Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Cellular Immunology 259 (2009) 150–156

Contents lists available at ScienceDirect

Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm

Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues Keon Hee Yoo a,c,1, In Keun Jang b,1, Myoung Woo Lee a, Hyo Eun Kim b, Mal Sook Yang b, Youngwoo Eom b, Jong Eun Lee b, Young Jin Kim b, Seong Kyu Yang a, Hye Lim Jung a, Ki Woong Sung a, Cheol Woo Kim c, Hong Hoe Koo a,* a b c

Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Biomedical Research Institute, LifeCord Inc., Suwon, Republic of Korea Department of Pathology, School of Medicine, Seoul National University, Seoul, Republic of Korea

a r t i c l e

i n f o

Article history: Received 13 February 2009 Accepted 12 June 2009 Available online 23 June 2009 Keywords: Mesenchymal stem cells Immunomodulation IFN-c TNF-a Indoleamine 2,3-dioxygenase

a b s t r a c t Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJMSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-c and tumor necrosis factor (TNF)-a secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-b1, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-c and/or TNF-a, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-c and/or TNF-a produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Mesenchymal stem cells (MSCs) were first characterized by Friedenstein and colleagues, who identified an adherent, fibroblast-like population from adult bone marrow (BM) [1,2]. MSCs have the capacity to differentiate into multiple tissues, including bone and cartilage [3,4], tendon [5], muscle [6], fat [7], and marrow stromal connective tissue which supports hematopoietic cell differentiation [8,9]. Human BM-MSCs constitutively secrete cytokines important for hematopoiesis and promote engraftment of hematopoietic stem cells (HSCs) in experimental animal models [10,11]. In addition, BM-MSCs have immunomodulatory properties and reduce inflammation, suppressing lymphocyte alloreactivity in vitro in mixed lymphocyte reaction (MLR) assays [12,13]. Intravenous administration

* Corresponding author. Address: Department of Pediatrics, Samsung Medical Center, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. Fax: +82 2 3410 0043. E-mail address: [email protected] (H.H. Koo). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2009.06.010

of murine MSCs improve the outcome of neural [14] and lung [15] injury in experimental animal models primarily through paracrine effects and a shift from the production of pro-inflammatory to anti-inflammatory cytokines at the site of injury. Murine BM-MSCs exposed to interferon (IFN)-c become activated and suppress graft versus host disease (GvHD) in vivo [16]. Thus, the immnuomodulatory effects of MSCs may be able to repair tissue damage caused by the immune system in autoimmune-induced inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, GvHD of the gut, liver, and skin after allogeneic HSC transplantation (HSCT), as well as prevent rejection of organ transplants. Finding a suitable cell source is a major challenge for cell therapy and tissue engineering. In addition to fulfilling the function of the reconstructed tissue, low immunogenicity is needed for clinical applications. Although BM has been the main source of MSCs [1– 7,17], the use of BM-derived cells is not always acceptable due to the high degree of viral exposure, the possibility of donor morbidity, and the significant decreases in cell number and proliferation/differentiation capacity with age [18]. A highly invasive procedure is used to obtain BM, and, thus, the need to find an alternative MSC source to provide MSCs with immunomodulatory properties has emerged.

