Original Paper Accepted after revision: August 30, 2014 Published online: November 29, 2014
Cells Tissues Organs DOI: 10.1159/000367986
The Immunomodulatory Properties of Periodontal Ligament Stem Cells Isolated from Inflamed Periodontal Granulation Chenghua Li a, b Xinwen Wang a Jun Tan c Tao Wang d Qintao Wang a
a
State Key Laboratory of Military Stomatology, Department of Periodontology, School of Stomatology, The Fourth Military Medical University, Xi’an, b Department of Stomatology, Beidaihe Sanatorium of Beijing Military Command, Qinhuangdao, c Department of Stomatology, General Hospital of Guangzhou Military Command of PLA, Guangzhou, and d Kong Jun Qing Dao Hang Kong Yi Xue Jian Ding Zhong Xin, Qingdao, PR China
Abstract Periodontitis is currently the main cause of tooth loss and as yet there is no appropriate method for establishing a functional and predictable periodontal regeneration. Tissue engineering involving seed cells provides a new prospect for periodontal regeneration. While periodontal ligament stem cells (PDLSCs) are a good choice for seed cells, it is not always possible to obtain the patients’ own PDLSCs. We and others have found a type of stromal cells from inflamed periodontal granulation. These cells displayed similar differentiation properties to PDLSCs. Inflammation has a profound influence on the immunomodulatory properties of mesenchymal stem cells, which may affect therapeutic outcome. In this study, we assessed the immunomodulatory characteristics of these inflamed human (ih)PDLSCs. Along with the similarity in cell surface marker expressions, they also displayed immunomodulatory properties comparable to those in healthy human (hh)PDLSCs. Both hhPDLSCs and ihPDLSCs can suppress the proliferation and secretion of IFN-γ in peripheral blood mononuclear cells by indirect soluble mediators and
© 2014 S. Karger AG, Basel 1422–6405/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cto
direct cell-cell contact. Albeit with some quantitative variances, the gene expressions of inducible nitric oxide synthases, indoleamine 2,3 dioxygenase, cyclooxygenase-2, TNF-α-induced protein 6 and IL-10 in ihPDLSCs displayed similar patterns as those in hhPDLSCs. Taken together, our results suggest that ihPDLSCs can provide a promising alternative to hhPDLSCs in terms of evident similarities in immunomodulatory properties as well as their easier accessibility and availability. © 2014 S. Karger AG, Basel
Introduction
Periodontitis is currently the main cause of tooth loss and is also associated with a number of systemic diseases such as diabetes and cardiovascular disease [Kinane and Marshall, 2001]. To date, no appropriate method has been developed to regenerate a functional and predictable periodontal tissue. The discovery of human periodontal ligament stem cells (PDLSCs), which form a cementum/ periodontal ligament (PDL)-like structure after in vivo transplantation [Seo et al., 2004], provides a new alternative for periodontal tissue regeneration [Seo et al., 2004]. Some animal tests have shown encouraging results for Prof. Qintao Wang, DDS, PhD The Fourth Military Medical University 145 West Changle Road Xi’an, Shaanxi 710032 (PR China) E-Mail lichenghua1 @ aliyun.com
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Key Words Mesenchymal stem cells · Immunomodulation · Periodontal ligament stem cells · Cell therapy
Abbreviations used in this paper CFSE Con A COX-2 FBS hhPDLSCs IDO ihPDLSCs iNOS MLR MSCs P PBMNCs PBS PDL PDLSCs PGE2 RPMI TNFAIP6
carboxyfluorescein diacetate succinimidyl ester concanavalin A cyclooxygenase-2 fetal bovine serum healthy human PDLSCs indoleamine 2,3 dioxygenase inflamed human PDLSCs inducible nitric oxide synthases mixed lymphocyte reaction mesenchymal stem cells passage peripheral blood mononuclear cells phosphate-buffered saline periodontal ligament periodontal ligament stem cells prostaglandin E2 Roswell Park Memorial Institute TNF-α-induced protein 6
2012]. In addition, it is well established that inflammatory cytokines have a profound influence on the immunomodulatory properties of MSCs [English et al., 2007; Ryan et al., 2007]. Like other MSCs, hhPDLSCs also possess immunomodulatory properties [Wada et al., 2009; Ding et al., 2010]. Nevertheless, it has yet to be elucidated whether ihPDLSCs, when isolated from an inflammatory environment, display similar immunomodulatory properties as hhPDLSCs. This is undoubtedly important in facilitating any future clinical applications. To address this, ex vivo expanded hhPDLSCs or ihPDLSCs were cocultured with activated peripheral blood mononuclear cells (PBMNCs). The results indicated that ihPDLSCs had similar immunomodulatory properties to hhPDLSCs. Both hhPDLSCs and ihPDLSCs can suppress the proliferation and cytokine secretion of activated PBMNCs via similar mechanisms.
