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Immunology Letters 162 (2014) 239–246

Contents lists available at ScienceDirect

Immunology Letters

j ourna l ho me page: www.elsev ier .com/ locate / immlet

enstrual blood-derived stromal stem cells inhibit optimaleneration and maturation of human monocyte-derivedendritic cells

ahmood Bozorgmehra,b, Seyed Mohammad Moazzenia, Mojdeh Salehniac,li Sheikhiand,∗, Shohreh Nikooe, Amir-Hassan Zarnanib,f

Immunology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IranNanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, IranAnatomy Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IranImmunology Department, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, IranReproductive immunology Research Center, Avicenna Research Institute, ACECR, Tehran, IranImmunology Research Center, Iran University of Medical Sciences, Tehran, Iran

r t i c l e i n f o

rticle history:vailable online 18 October 2014

eywords:enstrual blood stromal stem cellendritic cellonocyte

L-6L-10

a b s t r a c t

Introduction: Menstrual blood stromal stem Cells (MenSCs) have shown promising potential for futureclinical settings. Nonetheless, data regarding their interaction with immune cells is still scarce. Here, weinvestigated whether MenSCs could affect the generation and/or maturation of human blood monocyte-derived dendritic cells (DCs).Materials and methods: MenSCs were isolated from menstrual blood of normal women through cultureof adherent mononuclear cells. Magnetically-isolated peripheral blood monocytes were differentiatedtoward immature DCs (iDC) and mature DCs (mDCs) in the presence or absence of MenSCs. Monocyte-derived cells were assessed for the percentage of monocyte-, iDC-, and mDC-specific markers as well asthe expression of costimulatory molecules. IL-6 and IL-10 levels were also determined in supernatantsof MenSC-monocytes cocultures.Results: Optimal phenotypic differentiation of monocytes into iDCs was inhibited upon coculture withMenSCs. Moreover, higher levels of IL-6 and IL-10 were detected in these settings. Even though additionof MenSCs to iDC cultures could not prevent iDC maturation, coculture of MenSCs with monocytes fromthe beginning of differentiation process could effectively hinder generation of fully mature DCs.

Conclusion: This is the first study to address the inhibitory impact of MenSCs on generation and maturationof DCs. IL-6 and IL-10 could be partly held responsible for this effect. Given the central roles of DCsin regulation of immune responses, these results highlight the importance of further research on thepotential modulatory impacts of MenSCs, as rather easily accessible and expandable stem cells, on theimmune system-related cells.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Utilization of stem cells has provided new perspectives on treat-ent of serious diseases such as autoimmunity, graft versus host

isease (GVHD), and cancer. Stem cells are divided into two gen-ral categories: embryonic and adult stem cells. Embryonic stemells possess a high proliferative capacity; nevertheless, their usage

∗ Corresponding author at: Immunology Department, Faculty of Medicine,amalvand district, Khorramabad, Iran. Tel.: +98 9166616974.

E-mail address: alisheikhian@gmail.com (A. Sheikhian).

ttp://dx.doi.org/10.1016/j.imlet.2014.10.005165-2478/© 2014 Elsevier B.V. All rights reserved.

poses the risk of malignancies and therefore limits their clinicalusage [1]. Adult stem cells, on the other hand, have been widelyrecovered from sources such as bone marrow [2], umbilical cordblood [3], and adipose tissue [4], and are considered to be of highclinical value. Nonetheless, utilization of these cells has also provedto be troublesome due to several limitations including invasive anddifficult isolation methods, and rather limited proliferative and/ordifferentiating capacity in case of certain stem cell subsets.