K.H. Yoo et al. / Cellular Immunology 259 (2009) 150–156

Clinically, the immunomodulatory properties of MSCs can be used to enhance engraftment and to reduce the incidence the GvHD after allogeneic HSCT. While several groups have shown that MSCs from BM and fetal liver are capable of suppressing T-cell proliferation [12,13,19], we extended these studies to MSCs obtained from other adult human tissues to determine whether these MSCs inhibit T-cell proliferation. In this study, we compared the immunomodulatory properties of MSCs derived from adult human adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ) with BM-MSCs. 2. Materials and methods 2.1. Isolation of human BM, AT, CB, and WJ-derived MSCs The Institutional Review Board of Samsung Medical Center approved this study and all samples were obtained with informed consent. 2.1.1. BM- and CB-MSCs MSCs were isolated and cultured as previously described [20]. Briefly, mononuclear cells were isolated from normal bone marrow aspirates, or term cord blood of newborns, using Ficoll-Hypaque density gradient centrifugation (Histopaque-1077; Sigma–Aldrich, St. Louis, MO). Cells were seeded on uncoated T25 culture flasks (Nalge Nunc, Naperville, IL) at a density of 3  105 cells/cm2 in low glucose Dulbecco’s modified Eagle’s medium (LG-DMEM; Invitrogen-Gibco, Rockville, MD) containing 10% fetal bovine serum (FBS; Invitrogen-Gibco) and 100 U/ml penicillin/streptomycin (Invitrogen-Gibco). The cells were incubated in a humidified atmosphere at 37 °C with 5% CO2, and the medium was changed every 7 days until the adherent fibroblast-like cells reached confluence. Adherent cells were then resuspended with 0.05% trypsin–EDTA (Invitrogen-Gibco) and reseeded at 2  103 cells/cm2. 2.1.2. AT-MSCs MSCs were isolated and cultured according to a previous protocol [21]. Briefly, lipoaspirates were washed extensively with equal volumes of Dulbecco’s phosphate-buffered saline (DPBS; HyClone, Logan, UT), and the extracellular matrix was digested with 0.075% collagenase A (Roche Applied Science, Penzberg, Germany) at 37 °C for 30 min. Enzyme activity was neutralized with LG-DMEM containing 10% FBS, 100 U/ml penicillin/streptomycin; samples were centrifuged at 1200g for 10 min. The cell pellet was washed with DPBS and filtered through a 100 lm Nylon mesh (Cell strainer; Becton Dickinson, Franklin Lakes, NJ). After 24 h in culture, cells were washed with DPBS and incubated in a humidified atmosphere at 37 °C with 5% CO2 in medium. Adherent cells were resuspended with 0.05% trypsin–EDTA and reseeded at 2  103 cells/cm2.

151

CD105 were purchased from Ancell (Bayport, MN). A total of 5  105 cells were resuspended in 0.2 ml PBS and incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies for 20 min at room temperature. The fluorescence intensity of the cells was evaluated by flow cytometry (FACScan; Becton Dickinson) and the data were analyzed with the CELLQUEST software (Becton Dickinson). 2.3. T-cell proliferation: BrdU incorporation BM-, AT-, CB-, and WJ-MSCs were seeded at 1.25  104 cells per well in 96-well plates in high glucose DMEM, 10% FBS, 100 U/ml penicillin/streptomycin. After 24 h, 10 lg/ml mitomycin C (MMC; Sigma–Aldrich) was added to inhibit MSC proliferation, and cells were incubated for 2 h at 37 °C followed by five extensive washes with medium. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. 1  105 PBMCs per well were added and stimulated with 1 lg/ml phytohemagglutinin (PHA; Sigma–Aldrich). PHA-activated PBMCs were cultured in the presence or absence of MSCs. Cultures were plated in triplicate and incubated for 3–4 days before addition of 5-bromo-20 -deoxyuridine (BrdU). After 18 h, proliferation was assessed using the BrdU-Assay kit (Roche Applied Science) according to the manufacturer’s protocol. 2.4. Analysis of cytokines MSCs were seeded at 1  104 cells/cm2 in 6-well plates in 2 ml LG-DMEM. After 48 h, supernatants were collected and frozen at 70 °C. Multiplex human cytokine, chemokine, and growth factor detection (Upstate, Waltham, MA) was utilized to measure the production of interleukin (IL)-1a, IL-1b, IL-2, IL3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-c, IFN-a2, IFN-c inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, MIP-1b, RANTES (regulated upon activation normal T-cell expressed and secreted), tumor necrosis factor (TNF)-a, TNF-b, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, epidermal growth factor (EGF), Flt-3 ligand, and vascular endothelial growth factor (VEGF) in the culture supernatants. In addition, supernatants were collected from PBMCs cocultured with MSCs, stored at 70 °C, and production of the secretory factors listed above was assessed using multiplex human cytokine, chemokine, and growth factor detection (Upstate, Waltham, MA) according to the manufacturer’s instructions. 2.5. RNA extraction and RT-PCR analysis

2.1.3. WJ-MSCs MSCs were isolated using an explantation culture method [22]. Umbilical cords were washed in DPBS to remove blood components and cut into small pieces (0.5–1 cm). Vessels were removed to avoid endothelial cell contamination. Wharton’s jelly parts of the cord were cut in 0.5–1 cm3 pieces and placed directly into culture wells for culture expansion in LG-DMEM containing 10% FBS, 100 U/ml penicillin/streptomycin. When colonies of cells appeared and the wells reached 70% confluence, the cells were resuspended with 0.05% trypsin–EDTA and reseeded at 2  103 cells/cm2. 2.2. Flow cytometry Antibodies against the human antigens CD14, CD45, CD73, and CD90 were purchased from Becton Dickinson. Antibodies against

