Human Placental Mesenchymal Stem Cells (pMSCs ...

Stem Cell Rev and Rep DOI 10.1007/s12015-013-9455-2

Human Placental Mesenchymal Stem Cells (pMSCs) Play a Role as Immune Suppressive Cells by Shifting Macrophage Differentiation from Inflammatory M1 to Anti-inflammatory M2 Macrophages M. H. Abumaree & M. A. Al Jumah & B. Kalionis & D. Jawdat & A. Al Khaldi & F. M. Abomaray & A. S. Fatani & L. W. Chamley & B. A. Knawy

# Springer Science+Business Media New York 2013

Abstract Background Mesenchymal stem cells (MSCs) have a therapeutic potential in tissue repair because of capacity for multipotent differentiation and their ability to modulate the immune response. In this study, we examined the ability of human placental MSCs (pMSCs) to modify the differentiation of human monocytes into macrophages and assessed the influence of pMSCs on important macrophage functions. M. H. Abumaree : M. A. Al Jumah : D. Jawdat : A. Al Khaldi : A. S. Fatani : B. A. Knawy College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Mail Code 1515, Saudi Arabia M. H. Abumaree (*) : M. A. Al Jumah : D. Jawdat : A. Al Khaldi : F. M. Abomaray : B. A. Knawy King Abdullah International Medical Research Center, King Abdulaziz Medical City, National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Mail Code 1515, Saudi Arabia e-mail: [email protected] B. Kalionis Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, Victoria, Australia 3052 B. Kalionis Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia 3052 L. W. Chamley Department of Obstetrics & Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, 11, Private Bag 92019, Grafton, Auckland, New Zealand

Methods We used GM-CSF to stimulate the differentiation of monocytes into the M1 macrophage pathway and then co-cultured these cells with pMSCs in the early stages of macrophage differentiation. We then evaluated the effect on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry and ELISA. The mechanism by which pMSCs could mediate their effects on macrophage differentiation was also studied. Results The co-culture of pMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway resulted in a shift to anti-inflammatory M2-like macrophage differentiation. This transition was characterized by morphological of changes typical of M2 macrophages, and by changes in cell surface marker expression including CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b, which are distinctive of M2 macrophages. Coculture with pMSCs reduced the expression of the costimulatory molecules (CD40, CD80 and CD86) and increased the expression of co-inhibitory molecules (CD273, CD274 and B7-H4) as well as the surface expression of major histocompatibility complex (MHC-II) molecules. Furthermore, the secretion of IL-10 was increased while the secretion of IL-1β, IL-12 (p70) and MIP-1α was decreased; a profile typical of M2 macrophages. Finally, pMSCs induced the phagocytic activity and the phagocytosis of apoptotic cells associated with M2- like macrophages; again a profile typical of M2 macrophages. We found that the immunoregulatory effect of pMSCs on macrophage differentiation was mediated by soluble molecules acting partially via glucocorticoid and progesterone receptors.

Stem Cell Rev and Rep

Conclusions We have shown that pMSCs can transition macrophages from an inflammatory M1 into an antiinflammatory M2 phenotype. Our findings suggest a new immunosuppressive property of pMSCs that may be employed in the resolution of inflammation associated with inflammatory diseases and in tissue repair. Keywords Placenta mesenchymal stem cells . Immune suppression . Macrophages . Inflammation

Introduction Mesenchymal stem cells (MSCs) are the main subset of adult stem cells that can be isolated from different human tissues [1, 2]. MSCs are described as multipotent cells that can differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons and hepatocytes [1]. We have successfully isolated MSCs from the human term placenta, a tissue which is accessible, abundant and commonly discarded after normal delivery, capable of selfrenewal, capable of differentiation into lineages of mesenchymal tissues and with immunoregulation properties [2, 3]. These properties of placental MSCs (pMSCs) make them an attractive alternative source of MSCs for cell-based therapy. The mechanisms underlying the immunoregulatory functions of MSCs are complex and not fully understood. MSCs have immunomodulatory effects, particularly on T and B lymphocytes [3, 4]. However, relatively little attention has been paid to the potential interaction of MSCs with macrophages. A few studies reported that MSCs can induce anti-inflammatory phenotypes in macrophages in vitro and in vivo [5–11]. However, studies of the immunomodulatory effects of pMSCs on the functions of macrophages have not been undertaken. Macrophages comprise a heterogeneous population of cells that are widely distributed in many different tissues and are potent immune regulators [12]. Macrophages play a crucial role in the resolution of tissue injury and promotion of tissue repair in various human diseases [12]. In response to chemotactic signals, monocytes migrate into tissues and subsequently differentiate into macrophages to replace aged ones or to participate in the initial phases of tissue defense in response to harmful insults [12]. Macrophages also play an essential role in later phases of tissue homeostasis and repair, such as phagocytosis of apoptotic cells [12]. Functionally, macrophages can be polarized into distinct subpopulations of cells; each with specific functions related to the initiation and recovery phases of tissue injury [12]. The origin and activation state of macrophages and their microenvironment are critical determinants of their response to injury. Typically, macrophages are classified into two main groups: classicallyactivated macrophages (M1) and alternatively-activated macrophages (M2) [12].

