Allogeneic chondrogenically differentiated human bone marrow stromal cells do not induce dendritic cell maturation

Abstract Bone marrow stromal cell (BMSC)‐mediated endochondral bone formation may be a promising alternative to the current gold standards of autologous bone transplantation, in the development of novel methods for bone repair. Implantation of chondrogenically differentiated BMSCs leads to bone formation in vivo via endochondral ossification. The success of this bone formation in an allogeneic system depends upon the interaction between the implanted constructs and the host immune system. The current study investigated the effect of chondrogenically differentiated human bone marrow stromal cell (hBMSC) pellets on the maturation and function of dendritic cells (DCs) by directly coculturing bone forming chondrogenic hBMSC pellets and immature or lipopolysaccharide (LPS)‐matured DCs in vitro. Allogeneic chondrogenic hBMSC pellets did not affect the expression of CD80, CD86, or HLADR on immature or LPS‐matured DCs following 24, 48, or 72 hr of coculture. Furthermore, they did not induce or inhibit antigen uptake or migration of the DCs over time. IL‐6 was secreted by allogeneic chondrogenic hBMSC pellets in response to LPS‐matured DCs. Overall, this study has demonstrated that maturation of immature DCs was not influenced by allogeneic chondrogenic hBMSC pellets. This suggests that allogeneic chondrogenic hBMSC pellets do not stimulate immunogenic responses from DCs in vitro and are not expected to indirectly activate T cells via DCs. For this reason, allogeneic chondrogenic bone marrow stromal cell pellets are promising candidates for future tissue engineering strategies utilising allogeneic cells for bone repair.