MSCs were cultured in 6-well plates in the presence or absence 2000 U/ml IFN-c (LG chem, Seoul, Korea) and/or 10 ng/ml TNF-a (R&D Systems, Minneapolis, MN) for 48 h. Total RNA was extracted from cells using the TRIzol Reagent (Invitrogen-Gibco). RNA (2 lg) was reverse-transcribed with the AMV reverse transcriptase XL (TaKaRa Shuzo, Shiga, Japan) for 90 min at 42 °C in the presence of oligo(dT) primer. PCR was performed using Taq polymerase (BioQuest, Seoul, Korea). RNA was analyzed for the expression of human hepatocyte growth factor (HGF), indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor (TGF)-b1, cyclooxygenase (COX)-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Primer sequences are shown in Table 1.

152

K.H. Yoo et al. / Cellular Immunology 259 (2009) 150–156

Table 1 Primer sequences used for RT-PCR studies. Target

Primer sequencea 0

Product size (bp)

HGF

0

F 5 -ATGCATCCAAGGTCAAGGAG-3 R 50 -TTCCATGTTCTTTTGTCCCACA-30

349

IDO

F 50 -CGCTGTTGGAAATAGCTTC-30 R 50 -CAGGACGTCAAAGCACTGAA-30

234

IL-10

F 50 -ATCCAAGACAACACTACTAA-30 R 50 -TAAATATCCTCAAAGTTCC-30

588

TGF-b1

F 50 -CAGATCCTCTCCAAGCTG-S0 R 50 -TCGGAGCTCTGATGTGTT-30

270

COX-1

F 50 -GAGTTTGTCAATGCCACCT-30 R 50 -CAACTGCTTCTTCCCTTTG-30

215

COX-2

F 50 -TCCTTGCTGTTCCCACCCATG-30 R 50 -CATCATCAGACCAGGCACCAG-30

847

GAPDH

F 50 -ATCACCATCTTCCA-GGAGCG-30 R 50 -GTTCTTCCACCACTTCGTCC-30

573

HGF, hepatocyte growth factor; IDO, indoleamine 2,3-dioxygenase; IL-10, interleukin-10; TGF-b1, transforming growth factor; COX-l,2, cyclooxygenase-l,2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a Forward (F) and reverse (R) primers used to detect mRNA expression of the indicated targets.

NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate) with a protease inhibitor cocktail (Sigma–Aldrich). Cell lysates were centrifuged at 13,709g for 10 min at 4 °C. Supernatants were harvested, and protein concentrations were determined using a BCA protein assay kit (Pierce, IL). For electrophoresis, protein sample (30 lg each) were dissolved in sample buffer (60 mM Tris–HCl, pH 6.8, 14.4 mM b-mercaptoethanol, 25% glycerol, 2% SDS, 0.1% bromophenol blue), boiled for 5 min, and separated on 10% SDS–PAGE reducing gels. Separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, UK) using a trans-blot system (Invitrogen-Gibco). Blots were blocked for 1 h at room temperature in Tris-buffered saline (TBS) (10 mM Tris–HCl, pH 7.5, 150 mM NaCl) containing 5% non-fat dry milk (Becton Dickinson), washed three times with TBS, and incubated at 4 °C overnight with primary antibodies (1:1000 dilution) in TBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.02% Tween 20) containing 3% non-fat dry milk. The next day, blots were washed three times with TBST, and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:2000) in TBST containing 3% non-fat dry milk. After washing three times with TBST, antibody binding was visualized with an ECL detection system (Amersham Biosciences).

2.6. Immunoblotting 2.7. Statistical analysis Cells were washed with cold DPBS, and lysed in 300 ll of cold RIPA buffer (50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 150 mM

All results are expressed as means ± standard deviation (SD).

Fig. 1. Characteristics of MSCs derived from adult human tissues. Phase contrast images of BM-MSCs (A), AT-MSCs (B), CB-MSCs (C) and WJ-MSCs (D) are shown at passage 2– 4. All cells were plastic adherent with a spindle shaped or fibroblastic morphology. (E) Cells were labeled with FITC- and PE-conjugated antibodies and examined by flow cytometry. Histograms demonstrating the expression of surface antigens were plotted against control (anti-IgG). CD14, monocyte; CD45, leukocyte common; CD73, SH-3 and SH-4; CD90, Thy-1; CD105, endoglin (SH-2).