Menstrual blood stromal stem cells (MenSCs), originate fromthe endometrial layer of uterus, and are known as a type ofmesenchymal-like stem cell, with considerable potential for clin-ical applications; in fact, in some publications they are given

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ther names such as menstrual blood mesenchymal stem cellsnd endometrial regenerative cells [5–11]. With respect to theirmmunophenotypic features, MenSCs have been reported to beositive for markers such as CD9, CD10, CD29, CD44, CD73, CD90,D105, and OCT-4A, while negative for CD34, CD38, CD45, CD133,nd STRO-1 [5,7,10]. They are able to selfrenew and differentiatento cells of all three germ layers [5,7]. Ease of access and culture,igh proliferation rate, lack of karyotypic abnormality even after8 cell divisions and secretion of relatively high levels of angio-enic factors [5,10] are among the features proposing MenSCs as aotential tool to be used in the clinic.

In fact, the aforementioned MenSC capacities have been theocus of a number of recent studies. In this regard, based on theirigh angiogenic capacity, anti-inflammatory properties, and lackf immunogenicity, allogenic MenSCs were successfully utilized toreat an experimental model of critical limb ischemia [12]. Further-

ore, one recent study demonstrated that when injected into aat model of infarction, MenSCs could efficiently transdifferenti-te into cardiomyocytes and restore cardiac function resulting inecreased myocardial infarction areas [13]. Notably, it was shownhat this type of menstrual blood-derived stem cell could be useds a new source with the capacity to produce inducible pluripotenttem cells (iPSCs), which are well known as promising candidatesn regenerative medicine [11].

Interestingly, thus far, there are only a few published reportsnvestigating MenSC impacts on immune responses and/or cellsnd there is a great lack of information in this regard [14]. Onetudy demonstrated that MenSCs could modulate a mixed leuko-yte reaction (MLR) and shift the cytokine responses toward the

helper 2 (Th2) profile [12]. Moreover, our group showed thatepending on their concentration MenSCs could either inhibit ortimulate an allogenic MLR [15]. In general, MenSCs seem to beore similar to mesenchymal stem cells (MSCs) with respect to

mmunophenotypic and functional features.Dendritic cells (DCs) are known as the most professional antigen

resenting cells with a pivotal role in regulating immune responseshrough skewing T cells toward Th1, Th2, Th17, or Treg [16,17].mmature or semi-mature DCs express low levels of costimulatory

olecules and induce immunological tolerance, while perfectlyatured DCs increase the expression of costimulatory molecules

nd promote robust T cell responses [16,18].Given all the mentioned advantages, MenSCs could be regarded

s potential candidates to be used next to the previously discov-red stem cells subsets in clinical settings [14]. Nonetheless, akin tohat has been done in the case of other stem cell types, there is still

he need to unravel the probable mechanisms through which Men-Cs may affect different arms of the immune system. Hence, in theresent study, we investigated MenSC impacts on the generationnd maturation of human monocyte-derived DCs. The expressionf monocyte-, immature dendritic cell (iDC)-, and mature den-ritic cell (mDC)-specific-markers was investigated on the surfacef monocyte-derived cells recovered from MenSC-monocyte orenSC-iDC cocultures. Moreover, the level of interleukin-6 (IL-6)

nd IL-10, as key molecules known to inhibit monocyte to immatureendritic cell differentiation, was assessed in MenSC-monocyteocultures.

. Materials and methods

.1. Enrichment and culture of MenSCs

With respect to isolation procedure as well as marker expres-ion of menstrual blood-derived stromal stem cells, there exist aew differences between previously published studies. Menstruallood-derived cells investigated in this study and in our previous