Materials and Methods
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Cells Tissues Organs DOI: 10.1159/000367986
Cell Cultures Isolation of hhPDLSCs Healthy human third molars were extracted from 5 systemically healthy adults (35–45 years of age) at the Department of Periodontology and Oral Medicine, Stomatological Hospital of the Fourth Military Medical University, Xi’an, China. Informed consent and ethical approval was obtained from the school’s ethics committee. PDLSCs were isolated and cultured as we previously described [Wang et al., 2010; Zhang et al., 2012]. PDL tissues were scraped from the middle third of the root surface and then digested with collagenase I (3 mg/ml; Sigma-Aldrich, St. Louis, Mo., USA) for 2 h at 37 ° C to obtain single-cell suspensions. Cells were maintained in α-minimal essential medium (Sigma-Aldrich) with 10% fetal bovine serum (FBS; Thermo Electron, Melbourne, Australia), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Carlsbad, Calif., USA), and incubated at 37 ° C in 5% CO2. The medium was changed every 3 days. Single cell-derived colony cultures were obtained by limiting dilution technique, and different colonies were gathered as passage (P)0 cells. To avoid changes in cell behaviors caused by prolonged culture, only cells from P3 to P5 were used in this study.
Isolation of ihPDLSCs The inflammatory granulation tissues were isolated as previously described [Hung et al., 2011; Park et al., 2011] from 5 systemically healthy patients with moderate-to-severe chronic periodontitis (40–50 years of age) at the Department of Periodontology and Oral Medicine, Stomatological Hospital of the Fourth Military Medical University, Xi’an, China. All patients provided their informed consent. They had received periodontal nonsurgical treatments prior to a periodontal probing depth of >6 mm with bleeding on probing and radiographic evidence of intrabony pockets. All surgical procedures were performed for the purpose of treatment. After removing the unattached necrotic granulation tis-
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periodontal tissue regeneration by applying PDLSCs [Liu et al., 2008; Ding et al., 2010]. The hPDLSCs are mostly isolated from impacted third molars or extracted teeth during orthodontic therapy, but these are not always available. Excitingly, our lab and others have successfully isolated a type of stromal stem cell from periodontal granulation tissue [Hung et al., 2011; Park et al., 2011], which is usually discarded as medical waste. These cells, namely inflamed human (ih)PDLSCs, exhibited similar tissue regeneration functions as healthy human (hh)PDLSCs in an in vivo transplantation model [Park et al., 2011], and also improved new bone formation when transplanted into calvarial-defect mice [Hung et al., 2011]. A unique feature of mesenchymal stem cells (MSCs) is their ability to mediate potent immunosuppressive and immunoregulatory effects on nearly all kinds of immune cells – the so-called immunomodulatory properties [Marigo and Dazzi, 2011]. These properties are important for MSCs to exert their therapeutic effects and are the foundation for most of the clinical trials focusing on MSC application [Golemovic et al., 2012; Yi and Song, 2012]. Even with low immunogenicity, MSCs can still stimulate the immune responses of the recipient under certain circumstances [Chan et al., 2006; Nauta et al., 2006; Sbano et al., 2008]. Thus, MSCs with normal immunomodulatory properties would be necessary for allogeneic application. Although the mechanism is still largely unknown, only bone marrow MSCs with normal immunomodulatory properties showed therapeutic effects on systemic sclerosis and experimental colitis in mice [Akiyama et al.,
Flow Cytometric Analysis To identify the phenotype of hPDLSCs, approximately 5 × 105 hhPDLSCs or ihPDLSCs (5 lines for each type) were incubated with PE- or FITC-conjugated monoclonal antibodies for human CD31, CD29, CD44, CD90, CD105 (eBioscience, San Diego, Calif., USA), Alexa Fluor 647-conjugated STRO-1, FITC-conjugated CD34 and PE-conjugated CD146 (Biolegend, San Diego, Calif., USA) or isotype-matched control IgGs for 1 h on ice. After washing, cells were subjected to flow cytometric analysis using a Beckman Coulter Epics XL (Beckman Coulter, Fullerton, Calif., USA). PBMNC Proliferation Assay Using Mitogenic Stimulation Concanavalin A (Concanavalin A-Stimulated PBMNC Assay) and Mixed Lymphocyte Reaction Assay To investigate the immunomodulatory effects of hhPDLSCs or ihPDLSCs on lymphocyte proliferation, we established a PBMNC proliferation assay following mitogenic concanavalin A (Con A) stimulation (Con A-stimulated PBMNC assay) and allogeneic mixed lymphocyte reaction (MLR) in vitro. PBMNCs from healthy donors were obtained by the gradient centrifugation separation method. In brief, 20 ml of fresh heparinized peripheral blood was diluted with an equal volume of phosphate-buffered saline (PBS). Diluted blood (5 ml) was carefully layered on 5 ml Ficoll (1.077 g/ ml; Dingguo, Beijing, China) prior to centrifugation at 900 g for 20 min. The PBMNC layer was separated and washed with five volumes of PBS three times, and the