M1 macrophages are pro-inflammatory cells with potent antimicrobial activities and promote T helper 1 (Th1) cell responses and have also been implicated in many inflammatory diseases [12]. Conversely, M2 macrophages are immunosuppressive cells that can support helper 2 (Th2)-associated effector functions. M2 macrophages are characterized based on their low production of proinflammatory cytokines, such as IL-1ß and IL-12, high production of anti-inflammatory cytokines such as IL-10 and have high phagocytic activities [12]. M2 macrophages play a major role in the resolution of inflammation, tissue remodeling and in wound repair by releasing IL-10, secreting trophic factors and enhancing the clearance of apoptotic cells [12]. Macrophage heterogeneity is likely to reflect the plasticity and versatility of these cells in response to microenvironmental signals. The differentiation of macrophages and dendritic cells from monocytes is reversible and this is determined by the microenvironments and the differentiation signals [13]. Various signals induce the differentiation of M1 or M2 macrophages. The differentiation pathway of M1 macrophage is activated by different molecules, such as interferon gamma (IFN-γ) or granulocyte macrophage colony stimulating factor (GM-CSF) while the differentiation of M2 macrophages is promoted by different molecules, such as IL1 receptor antagonist, IL-4, IL-6, IL-10, IL-13, glucocorticoids, macrophage colony stimulating factors (MCSF), vascular endothelial growth factor (VEGF), transforming growth factor beta- 1 (TGF-β1), leukemia inhibitory factor (LIF) and prostaglandin E2 (PGE2) [14–18]. Several studies have demonstrated the protective role of macrophages in cell therapy. The beneficial therapeutical effects of macrophages were shown in various tissue injury, such as central nervous system (CNS), heart and lungs [8, 10, 11, 19, 20]. For example, the immune system is activated following injury to the CNS. This affects the ability of neurons to survive and to regenerate damaged axons. However, the role of the immune system in the CNS is determined by the type and duration of the immune response and the balance between destructive and protective factors that ultimately define the net result of the neuro-immune interaction [21]. Injury in the CNS rapidly induces the response of M1 macrophages and then this response is shifted into M2 macrophages [21]. The response of M1 macrophages induces neuronal damage, whereas M2 macrophage response promotes neuronal regeneration [21]. Therefore, switching macrophages from M1 into M2 phenotype can promote the repair of CNS. Accordingly, modulating immune response seems to be a promising strategy for successful tissue repair. This reversibility in macrophage functions will be one of the useful therapeutic targets for the design of an efficient cellular therapy utilizing macrophages to remodel and repair tissue injury. Several recent studies showed that macrophages can be switched from inflammatory into anti-inflammatory phenotypes following their interaction with MSCs; this interaction


resulted in the resolution of inflammation and tissue repair of heart, lung and skin [5–11]. These studies used MSCs from bone marrow, adipose tissues, gingiva and cord blood, but the interaction between pMSCs and macrophages has not been examined. pMSCs used in regenerative or reparative therapies are likely to interact with macrophages and their immunoregulatory abilities could potentially influence macrophage differentiation and/or function. Therefore, we examined whether human pMSCs can induce anti-inflammatory phenotypes in human macrophages and also studied the mechanism by which pMSCs can shift macrophage differentiation. Previous studies demonstrated that molecules with antiinflammatory properties can mediate MSC induction of M2 macrophage differentiation [15–18, 22], and therefore we examined the role of glucocorticoid receptor (GR) and progesterone receptor (PR) in mediating the anti-inflammatory effects of pMSCs on macrophages. These two receptors are involved in the production of anti-inflammatory proteins, promotion of phagocytosis of apoptotic cells and the differentiation of anti-inflammatory M2 macrophages [18, 23]. We found that human pMSCs can induce an anti-inflammatory phenotype in macrophages by shifting the differentiation of macrophages from M1 into M2 macrophages and also by down-regulating the expression of costimulatory molecules (CD40, CD80 and CD86) with induced expression on coinhibitory proteins (CD273, CD274 and B7H4). In addition, we found the immunoregulatory effects of pMSCs on human macrophages are mediated partially via GR and PR.