Letters 162 (2014) 239–246

works are more similar to those assessed by Meng et al. and Mur-phy et al. MenSCs were isolated from menstrual blood accordingto the protocol explained elsewhere by our group [15,19–23]. Inbrief, menstrual blood samples were obtained from normal womenusing Urine Cups (Diva Cup, Sweden) on day 2 of the menstrualphase and were immediately transferred into 15 mL conical tubes(Greiner, Germany) containing phosphate buffered saline (PBS)supplemented with 5 mM ethylenediaminetetraacetic acid (EDTA)(Merck, Germany), fungizone (0.25 �g/mL), penicillin (100 IU), andstreptomycin (100 �g/mL) (all from Sigma, USA). Mononuclear cellswere consequently separated using Ficoll-Hypaque density gradi-ent medium (Biosera, USA). Upon washing with PBS, cells wereresuspended in DMEM-F12 medium (Gibco, Germany) contain-ing the above-mentioned antibiotics plus 10% fetal bovine serum(FBS) (Gibco, Germany). Thereafter, the mononuclear cells wereplated into either 25 cm2 or 75 cm2 cell culture flasks (Greiner,Germany) and were cultured in a humidified incubator at 37 ◦Cand 5% CO2. Following an overnight culture, the non-adherent cellswere removed by washing and the adherent portion underwent2–4 passages to omit unwanted cells including monocytes andmacrophages. Finally, the firmly adhered MenSCs were detachedusing Tripsin-EDTA (Gibco, Germany) and frozen for future exper-iments. MenSCs used in all experiments were from passages 4–6.The purity of MenSCs was determined by flow cytometry. Informedwritten consent was obtained from all participants, and all studyprocedures were approved by the local ethical committee.

2.2. Generation of iDCs

DCs were generated in accordance with previously describedprotocols [24]. Briefly, to prepare a sufficient number of monocytes,buffy coat samples were obtained from Iran Blood TransfusionCenter. Mononuclear cells were isolated using Ficoll-Hypaqueand platelets were excluded via low-speed centrifugation. Mono-cytes were positively enriched using microbeads conjugated toanti-CD14 antibody (Ab) according to manufacturer’s instructions(Miltenyibiotec, Germany). The purity of monocytes was confirmedby flow cytometry. Freshly prepared monocytes were cultured in6-well plates containing RPMI-1640 medium supplemented withpenicillin (100 IU), streptomycin (100 �g/mL), l-glutamine (2 mM)(all from Sigma, USA), non-essential amino acids (1×), sodiumpyruvate (1×), and 10% FBS (all from Gibco, Germany) in thepresence of recombinant IL-4 (20 ng/mL) and granulocyte mono-cyte colony-stimulating factor (GM-CSF) (50 ng/mL) (both fromPeprotec, USA). Half of the medium was replaced with freshlycytokine-supplemented medium on day 3 of culture and the culturewas continued for another 2 days. On day 5, iDCs were collected bygentle aspiration and analyzed for the expression of iDC-specificmarkers by flow cytometry.

2.3. Differentiation of monocytes toward iDC in the presence ofMenSCs

MenSCs (not irradiated) were cultured in 6-well plates at4 × 105 cells/well in RPMI-1640 containing 10% FBS, 2 mM l-glutamine, non-essential amino acids (1×), and sodium pyruvate(1×) for 6–8 h. Thereafter, 1 × 106 freshly isolated monocytes (withthe mean purity of 94%) in the presence of proper concentra-tions of IL-4 and GM-CSF (described in the previous section)were plated with MenSCs to reach a MenSC/monocyte ratio of1:2.5. Additionally, a titration of cocultures was also carried out

at 1:5, 1:10, 1:20, and 1:50 (MenSC/monocyte) ratios. After fivedays, monocyte-derived cells generated in the presence of MenSCs(named MenSC-iDCs) were gently harvested from cocultures andassessed for the expression of monocyte- and iDC-specific markers.

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s the control group, monocytes were differentiated toward iDCsn the absence of MenSCs (Alone iDC).

.4. Differentiation of iDCs toward mDCs in the presence ofenSCs

Monocytes were differentiated toward iDCs as described previ-usly, in the absence of MenSCs. On day 5, MenSCs were added toDC cultures at 1:2.5 (MenSC/monocyte) ratio and maturation wasnduced with addition of LPS (Sigma, USA) for 2 additional days. Onay 7, DCs generated in the absence but matured in the presence ofenSCs (named iDC + [MenSC & LPS]) were gently harvested and

ssessed for the expression of DC- and monocyte-related mark-rs. As the control group, iDCs generated in the absence of MenSCere further matured by addition of LPS in the absence of MenSCs

named Alone iDC + LPS).