precipitated cells were resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Eugene, Oreg., USA). For the Con A-stimulated PBMNC assay, different numbers of γ-irradiated (30 Gy) hhPDLSCs or ihPDLSCs were cocultured with Con A-stimulated PBMNCs (10 μg/ml; Sigma-Aldrich), and the proliferation of PBMNCs was assessed after 3 days. To visualize the cell division cycles by flow cytometry (Epics XL flow cytometer; Beckman Coulter), PBMNCs were labelled with 2 mM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) for 15 min at 37 ° C in PBS and washed three times with RPMI-1640 with 10% FBS. The γ-irradiated hhPDLSCs or ihPDLSCs (1 × 105) were cocultured with the CFSE-labelled PBMNCs (1 × 105) from different donors following Con A stimulation (10 μg/ml). The CFSE-unlabeled and CFSE-labeled PBMNCs were applied as negative and positive controls, respectively. After 3 days of culture, the PBMNCs were analyzed by flow cytometry to detect green fluorescence (CFSE). To study the effects of hhPDLSCs or ihPDLSCs on a two-way MLR, hhPDLSCs or ihPDLSCs were added at the beginning of the MLR. PBMNCs (1 × 105) from two individuals were mixed before coculturing with hhPDLSCs or ihPDLSCs (1 × 105) from a third party. The proliferation of PBMNCs was assessed 5 days later. The proliferation of PBMNCs was measured by a method based on the tetrazolium salt WST-8 according to the manufacturer’s instructions (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). Briefly, at
The Immunomodulatory Properties of ihPDLSCs
the end of each time point (3 days for Con A-stimulated PBMNC assay and 5 days for MLR), the suspended PBMNCs were transferred to a new 96-well plate and 20 μl of WST-8 were added to each well. The plates were then incubated for an additional 4 h at 37 ° C. The absorbance, which represents a direct correlation with the cell number, of each plate was measured at 450 nm. Both assays were performed in 200 μl RPMI-1640 with 10% FBS in 96-well round-bottom plates (Corning Inc., Corning, N.Y., USA), using media alone as the blank control. All the 5-cell lines of hhPDLSCs or ihPDLSCs were used in these tests and all experiments were run in triplicate.
Cytokine Detection For the Con A-stimulated PBMNC assay, the cell-free supernatants were harvested and stored at –80 ° C for cytokine assays. The concentration of IFN-γ in the culture supernatants of PBMNCs with or without hPDLSCs (5 lines for each type) was measured by ELISA (Keyingmei, Beijing, China) according to the manufacturer’s instructions.
Transwell Cocultures hhPDLSCs or ihPDLSCs were added to Con A-stimulated PBMNC assay in a separated coculture system using Transwell plates (Nunc, Roskilde, Denmark). PBMNCs (2 × 105) were placed in the insert with 0.4-mm-pore-sized membrane at its bottom. hhPDLSCs or ihPDLSCs (2 × 105, 5 lines for each type), irradiated with 30 Gy, were seeded in the chamber of the 24-well flat-bottom plate. Cells were cultured in 500 μl RPMI-1640 with 10 μg/ml Con A for 3 days. The proliferation of PBMNCs was then assessed. Quantitative RT-PCR Analysis Total cellular RNA was extracted from hhPDLSCs or ihPDLSCs before or after coculture with the activated PBMNCs using Trizol (Invitrogen) according to manufacturer’s instructions. Isolated total RNA was then subjected to reverse transcription using Oligo dT primer and PrimeScript® RTase (Takara, Dalian, China) according to manufacturer’s instructions. Real-time PCR was performed with SYBR® Premix Ex TaqTM II (Takara, Dalian, China) using a C1000TM Thermal Cycler (Bio-Rad, Hercules, Calif., USA). The primers used in this study were as follows: IL-10: forward, 5′-GTGATGCCCCAAGCTGAGA-3′, and reverse, 5′-CACGGCCTTGCTCTTGTTTT-3′; inducible nitric oxide synthases (iNOS): forward, 5′-AAAGACCAGGCTGTCGTTGA-3′, and reverse, 5′-ACGGGACCGGTATTCATTCT-3′; TNF-α-induced protein 6 (TNFAIP6): forward, 5′-TCATGTCTGTGCTGCTGGATG-3′, and reverse, 5′-GGGCCCTGGCTTCACAA-3′; indoleamine 2,3 dioxygenase (IDO): forward, 5′-AGAGTCAAATCCCTCAGTCC-3′, and reverse, 5′-AAATCAGTGCCTCCAGTTCC3′; cyclooxygenase-2 (COX-2): forward, 5′-TGAAACCCACTCCAAACACAG-3′, and reverse, 5′-AGAGAAGGCTTCCCAGCTTTT-3′; β-actin: forward, 5′-CTCCACCCTGGCCTCGCTGT-3′, and reverse, 5′-GCTGTCACCTTCACCGTTCC-3′. The expression levels of the target genes were normalized to that of the housekeeping gene β-actin. Statistical Analysis All values are expressed as mean ± SD. To test the statistically significant differences between paired observations, the Student t test for paired data was used. A p value 0.05). We further investigated the cell division cycles of PBMNCs cocultured with or without hPDLSCs in Con A-stimulated PBMNC assay. Con Astimulated PBMNCs in the coculture groups showed that cell division peaked at the undivided phase (similar to that of PBMNCs without stimulation). However, the cell
The Immunomodulatory Properties of ihPDLSCs
Cells Tissues Organs DOI: 10.1159/000367986
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Fig. 3. The inhibitory effects of hhPDLSCs and ihPDLSCs on PBMNC IFN-γ secretion. Supernatant of Con A-stimulated PBMNCs cocultured with or without hPDLSCs was collected and detected using an ELISA kit for IFN-γ. * p < 0.05 (n = 5).