Materials and Methods Ethics of Experimentation This study was approved by the institutional research board at King Abdulla International Medical Research Centre/ King Abdulaziz Medical City, Riyadh, Saudi Arabia. All placentae were obtained with informed patient consent. Placentae Human placentae were obtained from uncomplicated pregnancies following normal vaginal delivery (38–40 weeks of gestation). The gestational age and fetal viability of all pregnancies were confirmed by early ultrasound examination before 20 weeks gestation. The placentae were used within 2 h of delivery. Isolation of Mesenchymal Stem Cells from Placental Explants in Vitro pMSCs were isolated from chorionic villi of human term placenta as described previously [2]. Briefly, placental tissues were dissected and then washed thoroughly with sterile

phosphate buffered saline (PBS), pH 7.4. After removing the maternal decidua, the chorionic villi from the fetal portion were cut into pieces of approximately 40 mg wet weight and washed with PBS and then incubated with 2.5 % trypsin (Invitrogen, Saudi Arabia) diluted in DMEM-F12 medium (Invitrogen, Saudi Arabia) containing (271 unit/ml) DNase (Invitrogen, Saudi Arabia), 100 μg/ml streptomycin and 100 U/l penicillin with gentle rotation overnight at 4 °C. Tissues were then washed thoroughly with PBS and allowed to adhere to the plastic in six well plates for 1 h at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air (a cell culture incubator). Then, DMEM-F12 medium containing 10 % Mesenchymal Stem Cell Certified fetal bovine serum (MSCFBS) (Invitrogen, Saudi Arabia), 100 μg/ml of Lglutamate, 100 μg/ml streptomycin and 100 U/l penicillin was gently added to the tissues and cultured at 37 °C in a cell culture incubator. Every 2 days, the medium was removed and replaced with fresh medium. On day 14, the tissues were removed and cells migrated out from the cut ends of the tissues were then harvested with TrypLE™ Express detachment solution (Invitrogen, Saudi Arabia) and characterized by flow cytometry using MSC positive markers (CD44, CD90, CD146, CD166 and CD105) and hematopoietic negative markers (CD19, CD45, HLA-DR, CD80, CD86, and CD40) as described previously [2]. Cells at a density of 1×105 cells in 75 cm2 flask (BD, Saudi Arabia) were re-cultured until they reached 75 % confluency and then used in subsequent experiments. Before any further experimental uses, pMSCs at passage 2 were assessed for differentiation into adipocytes, chondrocytes and osteocytes as described previously [2]. Adipogenic, osteogenic and chondrogenic differentiations were performed by incubating pMSCs in adipogenic (#390415), osteogenic (#390416) and chondrogenic (#390417) media, respectively. All differentiation media were purchased from R & D Systems (Saudi Arabia). Each differentiation medium was supplemented with 10 % MSCFBS, 100 μg/ml of Lglutamate, 100 μg/ml streptomycin and 100 U/l penicillin. Adipocytes, osteoblasts and chondrocytes were identified using LipidTOX™ Green, Alizarin Red S and Alcian Blue dyes, respectively as described previously [2]. All antibodies were from Beckman Coulter (Saudi Arabia). Thirty placentae were used in this study. Isolation of Human Monocytes Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood drawn from 30 normal healthy volunteers. PBMCs were isolated by centrifugation on a FicollPaque Leucosep™ density gradient (Greiner Bio-One, Saudi Arabia) and suspended in RPMI-1640 medium. Monocytes were obtained using a magnetic cell separation system as described previously [24]. PBMCs were incubated with antiCD14 monoclonal antibody-coated microbeads, and