.5. Generation of mDCs in the presence of MenSCs

Monocytes cocultured with MenSCs (1:2.5 MenSC/monocyteatio), were differentiated toward iDCs (IL-4 + GM-CSF) and thennduced for maturation using LPS. The cells which were differen-iated in the presence of MenSCs were harvested after 7 days ofoculture (named MenSC-mDC) and investigated for expression ofC- and monocyte-specific markers. As for the control group, bothroduction and maturation of DCs were performed in the absencef MenSCs (Alone mDC).

.6. Flow cytometry experiments

To investigate monocyte and DC immunophenotypes, FITC-onjugated monoclonal Abs against CD1a and HLA-DR, andE-conjugated monoclonal Abs against CD14, CD40, CD80, CD83,nd CD86 as well as isotype-matched control Abs (all from BD Bio-ciences, USA) were used. DCs were incubated in staining bufferPBS-FBS 2%) with fluorochrome-conjugated Abs at 4 ◦C. Unboundeagents were subsequently removed using extensive washing andhe cells were resuspended in staining buffer. Expression of DC-pecific markers was determined by flow cytometry. To comparehe density of CD40, CD80, CD83, CD86, and HLA-DR we used meanatio fluorescence intensity (MRFI) [24]. This parameter is the ratioetween the mean fluorescence intensity (MFI) of cells stained withhe Ab of interest and that of the same population of cells stainedith the isotype-matched control Ab. With respect to CD1a (DC

pecific marker) and CD14 (monocyte specific marker), the per-entage of positive cells was determined.

To analyze the immunophenotype of MenSCs, FITC-conjugatednti-CD9, CD34, CD38, and CD133 as well as PE-conjugatednti-CD10, CD29, CD44, CD45, CD73 monoclonal Abs were allurchased from BD Biosciences (USA). PE-conjugated anti-CD105as obtained from R&D Systems (USA). Staining for OCT-4Aas performed using a primary Ab (polyclonal rabbit-anti-humanct-4A; Abcam, USA) and a secondary Ab (FITC-conjugated goatnti-rabbit FC, from Sigma, USA). STRO-1 expression was alsoetermined using a primary monoclonal mouse-anti-human Stro-1b (R&D Systems, USA) and a secondary FITC-conjugated sheep-nti-mouse Ab (Avicenna Institute, Iran). All the flow cytometryxperiments were evaluated by a Partec Flow cytometer (Germany)nd the data were analyzed using the Flowjo software (version:.6.1.).

All MenSC-related staining procedures and analysis methods

ere carried out in accordance with a previous publication by our

roup [15] and flow cytometry results of the present study provedroper enrichment of MenSCs (Data not shown). Moreover, theapacity of MenSCs to differentiate into cells of different lineages

Letters 162 (2014) 239–246 241

has been previously shown by our colleagues who have used thesame cell source as the one used in the current study [19,22,25–27].

2.7. Cytokine assay

Monocytes were differentiated toward iDCs either in the pres-ence or absence of MenSCs. After 5 days the supernatants ofthe cultures were collected and frozen at −80 ◦C for cytokineassays. IL-6 and IL-10 levels were determined using Sandwich ELISAaccording to manufacturer’s instructions (BD Biosciences, USA). Thelowest detection limits for IL-6 and IL-10 were 3.1 and 7.8 pg/mL,respectively. All the experiments were carried out in duplicates.

2.8. Statistical analysis

All the statistical analyses and comparisons between groupswere performed using GraphPad Prism version 6.00 for Windows,GraphPad Software, La Jolla, CA, USA, www.graphpad.com. Eitherpaired sample t or Wilcoxon test was used to compare the results.Descriptive data were reported as mean ± SEM. P values less than0.05 were interpreted as statistically significant, and values lessthan 0.05, 0.01, 0.001, and 0.0001 were indicated with *, **, ***, and****, respectively.

3. Results

3.1. MenSCs effectively interfere with generation of iDCs frommonocytes

To generate iDCs from monocytes, CD14+ cells were purifiedfrom normal blood samples and differentiated into iDCs using acocktail of IL-4 and GM-CSF. The cultures were set either in thepresence or absence of MenSCs. On day 5, monocyte-derived cells(MenSC-iDC or Alone iDC) were assessed for the expression ofmonocyte- and iDC-specific markers. Fig. 1 indicates the result of arepresentative experiment.