division peak of Con A-stimulated PBMNCs shifted greatly to the left (fig. 2c). While the results showed that Con A led to extensive division in PBMNCs alone, coculture with PDLSCs inhibited this effect of Con A in PBMNC division. Cytokine Detection To assess whether ihPDLSCs can suppress the secretion of proinflammatory cytokine in Con A-stimulated PBMNCs, we detected the concentration of IFN-γ in the supernatant of PBMNCs cocultured with or without ihPDLSCs. In PBMNCs alone without Con A stimulation, the concentration of IFN-γ was 389 ± 50 pg/ml while, in the PBMNCs with Con A stimulation, the concentration of IFN-γ was increased to 767 ± 35 pg/ml (p < 0.05). With the existence of ihPDLSCs or hhPDLSCs in the culture, the concentration of IFN-γ in PBMNCs significantly declined to 480 ± 42 and 450 ± 60 pg/ml, respectively (p < 0.05, compared with the PBMNCs with Con A group). However, there was no statistical significance between the hhPDLSC and ihPDLSC groups (p > 0.05; fig. 3). Mechanisms Mediating Inhibitory Effects of hhPDLSCs and ihPDLSCs on PBMNC Proliferation To determine whether ihPDLSCs exert their immunosuppressive effects on activated PBMNCs by cell-cell contact, hhPDLSCs or ihPDLSCs were cocultured with Con A-stimulated PBMNCs using Transwell culture. In parallel studies, conditioned media of cultured hhPDLSCs or 6
Cells Tissues Organs DOI: 10.1159/000367986
ihPDLSCs was applied to Con A-stimulated PBMNCs to examine whether the immunomodulatory properties of ihPDLSCs were mediated via the secretion of inhibitory factors. The results showed that PBMNC proliferation was still significantly inhibited (p < 0.05) in the Transwell experiments, but to a somewhat lesser extent (fig. 4a). The expressions of iNOS, IDO, COX-2, TNFAIP6 and IL-10 by hhPDLSCs and ihPDLSCs before or after coculturing with Con A-stimulated PBMNCs for 3 days in Transwell culture were investigated. There were no detectable levels of iNOS, IDO, COX-2, TNFAIP6 and IL-10 expression in ihPDLSCs or hhPDLSCs prior to coculturing with Con A-activated PBMNCs (fig. 4b–f). However, expressions of these mediators were greatly upregulated following coculture (fig. 4b–f; p < 0.05). Moreover, hhPDLSCs illustrated statistically higher expressions of both iNOS and IDO than ihPDLSCs (p < 0.05). In addition, hhPDLSCs showed a distinctly lower level of TNFAIP6 expression than ihPDLSCs (p < 0.05). However, there was no statistical significance between hhPDLSCs and ihPDLSCs for iNOS and IL-10 expressions following coculture (p > 0.05; fig. 4b–f).
Discussion
While PDLSCs offer promising potential for periodontal regeneration with their unique abilities in forming cementum/PDL-like structures [Seo et al., 2004] and immunomodulatory properties [Ding et al., 2010], there are limited sources readily available. Consistent with others [Hung et al., 2011; Park et al., 2011], this study demonstrated the feasibility of isolating a type of stromal cell from granulation tissue, namely ihPDLSCs, which possess a similar phenotype to hhPDLSCs while also having the capacity to differentiate into osteogenetic or adipogenetic cells under special differentiation conditions (data not shown). It is also reported that these cells can form cementum/PDL-like structures after in vivo transplantation [Park et al., 2011]. Although ihPDLSCs exhibited decreased mineralized potential compared to hhPDLSCs [Park et al., 2011], we still consider them as potential alternative cells to hhPDLSCs owing to their easier availability. The differentiation and replacement into damaged cells is only a rare event in injuries [Quarto et al., 2001; Togel et al., 2005]. This strongly suggests that MSCs do not mainly contribute to tissue regeneration by differentiating into the appropriate cell type, but rather by promoting the regenerative potentials of residual host parenchymal stem cells. The immunomodulaLi /Wang /Tan /Wang /Wang
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Fig. 4. Mechanisms mediating the inhibitory effects of hPDLSCs. a PBMNCs (2 × 105) were placed in inserts with 0.4-mmpore-sized membrane at its bottom, and hhPDLSCs or ihPDLSCs (2 × 105) irradiated with 30 Gy were seeded in the chamber of a 24-well flat-bottom plate. Cells were cultured in 500 μl of RPMI-1640 with 10 μg/ml Con A for 3 days. The proliferation of PBMNCs was then assessed. * p < 0.05 (n = 5). The gene expression of iNOS (b), IDO (c), COX-2 (d), TNFAIP6 (e) and IL-10 (f) by hhPDLSCs and ihPDLSCs before and after coculture was investigated by quantitative RT-PCR analysis. * p < 0.05 (n = 5).
