCs. The percentages are indicated along with the fluorescence intensities. MSCs: mesenchymal stem cells, HLA: human leukocyte antigen, CP-MSCs: chorionic plate-derived MSCs,BM-MSCs: bone marrow-derive MSCs, AD-MSCs: adipose-derived MSCs, WI-38: normal fibroblast cell line.

Fig. 2. Immunomodulatory effects of MSCs derived from placenta, bone marrow, and adipose on activated T-cells depend on co-cultured MSCs in dose-dependent manner. (A)MNCs isolated from umbilical cord blood were cultured with or without 1 μg/ml anti-CD3 and anti-CD28 for 72 h, which are T-cell activating mAbs, and clustering of activated T-cells decreases with increasing number of co-cultured MSCs. Scale bar=50 μm. (B) Proliferation activities of T-cells in co-cultured MSCs were determined by BrdU ELISA analysis at72 h cultivation. With increasing number of co-cultured MSCs, the suppressive effects on T-cell activation increases. MSCs: mesenchymal stem cells, MNCs: mononuclear cells, CP-MSCs: chorionic plate-derived MSCs, BM-MSCs: bone marrow-derive MSCs, AD-MSCs: adipose-derived MSCs. WI-38: normal fibroblast cell line.

222 J.M. Lee et al. / International Immunopharmacology 13 (2012) 219–224

Fig. 4. Multiplex supernatant cytokine assay of co-culture of MSCs and activated T-cells shows association of cytokine concentration in dose-dependent manner of MSCs. MSCs:mesenchymal stem cells, CP-MSCs: chorionic plate-derived mesenchymal stem cells, BM-MSCs: bone marrow-derived MSCs, AD-MSCs: adipose-derived MSCs, WI-38: normalfibroblast cell line, GM-CSF: granulocyte macrophage colony-stimulating factor, IFN-γ: interferon-gamma, TNF-α: tumor necrosis factor-alpha, IL: interleukin.

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may play a role in mediating the inhibition of T-lymphocyteaggregation and proliferation and that large amount of cytokinessecreted specifically from CP-MSCs can more effectively enhanceimmune suppression than other MSCs.

4. Discussion

Cell therapy using stem cells has gained popularity since stemcells have potentials to overcome the limitations of conventionaltherapies. As such, several candidates of stem cells have beensuggested and are currently being investigated. Yet one importantquestion that remains to be answered is which cell is most effectiveand suitable among the various stem cells. Traditionally, BM-MSCshave been of most interest, but BM-MSCs have their own limitationsthat prevent wider application including necessity to accompanyinvasive procedure to procure and decreasing capacity and number ofcells to differentiate with increasing age [20,21]. AD-MSCs areanother rising candidate for stem cell therapy because they havesimilar ability and differentiation potentials as BM-MSCs [22]. Of noteare recent investigations and a review that have suggested a newhope for PDSCs because these stem cells are free from ethicalconcerns, easy to procure, available in large amount, and multipotentto differentiate into three germ layers [15]. The utility of PDSCs isbeing explored towards wider range of areas and is incorporated inclinical trials as well based on the reports of their superiorimmunomodulatory effects. Indeed, considering the fundamentalrole of placenta as a bridge between mother and fetus, theimmunomodulatory function might be inherited as well in the

PDSCs [23]. However, a comparative study of PDSCs with variousMSCs according to the origin and the localization has not beensystematically conducted yet in regard to the immunomodulatoryfunction [13]. Therefore, we herein report a distinctive and possiblysuperior immunoregulatory function of CP-MSCs in comparison withBM-MSCs and AD-MSCs.

T-cells are the primary cells in immune response, and suppressiveeffects of MSCs towards activated T-cells with highly potent monoclonalanti-CD3/CD8 antibodieswere evaluated and compared in this study [24].Co-culturedMSCs all showed inhibitory effectswith increasing number ofcells, and although a slightly earlier suppression with AD-MSCs thanBM-MSCs or CP-MSCswas observed in T-cell proliferation analysis, it wasnot evident in BrDU ELISA assay, in which dose-dependently decreasingratio was well observed with CP-MSCs.