As described earlier, a set of preliminary cocultures were per-formed at 1:2.5, 1:5, 1:10, 1:20, and 1:50 MenSC/monocytes ratiosto determine the ratio at which MenSCs exerted the highestinhibitory effects. Collectively, in terms of CD marker expression,MenSCs exerted their inhibitory impact on iDC generation at 1:2.5(MenSC/monocyte) ratio, and this effect was considerably dimin-ished at lower concentrations of MenSCs. In fact, a significantdecrease could still be observed at 1:5, 1:10, and 1:20 ratios forCD1a. However, no significant downreglulation in the percentageof CD14 as well as the expression of costimulatory molecules andHLA-DR on MenSC-iDC relative to Alone iDC at ratios less than 1:2.5was observed (data not shown).

Accordingly, 1:2.5 (MenSC/monocyte) was selected as the tar-get ratio for performing the following experiments. To the results,CD14+ cells (monocytes), cultured alone, properly differentiatedinto iDCs (Alone iDCs) as judged by the expression of CD14(monocyte-specific) and CD1a (iDC-specific) with the mean per-centages of 0.4 ± 0.1 and 88.6 ± 1.4, respectively. However, MenSCpresence resulted in a significant increase in the percentageof CD14+ cells (9.9 ± 2.4), and decrease in that of CD1a+ cells(55.2 ± 6.1) (Fig. 2A).

Furthermore, Alone iDCs could successfully express costimula-tory molecules as well as HLA-DR (MRFI for CD40: 1.6 ± 0.15, CD80:3.1 ± 0.12, CD86: 16.2 ± 1.98, HLA-DR: 70.1 ± 5), while the density

of all markers except for HLA-DR was significantly lower on Mens-iDCs (MRFI for CD40: 1.15 ± 0.12, CD80: 2.6 ± 0.17, CD86: 9.1 ± 0.6,HLA-DR: 64.2 ± 2.8) (Fig. 2B). It is of note that, cell viability did notdiffer between Alone iDCs and MenSC-iDCs (not shown) suggesting

242 M. Bozorgmehr et al. / Immunology Letters 162 (2014) 239–246

Fig. 1. MenSCs effect on generation and maturation of DCs. Results of representative flow cytometry experiments. Monocytes were differentiated toward iDCs either inthe absence (Alone iDC) or presence (MenSC-iDCs) of MenSCs. Moreover, iDCs produced in the absence of MenSCs were induced for maturation both in the absence (AloneiDC + LPS) and presence (Alone iDC + [MenSC & LPS]) of MenSCs. Additionally, MenSCs impact on the whole process of monocyte to mDC differentiation was investigated(Alone mDC versus MenSC-mDC). For CD14 and CD1a, the percentage of positive cells, and for CD40, CD80, CD83, CD86, and HLA-DR the mean fluorescence intensity (MFI)are indicated. Open histograms in each graph represent the isotype control.

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hat MenSCs inhibited iDC generation with no major effects on celliability.

.2. MenSCs show no pronounced impacts on DC maturation step

To assess whether MenSCs had any inhibitory effects on DCaturation step, iDCs produced in the absence of MenSCs were

nduced for maturation in MenSC presence. In our results, neitherhe percentage of CD14+ and CD1a+ cells nor the expression level ofostimulatory molecules and MHC-II was affected by the presencef MenSCs in DC maturation phase (Fig. 3).