monocytes were then isolated by passing the PBMCs through a column placed in a magnetic cell separator according to the manufacturer’s instructions. The purity of monocytes was assessed by flow cytometry using an antiCD14 monoclonal antibody. Samples with purity greater than 98 % were used for experiments. Culture of Monocyte Derived Macrophages with Different Treatments of pMSCs (Direct Contact, Supernatant and Conditioned Medium) Monocytes were differentiated into M1 macrophages as described previously [25]. Monocytes at a density of 1×106 were seeded in 6-well culture plates in M1 differentiation medium consisting of RPMI 1640 medium supplemented with 10 % FBS, 2 mM L-glutamine, 50 ng/mL GM-CSF (R and D Systems, Saudi Arabia), 50 U/mL penicillin and 50 μg/mL streptomycin and then incubated for 6 days at 37 °C in a cell culture incubator. For intercellular direct contact experiments (ICpMSC), the 0.4 μm pore size transwell chamber membrane culture system (Greiner Bio-One, Saudi Arabia) was used as described previously [26]. pMSCs were seeded on the reverse side of the membrane of the chamber until the cells were fully adhered and monocytes were seeded on the upper side of the membrane. For soluble factor experiments (SFpMSC), pMSCs were physically separated from monocytes by culturing them on the upper compartments while monocytes were cultured in the lower compartment. Both culture systems prevent the contamination of monocyte-derived macrophages with pMSCs and facilitate harvesting macrophages without pMSC contamination. In both systems, cells were cultured in M1 macrophage differentiation medium for 6 days as described previously [25]. For conditioned medium experiments (CMpMSC), supernatant from unstimulated pMSCs were added to monocytes cultured in M1 differentiation medium. To produce conditioned medium, 1×105 pMSCs were cultured in 75 cm2 flasks containing DMEM-F12 medium with 10 % MSCFBS, 100 μg/ml of L-glutamate, 100 μg/ml streptomycin and 100 U/l penicillin. Every 2 days, the medium was removed and replaced with fresh medium. When cells reached 75 % confluency, cells were cultured in fresh medium for 2 days and conditioned medium was then harvested, centrifuged at 500 × g for 10 min and stored at −80 °C until use. On day 7, macrophages and pMSCs were harvested with TrypLE™ Express detachment solution and characterized by flow cytometry technique. Macrophages were characterized for the expression of M2 macrophage markers (CD14, CD11b, CD36, CD163, CD204, CD206 and B7H4), costimulatory molecules (CD40, CD80, CD86, CD273 and CD274) and MHC class II molecule, HLA-DR while pMSCs were characterized for the expression of the intracellular proteins including IL1-ß, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor- alpha (TNF-α),

monocyte chemoattractant protein-1 (MCP-1), indoleamine 2,3-dioxygenase (IDO) and B7H4). In some experiments ICpMSC, SFpMSC, CMpMSC were added to M1 macrophage culture on either day 3 or day 7 and incubated for further 3 days and then characterized as described above. Before the addition of pMSCs to the coculture experiments (ICpMSC and SFpMSC), pMSCs were treated with 25 μg/ml Mitomycin C for 1 h at 37 °C to inhibit their proliferation and then followed by five extensive washes with RPMI-1640 medium containing FBS as described previously [27]. To assess whether pMSCs effects on macrophage differentiation were reversible, ICpMSC or SFpMSC or CMpMSC were removed after 3 days of co-culturing with monocytes and macrophages were washed thoroughly and then cultured again in M1 macrophage differentiation medium containing GM-CSF for another 3 days. Each experiment was performed in duplicate and repeated with 30 independent preparations of monocyte-derived macrophages and pMSCs. Monocytederived macrophages cultured in a GM-CSF medium without pMSCs were included as a negative control for all monocyte-derived macrophages cultured with pMSCs (ICpMSC, SFpMSC and CMpMSC). pMSC Induction of M2 Macrophage Differentiation CMpMSC experiments were performed in which 500 nM/mL Mifepristone (Tocris Bioscience, Saudi Arabia) in M1 macrophage differentiation medium containing 20 % CMpMSC was added to monocyte culture on day zero. The dose of 500 nM/mL Mifepristone was used because this dose was shown by others to result in a complete blockage of binding receptors [28]. Positive control was macrophages cultured in M1 macrophage differentiation medium containing 100 μM/ mL dexamethasone in the presence and absence of Mifepristone. After 6 days in culture, macrophages were then harvested as described above. The mean number of differentiated cells was quantified by counting the number of cells in ten random high-power fields (40×) on the culture plates. Monocytes cultured with CMpMSC without Mifepristone were included as a negative control. Each experiment was performed in duplicate and repeated with ten independent preparations of monocytes- derived macrophages and pMSCs. Phagocytic Function of Monocyte Derived Macrophages To study the phagocytic function of macrophages, the CytoSelect™ Phagocytosis kit was used (# CBA-224, Cell Biolabs, Saudi Arabia) according to the manufacturer’s instructions. Briefly, monocyte-derived macrophages were cultured for 6 days with ICpMSC or SFpMSC or CMpMSC in 24-well flat-bottom culture plates containing M1 differentiation medium, consisting of RPMI 1640