. It is unclear whether these immunomodulatory properties are maintained upon differentiation. Allogenic chondrogenically differentiated BMSCs could be used in a therapy as an alternative to the current gold standard for bone repair, autologous bone transplantation. For this to become a feasible option, the interaction between the implanted cells and the host immune system in the process of the bone formation needs to be fully elucidated.
Although there have been many studies investigating the effects of undifferentiated BMSCs on host immune cells, little is known about the effect of chondrogenically differentiated BMSC pellets. Undifferentiated BMSCs are known to have an immunosuppressive effect on T lymphocytes (Di Nicola et al., 2002;Krampera et al., 2003;Mougiakakos et al., 2011). In a recent study by our group, direct coculture of chondrogenically differentiated BMSC pellets and peripheral blood mononuclear cells (PBMCs) revealed that chondrogenically differentiated BMSC pellets did not induce the proliferation of naïve or stimulated T lymphocytes, suggesting that allogeneic chondrogenically differentiated BMSC pellets are non-immunogenic (Kiernan et al., 2016). However, it is possible that there might be an indirect stimulation of T cells by dendritic cells (DCs) that have encountered these BMSCs. DCs are the main antigen-presenting cells (APCs) of the innate immune system, with a primary role in activating T-cell-dependent immune responses (Guermonprez, Valladeau, Zitvogel, Thery, & Amigorena, 2002). Therefore, the aim of the current research was to determine if chondrogenically differentiated BMSC pellets affect DCs.
Immature DCs survey the peripheral tissues for potential threats to the immune system, have low levels of major histocompatibility complex (MHC) molecules (or human leukocyte antigen [HLA]) and the costimulatory molecules CD80 and CD86, and are not yet equipped to stimulate naïve T cells. Antigen uptake and processing by immature DCs, in addition to locally produced inflammatory cytokines, induce their maturation and migration to the draining lymph nodes. DC maturation is a prerequisite for the stimulation of naïve and memory T cells (Lutz & Schuler, 2002). Therefore, DCs present themselves as potential targets for BMSC-mediated immunosuppression. Undifferentiated BMSCs have been shown to inhibit the differentiation, maturation, and function of DCs (Jiang et al., 2005;Spaggiari, Abdelrazik, Becchetti, & Moretta, 2009;Zhang et al., 2004), however, little is known about the effect of chondrogenically differentiated BMSCs on DCs. Chondrogenically differentiated BMSCs have been reported to be more immunogenic compared with undifferentiated BMSCs (Chen et al., 2007;Ryan et al., 2014). However, few studies have directly cocultured chondrogenically differentiated BMSC pellets and immune cells. Undifferentiated BMSCs lack immunogenicity due to the absence of MHC Class I or costimulatory molecule expression on their surface, and their ability to secrete immunosuppressive molecules (PGE2, NO and IDO;Chen, Tredget, Wu, & Wu, 2008;Meisel et al., 2004;Ryan, Barry, Murphy, & Mahon, 2007;Sato et al., 2007). Chondrogenically differentiated BMSCs in contrast may lose this immunosuppressive capacity by upregulating costimulatory molecules on the surface, leading to induced DC maturation and enhanced lymphocyte proliferation (Chen et al., 2007). In addition to this, the anti-inflammatory mediators PGE2 and NO have been shown to be significantly reduced upon chondrogenic differentiation of BMSCs, inducing immunogenicity in these cells . Contradictory to these reports, Zheng, Li, Ding, Jia, and Zhu (2008)  Human bone marrow stromal cells (hBMSCs) were isolated as previously described (Kiernan et al., 2016) from heparinised femoral-shaft marrow aspirates of patients undergoing total hip arthroplasty (with informed consent after approval by Erasmus MC medical ethical committee protocol METC-2004-142). hBMSCs were expanded in standard medium consisting of minimum essential medium alpha medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heatinactivated fetal calf serum (Life Technologies; The Netherlands; lot number, 41Q2047K); 50-μg/ml gentamycin; 1.5-μg/ml fungizone (All Invitrogen); 1-ng/ml fibroblast growth factor-2 (Instruchemie B.V., Delfzijl, The Netherlands); and 0.1 mM of L -ascorbic acid 2-phosphate (Sigma-Aldrich, MO, USA). hBMSCs were cultured at 37°C under humidified condition and 5% carbon dioxide, refreshing medium every 3-4 days. Third to fourth passage cells were trypsinised using 0.05% trypsin (Life Technologies) when they reached 80-90% confluency and used for BMSC pellet cultures. hBMSCs (0.2 × 10 6 ) were added to 15-ml polypropylene tubes (Sarstedt) in 0.5 ml of standard chondrogenic medium consisting of high-glucose Dulbecco's modified Eagle's medium with 50-mg/ml gentamicin; 1.5-mg/ml Fungizone (Invitrogen); 100-mM sodium pyruvate (Sigma-Aldrich); 1:100 ITS (BD Biosciences); L -ascorbic acid 2-phosphate; 10-ng/ml TGF-β3 (Peprotech); and 100-nM dexamethasone (Sigma-Aldrich). Cells were cultured for 10 days with TGFβ3, refreshing the medium every 3-4 days.
The remaining layers above the filter were transferred to a new 50-ml falcon tube and centrifuged at 835g for 7 min with brake. Following four washing steps (in wash medium, 690× g for 7 min), monocytes were separated from freshly obtained PBMCs using the MACS Monocyte Isolation kit (Miltenyi, Biotech) according to manufacturer's instructions. Briefly, 500 × 10 6 PBMCs were incubated with 100-μl CD14+ MicroBeads for 20 min in the dark at 4°C, washed and resuspended in running buffer (phosphate buffered saline (PBS) with 0.5% BSA, Sigma, and 2-mM EDTA, Gibco). Subsequently, magnetically labelled cells were applied on a column (LS column, Miltenyi, Biotech), pre-washed with 5 ml of running buffer) and placed in a MidiMACS Separator (Miltenyi, Biotech). The CD14+ fraction was collected after removal of the column from the MidiMACS Separator while collecting the flow-through. Afterwards, CD14+ monocytes were washed with RPMI-1640 medium (supplemented with 50-μg/mL Gentamycin and 1.5-μg/mL Fungizone, both Invitrogen) and cultured in flat-bottomed six-well tissue culture plates at a concentration of 4 × 10 6 cells in 3ml RPMI-1640 medium supplemented with 10% FCS (heatinactivated, Gibco), 2-mM L-glutamine, 50-μg/ml Gentamycin, 1.5-μg/ml Fungizone (all Invitrogen), 50-ng/ml GM-CSF (Peprotech), and 10-ng/mL IL-4 (Peprotech) to induce DC differentiation. Every 2 days, half of the medium was refreshed, and monocyte-derived DCs were cultured for 6 days (immature DCs). For induction of DC maturation, LPS (Sigma) was added at 100 ng/ml on Day 5 for 24 hr (LPS-matured DCs).