3.3. Monocytes cocultured with MenSCs fail to differentiate intofully mature DCs

Monocytes were differentiated toward iDCs and then mDCsin the presence of MenSCs. Control group cells (Alone-mDC),generated and matured in the absence of MenSCs, showed thetypical phenotype of mDCs so that the percentage of CD14+

and CD1a+ cells were 0.9 ± 0.21 and 89.2 ± 2.21, respectively;

Alone-mDCs were also properly mature regarding the expressioncostimulatory molecules and HLA-DR (MRFI for CD40: 10.5 ± 0.34,CD80: 10.5 ± 0.80, CD83: 10.8 ± 0.35, CD86: 260 ± 10.41, HLA-DR: 190 ± 8.33); however, coculture with MenSCs throughout the

M. Bozorgmehr et al. / Immunology Letters 162 (2014) 239–246 243

Fig. 2. MenSCs impact on monocyte to iDC differentiation. Generation of monocytestoward iDCs was carried out either in the absence or presence of MenSCs. Cellsgenerated in the presence of MenSCs (MenSC-iDCs) were compared with Alone iDCswith respect to the percentage of CD14+ and CD1a+ cells (A) as well as the expressionCD40, CD80, CD86, and HLA-DR (B). Monocytes (from four donors) were randomlycaM

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Fig. 3. MenSC impact on the generation of mDC from iDC. iDCs generated alonewere induced for maturation either in the absence (Alone-iDC + LPS) or presence(Alone iDC + [MenSC & LPS]) of MenSCs. The two groups were compared for thepercentage of CD14+ and CD1a+ cells (A) as well as the expression CD40, CD80, CD83,CD86, and HLA-DR (B). Monocytes (from three donors) were randomly cocultured

ocultured with unrelated MenSCs (from six donors) and data of MenSC-iDC groupre obtained from 12 independent experiments. Data are indicated as mean ± SEM.RFI: mean ratio fluorescence intensity.

eneration of iDCs and then mDCs (MenSC-mDC) significantlyindered full maturation of DCs as judged by the percentage ofD1a+ and CD14+ cells (46.9 ± 3.24 and 3.88 ± 1.24, respectively).lso, the expression of other surface molecules except for HLA-R was markedly decreased on MenSC-mDCs (CD40: 6.1 ± 0.56,D80: 7.4 ± 0.39, CD83: 7.8 ± 0.58, CD86: 144 ± 24.19, HLA-DR:74 ± 9.67) (Fig. 4).

.4. Il-6 and IL-10 concentrations in culture supernatants

To determine the concentration of cytokines, the supernatantsollected from 5 day cocultures of MenSC/monocyte (1:2.5 ratio)ere analyzed for the levels of IL-6 and IL-10 by ELISA. As indi-

ated in Fig. 5A, when cultured alone, MenSCs produced ratheroticeable levels of IL-6 (660 ± 31 pg/mL); however, IL-6 concen-ration was significantly increased in MenSC/monocyte cocultures3665 ± 524 pg/mL). Moreover, monocytes differentiated towardDCs in the absence of MenSCs produced low amounts of IL-634 ± 6 pg/mL). Similarly, IL-10 concentration was markedly highern the supernatant of MenSC/monocyte cocultures (14.7 ± 1.3)ompared to that of Alone iDC or MenSC groups, with undetectableevels of IL-10 (Fig. 5B).

. Discussion

In the present study, for the first time, the effect of MenSCs onhe generation of DCs from peripheral blood monocytes was inves-igated. MenSCs could efficiently prevent DCs from fulfilling their

with unrelated MenSCs (from six donors) and data are the result of 6 independentexperiments. Results are indicated as mean ± SEM. MRFI: mean ratio fluorescenceintensity.

phenotypic maturation. This inhibition was majorly exerted on thefirst step of DC generation, differentiation of monocytes into iDCs.Based on the cytokines assays, we propose that IL-6 and IL-10 mightbe partly responsible for this phenomenon.