153

K.H. Yoo et al. / Cellular Immunology 259 (2009) 150–156

3.2. AT-, CB-, and WJ-MSCs suppress mitogen-induced T-cell proliferation as efficiently as BM-MSCs

3. Results 3.1. Isolation and characteristics of MSCs derived from adult human tissues Human MSCs were obtained from adult human tissues including BM, AT, CB, and WJ. The morphology of the adherent cells varied among cultures from different sources. Cells obtained from BM, CB, and WJ were fibroblastic in shape, while AT-derived cells were small and spindle shaped (Fig. 1A–D). The cell-surface antigen profiles of these cells after 3–4 passages in culture were analyzed by flow cytometry. These cells were strongly positive for MSC-specific surface markers, such as CD105, CD73, and CD90, but negative for CD14 and CD45 (Fig. 1E). These cells also exhibited mesenchymal differentiation potential, as assessed by culturing in adipogenic, osteogenic, or chondrogenic medium (data not shown). To determine which cytokines, chemokines, and growth factors were secreted by the MSCs, the culture supernatants were analyzed using a luminex multiplex detection system for analyzing 32 different factors. While many of the levels of secreted factors were not significantly different from other types of MSCs, IL-12, IL-15, and PDGF-AA were secreted only by CB-MSCs and WJ-MSCs. In addition, WJ-MSCs secreted RANTES, but not VEGF, distinct from other MSCs. With the exception of one WJ-MSCs isolate, the non-stimulated MSCs did not secrete IFN-c, which is correlated with the immunomodulatory properties of MSCs (Table 2).

To evaluate whether MSCs affect the proliferation of T-cells in vitro, we measured the incorporation of the thymidine analog BrdU in mitogen-induced proliferating T-cells. When PHA-activated PBMCs were incubated on a layer of MSCs from AT, CB, or WJ, T-cell proliferation decreased significantly, and the effect was dependent on the number of MSCs in the coculture. There were no significant differences between the effects of MSCs derived from different tissues (Fig. 2A). These data demonstrate that AT-, CBand WJ-MSCs were as efficient in suppressing T-cell proliferation as BM-MSCs, which are comparable to BM preparations commonly used in other studies. 3.3. The suppressive effect of MSCs on T-cell proliferation requires the presence of IFN-c and/or TNF-a, which acts by enhancing the IDO activity of MSCs In order to dissect the contribution of soluble factors to immunosuppression, the supernatants of mitogen-activated T-cells cultured in the absence or presence of each type of MSCs were examined for the production of two potent immunomodulatory mediators, INF-c and TNF-a. The levels of IFN-c and TNF-a secreted from activated T-cells increased over time, but these levels were significantly reduced when T-cells were cocultured with MSCs (Fig. 2B–C). In addition, the levels of HGF, IL-10, TGF-b1, COX-1, and COX-2 expression by MSCs were only minimally af-

Table 2 Levels of factors secreted from MSCs derived from adult human tissues. 1  106 Cells

BM-MSC

AT-MSC

CB-MSC

WJ-MSC

#1

#2

#3

#1

#2

#3

#1

#2

#3

#1

#2

#3

IL-la IL-lb IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-12(p40) IL-12(p70) IL-13 IL-15

— — + — — — ++ — + — — — — — —

— — + — — — ++ — + — — — — — —

— — + — — — +++ — + — — — — — —

— — + — — — +++ — + — — — — — —

— — + — — — ++ — + — — — — — —

— — + — — — +++ — + — — — — — —

— — + — — — ++ — + — — + — — +

— — + — — — ++ — + — — — — — +

— — + — — — ++ — + — — + — — +

— — + — — — +++ — +++ — — + — — +

— — + — — — +++ — ++ — — + — — +

— — + — — — +++ — +++ — — ++ — — +

Eotaxin GM-CSF IFN-c IFN-a2 IP-10 MCP-1 MlP-la MIP-lb RANTES TNF-a TNF-b

— — — — — ++ — — — — —

— — — — — + — + — — —

— — — — — ++ — + — — —

— — — — — + — — — — —

— — — — — ++ — — — — —

— — — — — + — — — — —

— — — — — ++ — + — + —

— — — — — +++ — + — + —

— — — — — +++ — + — — —

— — — — + +++ — — ++ — —

— — — — + ++ — + + — —

— + + — — ++ — + ++ + —

FGF PDGF-AA PDGF-BB EGF Flt-3 VEGF

— — — — — ++

— — — — — ++

+ — — — — +++

+ — — — — ++

— — — — — +

— — — — — +

+ ++ — — — ++

+ + — — — ++

— + — — — ++

— + — — — —

+ + — — — —

— + — — — —

Cell culture supernatants were recovered from non-stimulated MSC cultures and stored at 70 °C. Multiplex human cytokine detection was utilized to measure secretory factor levels of MSCs derived from adult human tissues including BM, AT, CB, and WJ. Results from three independent cultures are shown for each MSC. —, value <50 pg/ml; +, 50 pg/ml 6 value < 500 pg/ml; ++, 500 pg/ml 6 value < 5000 pg/ml; +++, 5000 pg/ml 6 value.