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tory property of MSCs is considered to play a very important role during this therapeutic process [Shi et al., 2010]. Periodontitis is associated with abnormal or imbalanced immune reaction. Anti-inflammatory mediators can counteract and attenuate disease progression [Gemmell et al., 2002; Bascones et al., 2005; Garlet, 2010]. Similar to bone marrow MSCs, hhPDLSCs were also reported to have immunomodulatory properties [Wada et
al., 2009; Ding et al., 2010]. Nonetheless, this is still unclear for ihPDLSCs. CD146, a 113- to 119-kDa transmembrane glycoprotein, was initially identified as a marker of melanoma progression and invasion, and is involved in cell-to-cell junctions [Ouhtit et al., 2009]. It is widely used as an important surface marker in PDLSC research [Seo et al., 2004]. CD146 expression differs significantly between
The Immunomodulatory Properties of ihPDLSCs
Cells Tissues Organs DOI: 10.1159/000367986
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the effects of the former on suppressing the proliferation of PBMNCs were weakened, though not eliminated. NO is involved in the MSC-mediated immune suppression and can only function locally due to its high instability [Sato et al., 2007; Ren et al., 2008]. NO production is catalyzed by NOS, of which there are three types in human: iNOS, inducible primarily in macrophages, nNOS, in neuronsand, and eNOS, in endothelial cells. iNOS expression is inducible and plays a major role in immune regulation. We found that after hhPDLSCs or ihPDLSCs were cocultured with PBMNCs for 3 days, the mRNA expression of iNOS was dramatically elevated, which indicates that NO may also take part in the immunosuppression of hPDLSCs. This can partly explain why the immunosuppression effects of hPDLSCs in Transwell culture were decreased; the separation by Transwell prohibits NO from fully exerting its immunosuppressive effects. Soluble mediators secreted by MSCs are necessary for their immunomodulatory properties and proinflammatory cytokine stimulation is a prerequisite for the secretion of these mediators. Conditioned media derived from either hhPDLSCs or ihPDLSCs showed no suppression on PBMNC proliferation at all concentrations examined (10, 20 and 50% of total media; data not shown), suggesting hPDLSCs require stimulating factors from activated PBMNC to exert their suppressive effects. In agreement with our results, IDO and PGE2, two important mediators of MSC immunosuppression [Meisel et al., 2004; Aggarwal and Pittenger, 2005], have also been reported to be associated with the immunomodulation of PDLSCs [Wada et al., 2009; Ding et al., 2010]. Following coculture, the mRNA expressions of IDO and COX-2 (the enzyme for PGE2 production) were highly increased, while ihPDLSCs showed less IDO expression than hhPDLSCs. TNFAIP6 is a 30-kDa glycoprotein. It can produce antiinflammatory effects through different mechanisms. TNFAIP6 expression in MSCs is highly upregulated following intravenous administration. After myocardial infarction, intravenous administration of MSCs, but not MSCs transduced with TNFAIP6 siRNA, exhibited therapeutic effects in mice [Lee et al., 2009]. Interestingly, we have found that TNFAIP6 may also play a role in PDLSCs immunosuppression, as shown by dramatically elevated gene expression after coculture. The gene expression of IL-10 in PDLSCs was also greatly upregulated after coculture with activated PBMNCs. IL-10 is a well-established immune suppressor and is also one of the reported soluble cytokines that take part in the immunomodulatory properties of MSCs [Jui et al., 2012]. However, we also discovered its possible involvement with PDLSCs. Li /Wang /Tan /Wang /Wang
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hhPDLSCs and ihPDLSCs in our results (58.4% for hhPDLSCs and 9.2% for ihPDLSCs). Consistently, Xu et al. [2009] also reported that the positive rate of CD146 varies in periodontal ligament, fluctuating from 9.33 to 60.42%. Since this study is limited by the small number of samples analyzed (five samples for each cell type), further investigation is needed to validate whether CD146 expression is lower in ihPDLSCs in comparison with hhPDLSCs. The inflammation environment can affect the cytokine profile of MSCs and their immunomodulatory properties. The proinflammatory cytokine IFN-γ upregulates HGF and TGF-β1 expressed by MSCs and thus promotes the immunosuppressive capacity of adult human MSCs [Ryan et al., 2007]. Meanwhile, COX-2 and prostaglandin E2 (PGE2) expression by MSCs can also be upregulated by both IFN-γ and TNF-α in murine MSCs [English et al., 2007]. It appears, therefore, that a proinflammatory environment will enhance the immunosuppressive effects elicited by MSCs. In order to better understand the immunomodulatory property changes in ihPDLSCs, we performed a PBMNC proliferation assay as previously described [Bartholomew et al., 2002; Jarvinen et al., 2008; Wada et al., 2009; Sun et al., 2011]. Our results indicated that the immunomodulatory effects of ihPDLSCs were comparable to those observed in hhPDLSCs. They can not only suppress the proliferation of PBMNCs in a dosedependent manner, but also suppress the secretion