There are a few published reports exploring MenSCs potentialeffects on different arms of the immune system. Murphy et al.[12] for the first time questioned the probable effects of MenSCson a mixed lymphocyte reaction (MLR) established between twoallogeneic peripheral blood mononuclear cell populations. MenSCscould effectively suppress cellular proliferation in MLR, and shift Tcell responses toward Th2. Wang et al. [28] also similarly reportedthat MenSCs, in high concentration, have the capacity to suppressallogenic MLR. In another study, our group demonstrated that theinhibitory impacts of MenSCs on allogeneic MLR were dependentupon the number of MenSCs entering the reaction. Accordingly,only high concentrations of MenSCs were capable of suppressingcell proliferation in MLR, while MenSCs rather stimulated lympho-cytic proliferation when used at low concentrations, suggestingthe differential impacts MenSCs could exert under different cir-cumstances [29]. However, no study has so far investigated theinteraction between MenSCs and DCs.

Dendritic cells are well-proved to play a critical role in shif-ting T cell-based immune responses under different circumstances[16]. Therefore, hereby we examined MenSC effects on generationand maturation of DCs from human blood monocytes. In cocul-tures set with a high concentration of MenSCs (1:2.5 ratio), thepercentage of CD14+ cells was significantly higher while that ofCD1a+ cells was lower among MenSC-iDCs compared to the con-

trol group (Alone iDCs). As CD14 and CD1a are the markers specificto human monocytes and iDCs, respectively, these results revealthe inhibition of iDC generation by MenSCs at the phenotypic level.In other words, monocytes cocultured with MenSCs were not able

244 M. Bozorgmehr et al. / Immunology

Fig. 4. MenSC impact on the whole process of mDC generation. Monocytes werecocultured with MenSCs, differentiated toward iDC and then stimulated for matu-ration (MenSC-mDC). As the control group, mDCs were generated in the completeabsence of MenSCs (Alone-mDC). Monocytes (from three donors) were randomlycifl

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ocultured with unrelated MenSCs (from six donors) and data are the result of 6ndependent experiments. Results are indicated as mean ± SEM. MRFI: mean ratiouorescence intensity.

o fulfill their perfect differentiation into iDCs. It is of note, that the

enSC-associated inhibitory effect was dose-dependent, so that itas decreased at MenSC/monocyte ratios less than 1:2.5; we had

bserved similar results in our previous publication regarding thenhibitory impact of MenSCs on allogenic MLR [12,15,28]. These

ig. 5. IL-6 and IL-10 concentration in supernatant of monocyte-MenSC coculture. Monocn day 5, the supernatants of cocultures were collected and investigated for the level oetermined in supernatants of control groups: Alone iDC and MenSC. Monocytes (from fouata of the coculture groups are obtained from 12 independent experiments. Data are ind

Letters 162 (2014) 239–246

parallel results suggest the idea that DCs might have been the keycells through which MenSCs have exerted their inhibitory effectson allogenic MLR.

Costimulatory molecules as well as HLA-DR play a crucial role inthe DC-T cell interaction and the ensuing immune responses [18].Thus, we explored whether the expression of these molecules onmonocyte-derived iDCs was influenced upon coculture with Men-SCs. Contrasting with Alone iDCs, with a typical upregulation ofcostimulatory molecules (CD40, CD80, and CD86) on the surface,MenSC-iDCs failed to express optimal levels of the costimulatorymolecules. However, MenSC presence did not significantly affectthe expression of HLA-DR by MenSC-iDCs as compared to iDCs.Although HLA-DR plays a central role in interaction between DCsand T cells, effective T cell stimulation could not be accomplishedwithout the essential help of costimulatory molecules and lack orlow expression of costimulatory molecules on DCs could resultin disturbed and/or modulatory immune responses [16,18,30]. Ingeneral, these results were in line with what we observed in theprevious section for the percentage of CD14+ and CD1a+ cells, sup-porting the inhibitory effect of MenSCs on the perfect generationof iDCs from monocytes.

IL-6 and IL-10 are well proved to have inhibitory impacts on thedifferentiation of monocytes to DCs [31–34]. Hence, to investigatethe mechanism through which MenSCs affected iDC generation, wefurther determined the concentration of IL-6, and IL-10 in MensSC-iDC cocultures. Accordingly, the concentration of both cytokineswas markedly higher in cocultures, holding IL-6 and IL-10 partlyresponsible for the observed inhibitory effect on the generation ofphenotypically perfect iDCs from blood monocytes. Nonetheless,there are additional molecules such as prostroglandin-E2 (PGE2)[24] and HLA-G [35] with inhibitory roles on the generation DCsfrom monocytes. In particular, it was shown that PGE2 played amore important role than IL-6 in inhibition of DC generation [24].Thus, it would be tempting to assess the potential involvement ofother well-known inhibitory molecules in the observed inhibitoryeffects.