154

K.H. Yoo et al. / Cellular Immunology 259 (2009) 150–156

Fig. 3. RT-PCR analysis of immunomodulatory factors in MSCs after treatment with IFN-c or TNF-a. Total RNA of control untreated MSCs, or MSCs treated with either IFN-c or TNF-a (B) for 48 h, was analyzed by RT-PCR with primers specific for HGF, IDO, IL-10, TGF-b1, COX-1, or COX-2. The expression levels of HGF, IL-10, TGF-b1, COX-1 and COX-2 were similar in both control and treated MSCs, while the expression of IDO increased in response to treatment with IFN-c or TNF-a.

Fig. 2. IFN-c and/or TNF-a are involved in the immunomodulatory properties of MSCs derived from adult human tissues. PHA-induced proliferation of T-cells in PBMCs in the absence or presence of different numbers of MSCs, was evaluated at day 3 as percentage of BrdU+ cells (A). The levels of IFN-c (B) and TNF-a (C) secreted from activated T-cells cultured in the absence or presence of MSCs were measured at different times by luminex multiple cytokine detection. The levels of IFN-c and TNF-a secreted from activated T-cell cultured in the absence of MSCs increased over time. In contrast, levels were significantly reduced at all time points when cocultured with MSCs. Mean values of three repetitive measures are shown. Statistically significant differences are specified by asterisks, compared to the fraction cocultured with MSCs (*P < 0.05, **P < 0.01).

fected by treatment with IFN-c and/or TNF-a for all MSCs examined, while IDO levels were increased by treatment with IFN-c and/or TNF-a (Figs. 3 and 4). The suppressive effect of IFN-c and/ or TNF-a produced by activated T-cells is correlated with the induction of IDO expression in MSCs, which in turn inhibit T-cell proliferation. 4. Discussion Based on their pioneering studies initiated more than forty years ago, Friedenstein et al. [1] were the first to propose that hu-

Fig. 4. Expression levels of IDO in MSCs after treatment with IFN-c and/or TNF-a. Total RNA of MSCs treated with IFN-c and/or TNF-a for 0, 12, 24, 48, or 72 h was analyzed by RT-PCR using primers specific for IDO (A). Total protein of MSCs treated with IFN-c for 72 h was analyzed by immunoblotting using antibodies specific for IDO (B). The expression of IDO increased significantly in MSCs treated with IFN-c and/or TNF-a.