of IFN-γ, which is the main proinflammatory cytokine secreted by activated PBMNCs. CFSE staining suggested that the suppression of PBMNC proliferation is mainly due to the inhibition of the cell cycle. It is unlikely that the reduction in PBMNC proliferation was caused by the exhaustion of culture medium. Firstly, the color of the RPMI-1640 medium in coculture groups was similar to that of control groups without PDLSCs. Secondly, we have also tried to replace half the amount of the culture medium every day or using culture medium containing higher levels of FBS (20% instead of 10% v/v) with similar results (data not shown). The mechanism underlying the immunomodulatory properties of MSCs is complex and still not fully understood. Some reports have shown that soluble factors are the major mediators of suppression [Di Nicola et al., 2002; Meisel et al., 2004; Aggarwal and Pittenger, 2005], whereas others have demonstrated that T cell-MSC contact is required for this suppression [Djouad et al., 2003; Ren et al., 2008; Akiyama et al., 2012]. We have found that both cell-cell contact and soluble factors contribute to the immunomodulatory properties of hPDLSCs. When using Transwell culture to separate the hPDLSCs and PBMNCs,
The mechanism of MSC immunosuppression is complex and there are many mediators involved in this process. Immunosuppression of PDLSCs is more likely a combination effect of all the molecules. Though there are some significant differences between hhPDLSCs and ihPDLSCs in terms of the gene expressions of immune mediators, such as iNOS, IDO and TNFAIP6, their potency in suppressing PBMNC proliferation and secreting IFN-γ shows no apparent differences. However, because of a lack of blocking experiments, our research indicates a possible involvement of the mediators when tested during the immunosuppression of hhPDLSCs or ihPDLSCs. It therefore merits further investigation and clarification. In conclusion, ihPDLSCs have similar immunomodulatory properties as hhPDLSCs. Although different in
dose, the gene expression of some immune mediators in ihPDLSCs displayed a similar pattern when compared with hhPDLSCs. Along with their easier accessibility, our findings suggest that ihPDLSCs offer considerable potential as a valuable alternative to hhPDLSCs.
Acknowledgements This work was supported by grants from the Nature Science Foundation of China (81170963 and 81271137).
Disclosure Statement There are no conflicts of interest.
References
The Immunomodulatory Properties of ihPDLSCs
Ding, G., Y. Liu, W. Wang, F. Wei, D. Liu, Z. Fan, Y. An, C. Zhang, S. Wang (2010) Allogeneic periodontal ligament stem cell therapy for periodontitis in swine. Stem Cells 28: 1829– 1838. Djouad, F., P. Plence, C. Bony, P. Tropel, F. Apparailly, J. Sany, D. Noel, C. Jorgensen (2003) Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 102: 3837–3844. English, K., F.P. Barry, C.P. Field-Corbett, B.P. Mahon (2007) IFN-γ and TNF-α differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 110: 91–100. Garlet, G.P. (2010) Destructive and protective roles of cytokines in periodontitis: a re-appraisal from host defense and tissue destruction viewpoints. J Dent Res 89: 1349–1363. Gemmell, E., K. Yamazaki, G.J. Seymour (2002) Destructive periodontitis lesions are determined by the nature of the lymphocytic response. Crit Rev Oral Biol Med 13: 17–34. Golemovic, M., M. Skific, B.G. Cepulic (2012) Mesenchymal stem cells: immunomodulatory properties and clinical application (in Croatian). Lijec Vjesn 134: 42–49. Hung, T.Y., H.C. Lin, Y.J. Chan, K. Yuan (2011) Isolating stromal stem cells from periodontal granulation tissues. Clin Oral Investig 16: 1171–1180. Jarvinen, L., L. Badri, S. Wettlaufer, T. Ohtsuka, T.J. Standiford, G.B. Toews, D.J. Pinsky, M. Peters-Golden, V.N. Lama (2008) Lung resident mesenchymal stem cells isolated from human lung allografts inhibit T cell proliferation via a soluble mediator. J Immunol 181: 4389–4396.
Cells Tissues Organs DOI: 10.1159/000367986
Jui, H.Y., C.H. Lin, W.T. Hsu, Y.R. Liu, R.B. Hsu, B.L. Chiang, W.Y. Tseng, M.F. Chen, K.K. Wu, C.M. Lee (2012) Autologous mesenchymal stem cells prevent transplant arteriosclerosis by enhancing local expression of interleukin-10, interferon-γ, and indoleamine 2,3-dioxygenase. Cell Transplant 21: 971– 984. Kinane, D.F., G.J. Marshall (2001) Periodontal manifestations of systemic disease. Aust Dent J 46: 2–12. Lee, R.H., A.A. Pulin, M.J. Seo, D.J. Kota, J. Ylostalo, B.L. Larson, L. Semprun-Prieto, P. Delafontaine, D.J. Prockop (2009) Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5: 54–63. Liu, Y., Y. Zheng, G. Ding, D. Fang, C. Zhang, P.M. Bartold, S. Gronthos, S. Shi, S. Wang (2008) Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells 26: 1065–1073. Marigo, I., F. Dazzi (2011) The immunomodulatory properties of mesenchymal stem cells. Semin Immunopathol 33: 593–602. Meisel, R., A. Zibert, M. Laryea, U. Gobel, W. Daubener, D. Dilloo (2004) Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenasemediated tryptophan degradation. Blood 103: 4619–4621. Nauta, A.J., G. Westerhuis, A.B. Kruisselbrink, E.G. Lurvink, R. Willemze, W.E. Fibbe (2006) Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 108: 2114–2120.