| Phenotypic analysis of DC populations using flow cytometry
Following above-described coculture regimes, DCs were harvested by pipette aspiration. The chondrogenic hBMSC pellets were removed prior to the DC harvest. DCs were centrifuged for 8 min at 248g.
The cells were resuspended in FACSflow (BD Biosciences), and 2 × 10 5 cells per sample were transferred to FACS tubes. Cells centrifuged for 5 min at 689g were resuspended in 100 μl of FACSflow con-

| Analysis of DC phagocytic uptake
To study the effect of chondrogenically differentiated hBMSC pellets on DC phagocytic uptake capacity, 5 × 10 5 DCs were harvested and resuspended in 1 ml of PBS containing FITC-Dextran (1 mg/ml; Sigma). Cells were transferred back to the 24-well plates and incubated for 1 hr at 37°C, or at 4°C as a control. The DCs were then harvested; centrifuged (248g, 8 mins); and resuspended in ice-cold PBS.
Samples were washed twice with ice-cold PBS and resuspended in 500 μl of FACSflow. Quantitative uptake of FITC-Dextran was analysed by flow cytometry as above.

| Analysis of DC migratory capacity
Cell migration was performed using 24-well Transwell chambers (Corning Costar, Cambridge, MA) using 5-μm pore size polycarbonate membranes. Immature and LPS-matured DCs (1 × 10 5 in 100 μl of supplemented RPMI-1640 medium) cultured with chondrogenic hBMSC pellets were seeded into the upper chamber. Supplemented RPMI-1640 medium (600 μl per well) with and without CCL21 (250 ng/ml) was added to the lower chamber. Cells were incubated for 3 hr at 37°C. The number of migrated cells was quantified using Accucheck counting beads according to the manufacturer's instructions (Invitrogen). Briefly, cells were resuspended in 100 μl of FACSflow and an equal volume of Accucheck beads. The amount of counting beads was determined. Because the amount of counting beads in each samples is equal, the number of migrated cells was calculated. The number of DCs that migrated to normal medium was subtracted from those that migrated to the medium containing CCL21.
2.7 | IL-6, IL-10, and IL-12 secretion from supernatants from coculture Supernatants from the cocultures were centrifuged at 690g for 10 min and stored at −80°C for later analysis. IL-6 (Peprotech), IL-10 (R&D Systems), and IL-12 (Peprotech) secretion was determined in the supernatants from the cocultures using enzyme-linked immunosorbent assay measurements. The measurements were performed and calculated according to manufacturer's instructions.
Sectioned slides were stained using a rabbit monoclonal anti-CD11 (EP1347Y, Genetex) or rabbit IgG as a negative control antibody (X0903, Dako Cytomation) and labelled using an alkaline phosphatase link and label (Biogenex) to identify the presence of DCs within the matrix of the cells.

| Quantitative real-time reverse transcription polymerase chain reaction
Immature and LPS-matured DCs were removed from the coculture, washed with PBS, and resuspended in TRIzol reagent (Thermo Scientific). Similarly, chondrogenically differentiated hMSCs were removed from the coculture, washed with PBS, and crushed in TRIzol reagent.
RNA was isolated from all samples using RNeasy mini kit (Qiagen).
Complementary DNA was synthesised from isolated RNA using firststrand complementary DNA synthesis kit (Thermo Scientific) and used for real-time reverse transcription polymerase chain reaction (PCR).

| Statistics
Statistical analysis was performed using IBM SPSS Version 21 using a linear mixed model with Bonferroni post-test, or GraphPad Prism v.5 for a paired t test or an unpaired t test as indicated in figures. Values are presented as mean ± standard deviation where p < .05 was considered statistically significant.

| RESULTS
3.1 | Chondrogenically differentiated hBMSC pellets do not induce DC maturation following 24 hr of coculture Chondrogenically differentiated hBMSCs cultured in 3D for 10 days were added to immature or LPS-matured DCs for 24 hr (Figure 1a) at a ratio of one BMSC to five DCs. Thionine staining performed on hBMSC pellets confirmed that they were chondrogenic ( Figure S1).
Chondrogenically differentiated hBMSC pellets did not affect the expression of CD80, CD86, or HLADR in immature DCs (Figure 1b
Chondrogenically differentiated hBMSC pellets have been shown to form bone in both immunodeficient and immunocompetent animals (Farrell et al., 2011). DCs are known to express the important costimulatory molecules CD80 and CD86 upon maturation, which are ligands for CD28 to induce T cell activation (Sharpe & Freeman, 2002). HLADR (an MHC Class II cell surface receptor) is also expressed to present antigen to