K.H. Yoo et al. / Cellular Immunology 259 (2009) 150–156

man BM cells contained a precursor for multiple mesenchymal cell lineages. Over the ensuing decades, BM-MSCs have been characterized and have proven to have the capacity to differentiate into several mesenchymal lineages under appropriate conditions [2–4,17]. With the capacity to give rise to mesenchymal tissues, MSCs have been used in clinical applications and tissue engineering for repairing or regenerating damaged or mutated tissues. Allogeneic HSCT is widely used for the treatment of several pathological conditions, despite the lack of suitable HSC donors, which represents a limit to further expansion of this kind of therapeutic approach. Donor–recipient histo-incompatibility is associated with a high risk of both graft rejection and GvHD, situations in which strategies to diminish the immune response after transplantation are mandatory. Recently, several reports have suggested that MSCs not only have a multipotential differentiation capacity [23], but also seem to be capable of modulating immune responses, both in vitro and in vivo [12,24,25]. MSCs are able to prolong skin graft survival in nonhuman primates, and their co-infusion with HSCs has been found to reduce the incidence of GvHD in patients with hematological malignancies [24,25]. Thus, the infusion of MSCs in conjunction with a donor organ or BM may provide a useful tool for favoring the engraftment and reducing the incidence and/or the intensity of GvHD. Although BM has been represented the main available source of MSCs [1–7,17], the use of BM-derived cells is not always acceptable due to the high degree of viral exposure and the significant decreases in cell number and proliferation/differentiation capacity with age [18]. In addition, obtaining a BM sample requires a painful, invasive procedure. These facts have led investigators to search for good substitutes for BM as an MSC source. In this study, we isolated and cultured MSCs from adult human tissues, including BM, AT, CB, and WJ, and showed that these MSCs were positive for cell surface markers of typical MSCs. We have also demonstrated the potential of AT-MSCs, CB-MSCs, and WJMSCs to suppress mitogen-induced T-cell proliferation, at levels similar to that of BM-MSCs. AT is a particularly interesting alternative source of MSCs because AT can be obtained with a less invasive procedure, generally yields high amounts of MSCs [26], and, thus, may potentially be a better source of MSCs for clinical applications than the conventionally used BM. On the other hand, using CB- or WJ-derived MSCs harbors an advantage in that they do not require any invasive procedure which may be harmful to the donor. In addition, CB and WJ are rarely exposed to infectious agents. Therefore, CB or WJ might be a better source of MSCs as a third-party source, which can be used as a universal donor across the HLA barrier. Several studies have shown that MSCs possess the intrinsic homing ability to migrate to injured tissues and actively participate in tissue repair. MSCs can repair injured tissue by differentiating into the damaged cell types, by secreting appropriate cytokines and growth factors, and by undergoing cell fusion [27–29]. In addition, MSCs possess the unique ability to suppress immune responses, both in vitro [12,13] and in vivo [14–16]. Previous studies have examined the mechanisms by which MSCs mediate immunosuppression. However, there is controversy as to whether the suppressive effects of MSCs are contact-dependent or mediated through the release of soluble factors. In this study, the levels of IFN-c and/or TNF-a secretion from activated T-cells increased over time, but levels were significantly reduced when T-cells were cocultured with each type of MSCs. In addition, the levels of HGF, IL-10, TGF-b1, COX-1, and COX-2 expression were only minimally affected by treatment of MSCs with IFN-c and/or TNF-a, while expression of IDO increased in response to treatment with IFN-c and/or TNF-a. We also observed that T-cell proliferation is partially inhibited by MSCs in trans-well cultures (data not shown), indicating the possibility that soluble

155

factors, as well as cell to cell contact, may be involved in the immunosuppressive properties of MSCs. Some studies have implicated contact-dependent mechanisms, including the expression of B7H1 on MSCs [12,30]. Other studies reported that soluble factors secreted by MSCs, or by immune cells in response to MSCs, play a major role in MSCs-mediated immune suppression. Soluble factors such as HGF, the COX-1, 2 generated prostaglandins E2 (PGE2), TGF-b1, IDO, nitric oxide, and IL-10 have been implicated, while other factors remain unknown [13,31–34]. Antibodies against HGF and TGF-b1 partially restore proliferation of purified T-cells, but not PBMCs [35–37]. Cytokines play a crucial role in regulating MSCs-mediated immunosuppression. Aggarwal and Pittenger [32] reported that TNF-a can enhance the production of immunosuppressive prostaglandins by MSCs by as much as 100-fold. Zappia and colleagues have shown that MSCs are able to reduce the serum levels of INF-c and TNF-a produced by activated T-cells [14]. The reduction in TNF-a can be explained by secretion of the soluble TNF receptor II, which acts as an antagonist for TNF-a and TNF-b [38]. IFN-c is another important cytokine that regulates the immunomodulatory functions of MSCs. Compelling studies have shown that IFN-c plays an active role in MSCs-mediated immunosuppression [39,40]. IDO, which is induced by IFN-c, catalyses the conversion of tryptophan to kynurenine and inhibits T-cell proliferation by tryptophan depletion [41]. IDO is not expressed constitutively by MSCs, but can be induced by IFN-c, and expression contributed to immunomodulation [42]. In conclusion, our data indicate that AT-MSCs, CB-MSCs, and WJ-MSCs from adult human tissues effectively suppress mitogeninduced T-cell proliferation, to levels comparable to BM-MSCs induced suppression. The suppressive effect of IFN-c and/or TNF-a produced by activated T-cells was correlated to the ability of the factor to stimulate production of IDO by MSCs, which, in turn, inhibited T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts. Acknowledgment This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (Project No: 0720230). References [1] A.J. Friedenstein, K.V. Petrakova, A.I. Kurolesova, G.P. Frolova, Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation 6 (1968) 230–247. [2] A.J. Friedenstein, R.K. Chailakhyan, U.V. Gerasimov, Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers, Cell Tissue Kinet. 20 (1987) 263–272. [3] M. Owen, Marrow stromal stem cells, J. Cell Sci. Suppl. 10 (1988) 63–76. [4] A.I. Caplan, Mesenchymal stem cells, J. Orthop. Res. 9 (1991) 641–650. [5] R.G. Young, D.L. Butler, W. Weber, A.I. Caplan, S.L. Gordon, D.J. Fink, Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair, J. Orthop. Res. 16 (1998) 406–413. [6] S. Wakitani, T. Saito, A.I. Caplan, Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine, Muscle Nerve 18 (1995) 1417–1426. [7] J.E. Dennis, A.I. Caplan, Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse, J. Cell. Physiol. 167 (1996) 523–538. [8] T.M. Dexter, N.G. Testa, Differentiation and proliferation of hemopoietic cells in culture, Methods Cell Biol. 14 (1976) 387–405. [9] C. Friedrich, E. Zausch, S.P. Sugrue, J.C. Gutierrez-Ramos, Hematopoietic supportive functions of mouse bone marrow and fetal liver microenvironment: dissection of granulocyte, B-lymphocyte, and hematopoietic progenitor support at the stroma cell clone level, Blood 87 (1996) 4596–4606. [10] M. Angelopoulou, E. Novelli, J.E. Grove, H.M. Rinder, C. Civin, L. Cheng, D.S. Krause, Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice, Exp. Hematol. 31 (2003) 413–420.