Type and expression of HLA is an important phenotypewith regard toimmune regulation. In this study, CP-MSCs were positive for HLA-ABCand negative for HLA-DR, which are similarly expressed in BM-MSCs andAD-MSCs [25–27]. Interestingly, in contrast to BM-MSCs and AD-MSCs,HLA-G was strongly positive only in CP-MSCs, consistent with previousreport [14]. HLA-G is a specific MHC-I antigen that is critical to maintainimmune-tolerant state of pregnancy and is indicated as a contributingfactor to inducing a stronger immunosuppression [28]. Moreover, severalstudies of transplantation have documented the role of HLA-G as aprognostic indicator of graft tolerance [29]. Taken together, these datasuggest that immunophenotype of CP-MSCs may be superior to otherMSCs in terms of immunosuppressive function.

An important mechanism through which MSCs have beneficialeffects in cell therapy is by secretion of cytokines in paracrine

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manner. In this study, major cytokines in association with pro-inflammatory and anti-inflammatory function were analyzed andcompared. IL-4 and IL-13 are major anti-inflammatory cytokines, andit is intriguing that these two cytokines were increasing in dose-dependent manner with CP-MSCs. On the other hand, IFN-γ is amajor pro-inflammatory cytokine, and was detected in lower levelwith all MSCs. A noteworthy finding was a distinctly high concentra-tion of IL-2 in CP-MSCs in comparison with BM-MSCs and AD-MSCs.Although IL-2 is historically described as a T-cell growth factor invitro, it is also known to predominantly mediate tolerance in vivo, andthe high amount detected with CP-MSCs may be attributed to suchfunction of IL-2 [30]. Another higher concentration finding with CP-MSCs was noted in the analysis of TNF-α, and we assume that thisphenomenon is related to the function of TNF-α that it can induce theproduction of immunosuppressive prostaglandins in MSCs by asmuch as 100-fold [31]. All these results taken together demonstratethat CP-MSCs secrete cytokines in different fashion compared to BM-MSCs and AD-MSCs with possibly a better profile from immunosuppres-sive point of view, although actual function of cytokine networks shouldbe further validated and understood in the context of in vivo condition.

Currently, the major properties of stem cells that are consideredimportant in cell therapy are regenerative capacity including differenti-ation into appropriate tissue and immunomodulatory effects. CP-MSCshave been shown to have not only abovementionedmajor properties butalso anti-fibrotic effects [18,32], Although our study was conducted onlyin ex vivo setting, which is thus a limitation of our study, we furtherdemonstrate that CP-MSCs may have superior aspects in terms ofimmunomodulation compared to BM-MSCs and AD-MSCs, all of whichprovide good evidences of CP-MSCs as a useful stem cell source. Notsurprisingly, based on these grounds, several groups already initiatedclinical studieswith PDSCs in several diseases including Crohn's disease,rheumatoid arthritis, and limb ischemia (http://www.clinicaltrials.gov). It is intriguing that PDSCs can be applied to expect immunosup-pressive role in allogenic transplantation setting, which, if successful,would mean a competitive edge of PDSCs against the BM-MSCs orAD-MSCs of autologous transplantation. It is therefore anticipated thatif positive results are drawn from these clinical trials, the impact ofPDSCs in cell therapy will be remakarble.

In conclusion, CP-MSCs, BM-MSCs, and AD-MSCs all have immu-nomodulatory effects, and CP-MSCs may have advantage over theother MSCs. In conjunction with other previous studies, CP-MSCs aresuggested to be a useful stem cell source. Further studies should beconducted to validate these data.

Acknowledgments

Authors are grateful to Hey-Sun Kim at FACS Core Facility at CHAStem Cell Research Institute, CHA University for excellent technicalassistance and special thanks to Dr. Jong-Hyuk Sung (CHA University,Korea), who has provided the adipose-derived mesenchymal stemcells (AD-MSCs). This study was supported by The Korea HealthcareTechnology R&D Project, Ministry for Health Welfare & Family Affairs,Republic of Korea (Grant number: A084633) and the Korea ResearchFoundation Grant funded by the Korean Government (MEST) (Grantnumber: KRF-2008-313-E00247).

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