9
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Aggarwal, S., M.F. Pittenger (2005) Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105: 1815–1822. Akiyama, K., C. Chen, D. Wang, X. Xu, C. Qu, T. Yamaza, T. Cai, W. Chen, L. Sun, S. Shi (2012) Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell 10: 544– 555. Bartholomew, A., C. Sturgeon, M. Siatskas, K. Ferrer, K. McIntosh, S. Patil, W. Hardy, S. Devine, D. Ucker, R. Deans, A. Moseley, R. Hoffman (2002) Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30: 42–48. Bascones, A., S. Noronha, M. Gomez, P. Mota, M.A. Gonzalez Moles, M. Villarroel Dorrego (2005) Tissue destruction in periodontitis: bacteria or cytokines fault? Quintessence Int 36: 299–306. Chan, J.L., K.C. Tang, A.P. Patel, L.M. Bonilla, N. Pierobon, N.M. Ponzio, P. Rameshwar (2006) Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-γ. Blood 107: 4817–4824. Chen, F.M., H.H. Sun, H. Lu, Q. Yu (2012) Stem cell-delivery therapeutics for periodontal tissue regeneration. Biomaterials 33: 6320– 6344. Di Nicola, M., C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, S. Grisanti, A.M. Gianni (2002) Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99: 3838–3843.
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Sato, K., K. Ozaki, I. Oh, A. Meguro, K. Hatanaka, T. Nagai, K. Muroi, K. Ozawa (2007) Nitric oxide plays a critical role in suppression of Tcell proliferation by mesenchymal stem cells. Blood 109: 228–234. Sbano, P., A. Cuccia, B. Mazzanti, S. Urbani, B. Giusti, I. Lapini, L. Rossi, R. Abbate, G. Marseglia, G. Nannetti, F. Torricelli, C. Miracco, A. Bosi, M. Fimiani, R. Saccardi (2008) Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model. Arch Dermatol Res 300: 115–124. Seo, B.M., M. Miura, S. Gronthos, P.M. Bartold, S. Batouli, J. Brahim, M. Young, P.G. Robey, C.Y. Wang, S. Shi (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364: 149–155. Shi, Y., G. Hu, J. Su, W. Li, Q. Chen, P. Shou, C. Xu, X. Chen, Y. Huang, Z. Zhu, X. Huang, X. Han, N. Xie, G. Ren (2010) Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Res 20: 510– 518. Sun, Z., Q. Han, Y. Zhu, Z. Li, B. Chen, L. Liao, C. Bian, J. Li, C. Shao, R.C. Zhao (2011) NANOG has a role in mesenchymal stem cells’ immunomodulatory effect. Stem Cells Dev 20: 1521–1528.
Togel, F., Z. Hu, K. Weiss, J. Isaac, C. Lange, C. Westenfelder (2005) Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol 289: F31–F42. Wada, N., D. Menicanin, S. Shi, P.M. Bartold, S. Gronthos (2009) Immunomodulatory properties of human periodontal ligament stem cells. J Cell Physiol 219: 667–676. Wang, L., H. Shen, W. Zheng, L. Tang, Z. Yang, Y. Gao, Q. Yang, C. Wang, Y. Duan, Y. Jin (2010) Characterization of stem cells from alveolar periodontal ligament. Tissue Eng Part A 17: 1015–1026. Xu, J., W. Wang, Y. Kapila, J. Lotz, S. Kapila (2009) Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells. Stem Cells Dev 18: 487–496. Yi, T., S.U. Song (2012) Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications. Arch Pharm Res 35: 213–221. Zhang, J., Y. An, L.N. Gao, Y.J. Zhang, Y. Jin, F.M. Chen (2012) The effect of aging on the pluripotential capacity and regenerative potential of human periodontal ligament stem cells. Biomaterials 33: 6974–6986.
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Ouhtit, A., R.L. Gaur, Z.Y. Abd Elmageed, A. Fernando, R. Thouta, A.K. Trappey, M.E. Abdraboh, H.I. El-Sayyad, P. Rao, M.G. Raj (2009) Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146. Biochim Biophys Acta 1795: 130– 136. Park, J.C., J.M. Kim, I.H. Jung, J.C. Kim, S.H. Choi, K.S. Cho, C.S. Kim (2011) Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations. J Clin Periodontol 38: 721–731. Quarto, R., M. Mastrogiacomo, R. Cancedda, S.M. Kutepov, V. Mukhachev, A. Lavroukov, E. Kon, M. Marcacci (2001) Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344: 385– 386. Ren, G., L. Zhang, X. Zhao, G. Xu, Y. Zhang, A.I. Roberts, R.C. Zhao, Y. Shi (2008) Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2: 141–150. Ryan, J.M., F. Barry, J.M. Murphy, B.P. Mahon (2007) Interferon-γ does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol 149: 353–363.