156

K.H. Yoo et al. / Cellular Immunology 259 (2009) 150–156

[11] P.S. in ’t Anker, W.A. Noort, A.B. Kruisselbrink, S.A. Scherjon, W. Beekhuizen, R. Willemze, H.H. Kanhai, W.E. Fibbe, Nonexpanded primary lung and bone marrow-derived mesenchymal cells promote the engraftment of umbilical cord blood-derived CD34(+) cells in NOD/SCID mice, Exp. Hematol. 31 (2003) 881–889. [12] M. Krampera, S. Glennie, J. Dyson, D. Scott, R. Laylor, E. Simpson, F. Dazzi, Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide, Blood 101 (2003) 3722–3729. [13] W.T. Tse, J.D. Pendleton, W.M. Beyer, M.C. Egalka, E.C. Guinan, Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation, Transplantation 75 (2003) 389–397. [14] E. Zappia, S. Casazza, E. Pedemonte, F. Benvenuto, I. Bonanni, E. Gerdoni, D. Giunti, A. Ceravolo, F. Cazzanti, F. Frassoni, G. Mancardi, A. Uccelli, Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy, Blood 106 (2005) 1755–1761. [15] L.A. Ortiz, F. Gambelli, C. McBride, D. Gaupp, M. Baddoo, N. Kaminski, D.G. Phinney, Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects, Proc. Natl. Acad. Sci. USA 100 (2003) 8407–8411. [16] D. Polchert, J. Sobinsky, G. Douglas, M. Kidd, A. Moadsiri, E. Reina, K. Genrich, S. Mehrotra, S. Setty, B. Smith, A. Bartholomew, IFN-c activation of mesenchymal stem cells for treatment and prevention of graft versus host disease, Eur. J. Immunol. 38 (2008) 1745–1755. [17] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craiq, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [18] P. Rubinstein, R.E. Rosenfield, J.W. Adamson, C.E. Stevens, Stored placental blood for unrelated bone marrow reconstitution, Blood 81 (1993) 1679–1690. [19] C. Gotherstrom, O. Ringden, M. Westgren, C. Tammik, K. Le Blanc, Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells, Bone Marrow Transplant. 32 (2003) 265–272. [20] M.W. Lee, J. Choi, M.S. Yang, Y.J. Moon, J.S. Park, H.C. Kim, Y.J. Kim, Mesenchymal stem cells from cryopreserved human umbilical cord blood, Biochem. Biophys. Res. Commun. 320 (2004) 273–278. [21] P.A. Zuk, M. Zhu, H. Mizuno, J. Huang, J.W. Futrell, A.J. Katz, P. Benhaim, H.P. Lorenz, M.H. Hedrick, Multilineage cells from human adipose tissue: implications for cell-based therapies, Tissue Eng. 7 (2001) 211–228. [22] I.K. Jang, J.I. Ahn, J.S. Shin, Y.S. Kwon, Y.H. Ryu, J.K. Lee, J.K. Park, K.Y. Song, E.K. Yang, J.C. Kim, Transplantation of reconstructed corneal layer composed of corneal epithelium and fibroblasts on a lyophilized amniotic membrane to severely alkali-burned cornea, Artif. Organs 30 (2006) 424–431. [23] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. OrtizGonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Larqaespada, C.M. Verfaillie, Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418 (2002) 41–49. [24] A. Bartholomew, C. Sturgeon, M. Siatskas, K. Ferrer, K. McIntosh, S. Patil, W. Hardy, S. Devine, D. Ucker, R. Deans, A. Moseley, R. Hoffman, Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo, Exp. Hematol. 30 (2002) 42–48. [25] O. Ringdén, M. Labopin, A. Bacigalupo, W. Arcese, U.W. Schaefer, R. Willemze, H. Koc, D. Bunjes, E. Gluckman, V. Rocha, A. Schattenberg, F. Frassoni, Transplantation of peripheral blood stem cells as compared with bone marrow from HLA-identical siblings in adult patients with acute myeloid leukemia and acute lymphoblastic leukemia, J. Clin. Oncol. 20 (2002) 4655– 4664. [26] W.J. Jurgens, M.J. Oedayrajsingh-Varma, M.N. Helder, B. Zandiehdoulabi, T.E. Schouten, D.J. Kuik, M.J. Ritt, F.J. van Milligen, Effect of tissue-harvesting site on