medium supplemented with 10 % FBS, 2 mM L-glutamine, 50 ng/mL GM-CSF, 50 U/mL penicillin and 50 μg/mL streptomycin. On day 7, pre-labelled- Zymosan suspension was added to each well and then incubated for 2 h at 37 °C in a cell culture incubator. Negative controls of macrophages without ICpMSC or SFpMSC or CMpMSC and cells without Zymosan particles were also included. Following removal of the culture medium, cells were washed, fixed and incubated with a blocking solution for 1 h at room temperature. Cells were then washed, permeabilized and incubated with a detection reagent for 1 h at room temperature. Following washing, cells were incubated with a detection buffer for 10 min at room temperature and then incubated with a substrate to initiate the reaction for 20 min at 37 °C. Finally, the reaction was stopped and the optical density was read at 405 nm. Each experiment was performed in duplicate. Ten individual preparations of monocyte-derived macrophages and 10 pMSCs were used in this study. Phagocytosis of Apoptotic Cells by Macrophages Following 6 days of coculture of monocyte- derived macrophages with CMpMSC, macrophages were labelled with

5 μM green fluorescent cell tracker stain (5-chloromethylfluorescin diacetate; CMFDA; Molecular Probes, Invitrogen, Saudi Arabia) in DMEM/F12 Medium for 4 h. Apoptotic pMSCs were generated by freezing and thawing method as described previously [29] and labelled with 5 μM red fluorescent cell tracker stain 5-(and-6)-carboxy SNARF®-1, acetoxymethyl ester, acetate (SNARF; Molecular Probes, Invitrogen, Saudi Arabia) for 4 h and then added to macrophage culture at a ratio of 1 (Apoptotic pMSCs): 10 (macrophages) for 24 h in the presence or absence of 10 μg/mL cytochalasin B (Invitrogen, Saudi Arabia). Apoptotic pMSCs from each placenta were added to macrophages isolated from an individual (independent) donor. Remaining apoptotic cells were removed by extensive washing with PBS, and macrophages were fixed with 4 % (w/v) paraformaldehyde and visualized on an inverted fluorescent microscope (Nikon ECLIPSE Ti U) and photomicrographs recorded using a Nikon DS-Qi1 camera and software. The percentage of macrophages containing apoptotic cells was reported as the phagocytic index using the following formula:

. Phagocytic index ðPIÞ ¼ Apoptotic cells associated with macrophages Total number of macrophages 100

Comparison Between the Effects of pMSCs and Bone Marrow Derived MSCs on the Differentiation of Macrophages To compare between the effects of pMSCs and BMMSCs on macrophage differentiation, we used BMMSCs (Catalogue # C-12974, Promocell, Saudi Arabia). BMMSCs (1×105 cells) were cultured in 75 cm 2 flask containing αMEM supplemented with 10 % FBS, 100 μg/ml of L-glutamate, 100 μg/ml streptomycin and 100 U/l penicillin. Cells were then harvested and characterized by flow cytometry and used for co-culture studies. BMMSCs were positive for MSC markers (CD44, CD90 and CD105) and negative for hematopoietic markers (CD45) (data not shown). Intracellular contact (IC) and soluble factor (SF) experiments were performed as described above. Macrophage-like cells were characterized for the expression of M2 macrophage markers (CD163 and CD206) by flow cytometry. Soluble factors [IL-10 and IL12 (p70)], which are secreted bymacrophages, were measured by ELISA. Each experiment was performed in duplicate. Three independent experiments were performed in this study.

Quantification of Human Cytokines ELISA Kits (R & D Systems or MyBiosource, Saudi Arabia) were used according to the manufacturer’s instructions to quantify IL-1β, IL10, IL-12 (p70), MIP-1α and MCP-1 in the supernatants obtained from macrophage culture, whilst MCSF, TGFß-1, VEGF, LIF, B7-H4, and PEGE2 were quantified in the CM of unstimulated pMSCs or pMSC stimulated with macrophages. Both unstimulated and stimulated pMSCs were cultured at confluency for 2 days. To produce CM of stimulated pMSCs, soluble factor experiments (SFpMSC) were performed as described above. On day 7, the transwell coated pMSCs were washed thoroughly with PBS and then cultured in DMEM-F12 medium containing 10 % MSCFBS, 100 μg/ml of L-glutamate, 100 μg/ml streptomycin and 100 U/l penicillin for 2 days. Complete RPM-1640 and DMEM-F12 were included as a negative control. Flow Cytometry Cells were harvested using TrypLE™ Express detachment solution. For analysis, 1×105 of cells were stained with