Next, we intended to investigate whether MenSCs could haveany inhibitory effects on DC maturation as a separate step. Spag-

giari et al. [24] have demonstrated that MSCs exert their inhibitoryimpacts mainly on the first step, which is the generation of iDCsfrom monocytes, rather than the second step, maturation of iDCstoward mDCs. Hence, to address the same issue in case of MenSCs,

ytes from normal donors were differentiated toward iDC in the presence of MenSCs.f IL-6 (A) and IL-10 (B) using ELISA. The concentration of both cytokines was alsor donors) were randomly cocultured with unrelated MenSCs (from six donors) and

icated as mean ± SEM. MRFI: mean ratio fluorescence intensity. ND: not detectable.

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e performed similar experiments. Interestingly, no marked hin-rance in DC maturation was observed when MenSCs were addedo iDC cultures. The results of our study reveal for the first time that

enSCs inhibitory impact is majorly exerted on the first step thats monocyte to iDC differentiation.

As DCs’ state of maturation is believed to highly affect theirmmunoregulatory functions [16], we further explored whether

enSC presence throughout the whole process of mDC genera-ion would affect their immunophenotypic features. To this end,

onocytes were differentiated toward iDCs and induced for matu-ation in the complete presence of MenSCs. The monocyte-derivedells (MenSC-mDC) were compared with typically matured DCsAlone mDC), generated in the complete absence of MenSCs.

enSC-mDCs failed to transform into phenotypically fully matureCs, as determined by the percentage of CD1a+ and CD14+ cells

ogether with the expression level of costimulatory molecules.otably, comparable to what was observed in the case of MenSC-

DCs, no significant downregulation of HLA-DR was detectedn MenSC-mDCs. Nonetheless, the non-sufficient expression ofostimulatory molecules on MenSC-mDCs could be a sign of sub-ptimal maturation. Since, semi-mature DCs are known to presentore tolerogenic rather than stimulatory features [16,18,36], these

esults could further substantiate the modulatory effects of MenSCsn DC generation.

Collectively, in the present study, the potential effects of MenSCsn generation and also maturation of human blood-derived mono-ytes was assessed. Accordingly, as judged by the expression ofonocyte-, iDC-, mDC-specific markers, MenSCs did inhibit max-

mal maturation of DCs. Moreover, this impact was shown to beainly exerted during the differentiation of monocytes into iDCs.

n addition, IL-6 and IL-10 could partly be responsible for thesenhibitory effects. There are several studies published on utiliza-ion of MenSCs in animals models of various diseases; however,hus far, data regarding the possible impacts of these cells onmmune system cells are scarce. The evidence provided in the cur-ent study could be useful in interpretation of the so far observedmmunomodulatory impacts of MenSC, in vivo. Given the centralole DCs could play in modulation of immune responses, this studyurther endorses the idea of employing MenSCs as a rather easilyccessible and expandable immunomodulatory tool to be appliedo pre-clinical models of various diseases.

It should be noted, however, that here we explored how Men-Cs could affect DC generation at the phenotypic level. Even thoughnsufficient expression of costimulatory molecules could poten-ially shift DCs toward the immunomodulatory profile, it seemshat additional data addressing the functional maturation of DCsould further support the present results.

cknowledgments

This work was financially supported by Tarbiat Modares Uni-ersity of Medical Sciences (Grant number: 5066438) and Lorestanniversity of Medical Sciences (Grant number: 5066438). Theresent study was a part Mahmood Bozorgmehr’ Ph.D. dissertationntitled “The effect of normal and endometriotic patients’ men-trual blood stromal stem cells on dendritic cell generation andunction”.

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