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

yield of stem cells derived from adipose tissue: implications for cell-based therapies, Cell Tissue Res. 332 (2008) 415–426. D.J. Prockop, C.A. Gregory, J.L. Spees, One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues, Proc. Natl. Acad. Sci. USA 100 (Suppl. 1) (2003) 11917–11923. N. Terada, T. Hamazaki, M. Oka, M. Hoki, D.M. Mastalerz, Y. Nakano, E.M. Meyer, L. Morel, B.E. Petersen, E.W. Scott, Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion, Nature 416 (2002) 542– 545. J.L. Spees, S.D. Olson, J. Ylostalo, P.J. Lynch, J. Smith, A. Perry, A. Peister, M.Y. Wang, D.J. Prockop, Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma, Proc. Natl. Acad. Sci. USA 100 (2003) 2397–2402. A. Augello, R. Tasso, S.M. Negrini, A. Amateis, F. Indiveri, R. Cancedda, G. Pennesi, Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway, Eur. J. Immunol. 35 (2005) 1482–1490. F. Djouad, P. Plence, C. Bony, P. Tropel, F. Apparailly, J. Sany, D. Noël, C. Jorgensen, Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals, Blood 102 (2003) 3837–3844. S. Aggarwal, M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses, Blood 105 (2005) 1815–1822. S. Beyth, Z. Borovsky, D. Mevorach, M. Liebergall, Z. Gazit, H. Aslan, E. Galun, J. Rachmilewitz, Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness, Blood 105 (2005) 2214–2219. K. Sato, K. Ozaki, I. Oh, A. Meguro, K. Hatanaka, T. Nagai, K. Muroi, K. Ozawa, Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells, Blood 109 (2007) 228–234. K. Le Blanc, L. Tammik, B. Sundberg, S.E. Haynesworth, O. Ringdén, Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex, Scand. J. Immunol. 57 (2003) 11–20. D. Angoulvant, A. Clerc, S. Benchalal, C. Galambrun, A. Farre, Y. Bertrand, A. Eljaafari, Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens, Biorheology 41 (2004) 469–476. M. Di Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, S. Grisanti, A.M. Gianni, Human bone marrow stromal cells suppress Tlymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli, Blood 99 (2002) 3838–3843. A. Diez-Ruiz, G.P. Tilz, R. Zangerle, G. Baier-Bitterlich, H. Wachter, D. Fuchs, Soluble receptors for tumour necrosis factor in clinical laboratory diagnosis, Eur. J. Haematol. 54 (1995) 1–8. M. Krampera, L. Cosmi, R. Angeli, A. Pasini, F. Liotta, A. Andreini, V. Santarlasci, B. Mazzinghi, G. Pizzolo, F. Vinante, P. Romagnani, E. Maggi, S. Romagnani, F. Annunziato, Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells, Stem Cells 24 (2006) 386–398. J.L. Chan, K.C. Tang, A.P. Patel, L.M. Bonilla, N. Pierobon, N.M. Ponzio, P. Rameshwar, Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma, Blood 107 (2006) 4817–4824. D.H. Munn, M. Zhou, J.T. Attwood, I. Bondarev, S.J. Conway, B. Marshall, C. Brown, A.L. Mellor, Prevention of allogeneic fetal rejection by tryptophan catabolism, Science 281 (1998) 1191–1193. R. Meisel, A. Zibert, M. Laryea, U. Göbel, W. Däubener, D. Dilloo, Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation, Blood 103 (2004) 4619–4621.