ntibodies (Table 1) for 30 min and then washed twice with cold PBS by centrifugation at 150 × g for 5 min at 8 °C. To analyse intracellular expression of IL1-ß, IL-6, IL-8, IL-10, IL-12, TNF-α, MCP-1, Indoleamine-pyrrole 2,3-dioxygenase (IDO) and B7H4, cells were fixed with 4 % paraformaldehyde in sterile PBS, pH 7.4 for 10 min at room temperature and then permeabilized using a sterile PBS, pH 7.4 containing 0.1 % saponin for 5 min at room temperature. The expression of the corresponding intracellular and cell surface proteins was assayed by an FC500 (Beckman Coulter, Saudi Arabia) flow cytometer. As a negative control for analysis, cells in a separate tube were treated with FITC or PE-labeled mouse IgG or IgM antibody Statistical Analysis Data were analyzed using the Mann-Witney U and KruskalWallis tests for non-parametric data. These analyses were Table 1 Antibodies used in this study to characterize the isolated placental mesenchymal stem cells (pMSCs) and monocyte derived macrophages Antibody

Conjugate

Dilution

Clone

Manufacturer

CD45 CD19 CD90

FITC FITC FITC

1:10 1:10 1:10

J33 J3-119 F15-42-1-5

Beckman Coulter Beckman Coulter Beckman Coulter

CD44 HLA-ABC CD105 CD146 CD166 CD14 CD11B CD163 CD204 CD206 B7H4 CD80 CD86 CD40 HLA-DR CD273 CD274 IL1ß

FITC FITC PE PE PE PE PE PE PE PE PE PE PE PE FITC PC7 PC7 PE

1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10

J.173 B9.12.1 1G2 TEA 1/34 3A6 RMO52 M1/70 215927 351615 685641 MIH43 MAB104 HA5.2B7 MAB89 B8.12.2 PD-L2 PDL1.3.1 8516

Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter R & D Systems R & D Systems R & D Systems R& D Systems Abcam Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter R & D Systems

IL-6 IL-8 IL-12 IL-10 MCP-1 TNF-α IDO IgG/IgM

PE PE PE PE PE PE PE FITC/ PE

1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10

1936 6217 27537 127107 2H5 6402 700838

R & D Systems R & D Systems R & D Systems R & D Systems Abacm R & D Systems R & D Systems Beckman Coulter

Fig. 1 Morphological effects of human pMSCs on the GM-CSF–stimulated human monocyte to macrophage differentiation analysed by microscopic examination (Panels A and B) and flow cytometric analysis of cell surface molecules (CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b). Representative phase contrast microscopic images revealing a M1-like macrophages (fried egg-shaped morphology, scale bar 10 μm) differentiated from monocytes cultured for 6 days in medium containing GM-CSF and b elongated, spindle-like morphology (M2-like anti-inflammatory macrophages, scale bar 100 μm) differentiated from monocytes cultured for 6 days in medium containing GM-CSF and pMSCs. The expression profiles of 30 individual experiments demonstrating that pMSCs [ICpMSC (Intracellular Direct Contact), SFpMSC (Soluble Factor), CMpMSC (Conditioned Medium)] as compared to untreated (GMCSF treated monocytes without pMSCs) induced changes in membrane expression of c–i CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b on macrophages at day 7 of the culture of monocytes with GMCSF. ICpMSC experiments were conducted using 0.4 μm pore size transwell chamber membrane culture system in which pMSCs were seeded on the reverse side of the membrane of the chamber and monocytes were seeded on the upper side of the membrane. For SFpMSC experiments, pMSCs were cultured on the upper compartments while monocytes were cultured in the lower compartment. For CMpMSC experiments, supernatant from pMSCs were added to monocytes cultured in M1 differentiation medium. pMSCs were added to the monocyte cultures on day 1. The levels of expression are presented as median fluorescent intensity (MFI) or median percentage of B7-H4 positive cells as determined by flow cytometry. Experiments were conducted in triplicate using the indicated pMSC treatments. Thirty independent placentae were used to prepare pMSCs and monocytes were from the peripheral blood of 30 individual healthy donors. *P, **P and ***P