T cells via their T cell receptor. Low levels of MHC expression on
DCs are known to result in T cell anergy (Chung, Ysebaert, Berneman, & Cools, 2013 (Abdi, Singh, & Matzinger, 2012;Iwamoto, Ishida, Takahashi, Takeda, & Miyazaki, 2005). LPS has also been shown to induce a population of DCs lowly expressing CD11c that are unable to stimulate T cells but have the capacity to induce regulatory T cells (Wang et al., 2015). We hypothesize that the LPS-matured DCs cul- The level of IL6 gene expression was measured in immature and LPS-matured DCs cultured alone or with chondrogenically differentiated hBMSC pellets (black) and also in chondrogenic hBMSC pellets cultured alone or with immature and LPSmatured DCs (grey) over time. Chondrogenic hBMSC pellets expressed higher levels of IL-6 compared with immature and LPS-matured DCs. n = 3 (one different hBMSC and DC donors in triplicate at three timepoints) ± SD. Unpaired t test * p < .05, ** p < .005, and *** p < .001 hBMSCs (Jiang et al., 2005) Interestingly, it appears from this study that IL-6 appears to play an important role in the interaction between allogeneic chondrogenic hBMSCs and DCs. IL-6 is a pleiotropic cytokine known to be essential in the development of APCs (Chomarat, Banchereau, Davoust, & Palucka, 2000;Xing et al., 1998). It has been previously shown that IL-6 could be responsible for the ability of undifferentiated BMSCs to inhibit DC functions (Djouad et al., 2007;Jiang et al., 2005). Following 24 hr of coculture, it was found that IL-6 was significantly secreted in supernatants from cultures of LPS-matured DCs and allogenic chondrogenic hBMSC pellets and this continued over 48 and 72 hr of coculture. At mRNA level, IL-6 expression was found to be significantly increased by allogeneic chondrogenic hBMSC pellets cultured with LPS-matured DCs demonstrating that the allogeneic chondrogenic hBMSCs were responsible for the production of the IL-6. It has been hypothesised that IL-6 secretion by undifferentiated BMSCs maintains the CD14 monocyte population as opposed to specifically inhibiting the DCs (Jiang et al., 2005). However, all of the DCs were CD14 negative in coculture with chondrogenic hBMSC pellets.
Although there was no clear effect of allogeneic chondrogenic hBMSC pellets on maturation or function of LPS-matured DCs at 24 or 48 hr, IL-6 production appears to be produced by the pellets in response to LPS-matured DCs. It is possible that this was why there was no altered behaviour of the DCs. The secretion of IL-10 and IL-12 was also measured in the supernatants. IL-10 was not secreted by either immature DCs or immature DCs cocultured with allogeneic hBMSC pellets after 24 hr ( Figure S4). It was secreted by chondrogenically differentiated BMSC pellets cultured alone, LPS-matured DCs and LPS-matured DCs cocultured with chondrogenic hBMSC pellets. However, there was no significant difference in the secretion of IL-10 between cocultures. IL-12 is known to be involved in the differentiation and function of naïve T cells into effector cells (Hsieh et al., 1993). IL-12 was found to be secreted in both immature and LPS-matured DCs cocultured with allogeneic chondrogenically differentiated hBMSCs for 24 hr.
However, there was no significant difference in IL-12 secretion between conditions. Over time, the level of IL-12 secretion was also measured in supernatants ( Figure S5). It was found that IL-12 was secreted at higher levels in supernatants from cocultured LPS-matured DCs and chondrogenic hBMSC pellets following 48 and 72 hr, although at a much lower concentration in comparison to IL-6. The secretion of both IL-6 and IL-12 might lead ultimately to the induction of specific T cell responses, or the balance between these cytokines might prevent T cell activation by DCs.
In conclusion, we show for the first time that allogeneic chondrogenic hBMSC pellets do not induce maturation of immature

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.