Cells Tissues Organs DOI: 10.1159/000367986
The Immunomodulatory Properties of Periodontal Ligament Stem Cells Isolated from Inflamed Periodontal Granulation Chenghua Li a, b Xinwen Wang a Jun Tan c Tao Wang d Qintao Wang a
a
State Key Laboratory of Military Stomatology, Department of Periodontology, School of Stomatology, The Fourth Military Medical University, Xi’an, b Department of Stomatology, Beidaihe Sanatorium of Beijing Military Command, Qinhuangdao, c Department of Stomatology, General Hospital of Guangzhou Military Command of PLA, Guangzhou, and d Kong Jun Qing Dao Hang Kong Yi Xue Jian Ding Zhong Xin, Qingdao, PR China
Abstract Periodontitis is currently the main cause of tooth loss and as yet there is no appropriate method for establishing a functional and predictable periodontal regeneration. Tissue engineering involving seed cells provides a new prospect for periodontal regeneration. While periodontal ligament stem cells (PDLSCs) are a good choice for seed cells, it is not always possible to obtain the patients’ own PDLSCs. We and others have found a type of stromal cells from inflamed periodontal granulation. These cells displayed similar differentiation properties to PDLSCs. Inflammation has a profound influence on the immunomodulatory properties of mesenchymal stem cells, which may affect therapeutic outcome. In this study, we assessed the immunomodulatory characteristics of these inflamed human (ih)PDLSCs. Along with the similarity in cell surface marker expressions, they also displayed immunomodulatory properties comparable to those in healthy human (hh)PDLSCs. Both hhPDLSCs and ihPDLSCs can suppress the proliferation and secretion of IFN-γ in peripheral blood mononuclear cells by indirect soluble mediators and
© 2014 S. Karger AG, Basel 1422–6405/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cto
direct cell-cell contact. Albeit with some quantitative variances, the gene expressions of inducible nitric oxide synthases, indoleamine 2,3 dioxygenase, cyclooxygenase-2, TNF-α-induced protein 6 and IL-10 in ihPDLSCs displayed similar patterns as those in hhPDLSCs. Taken together, our results suggest that ihPDLSCs can provide a promising alternative to hhPDLSCs in terms of evident similarities in immunomodulatory properties as well as their easier accessibility and availability. © 2014 S. Karger AG, Basel
Introduction
Periodontitis is currently the main cause of tooth loss and is also associated with a number of systemic diseases such as diabetes and cardiovascular disease [Kinane and Marshall, 2001]. To date, no appropriate method has been developed to regenerate a functional and predictable periodontal tissue. The discovery of human periodontal ligament stem cells (PDLSCs), which form a cementum/ periodontal ligament (PDL)-like structure after in vivo transplantation [Seo et al., 2004], provides a new alternative for periodontal tissue regeneration [Seo et al., 2004]. Some animal tests have shown encouraging results for Prof. Qintao Wang, DDS, PhD The Fourth Military Medical University 145 West Changle Road Xi’an, Shaanxi 710032 (PR China) E-Mail lichenghua1 @ aliyun.com
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Key Words Mesenchymal stem cells · Immunomodulation · Periodontal ligament stem cells · Cell therapy
Abbreviations used in this paper CFSE Con A COX-2 FBS hhPDLSCs IDO ihPDLSCs iNOS MLR MSCs P PBMNCs PBS PDL PDLSCs PGE2 RPMI TNFAIP6
carboxyfluorescein diacetate succinimidyl ester concanavalin A cyclooxygenase-2 fetal bovine serum healthy human PDLSCs indoleamine 2,3 dioxygenase inflamed human PDLSCs inducible nitric oxide synthases mixed lymphocyte reaction mesenchymal stem cells passage peripheral blood mononuclear cells phosphate-buffered saline periodontal ligament periodontal ligament stem cells prostaglandin E2 Roswell Park Memorial Institute TNF-α-induced protein 6
2012]. In addition, it is well established that inflammatory cytokines have a profound influence on the immunomodulatory properties of MSCs [English et al., 2007; Ryan et al., 2007]. Like other MSCs, hhPDLSCs also possess immunomodulatory properties [Wada et al., 2009; Ding et al., 2010]. Nevertheless, it has yet to be elucidated whether ihPDLSCs, when isolated from an inflammatory environment, display similar immunomodulatory properties as hhPDLSCs. This is undoubtedly important in facilitating any future clinical applications. To address this, ex vivo expanded hhPDLSCs or ihPDLSCs were cocultured with activated peripheral blood mononuclear cells (PBMNCs). The results indicated that ihPDLSCs had similar immunomodulatory properties to hhPDLSCs. Both hhPDLSCs and ihPDLSCs can suppress the proliferation and cytokine secretion of activated PBMNCs via similar mechanisms.
Materials and Methods
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Cells Tissues Organs DOI: 10.1159/000367986
Cell Cultures Isolation of hhPDLSCs Healthy human third molars were extracted from 5 systemically healthy adults (35–45 years of age) at the Department of Periodontology and Oral Medicine, Stomatological Hospital of the Fourth Military Medical University, Xi’an, China. Informed consent and ethical approval was obtained from the school’s ethics committee. PDLSCs were isolated and cultured as we previously described [Wang et al., 2010; Zhang et al., 2012]. PDL tissues were scraped from the middle third of the root surface and then digested with collagenase I (3 mg/ml; Sigma-Aldrich, St. Louis, Mo., USA) for 2 h at 37 ° C to obtain single-cell suspensions. Cells were maintained in α-minimal essential medium (Sigma-Aldrich) with 10% fetal bovine serum (FBS; Thermo Electron, Melbourne, Australia), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Carlsbad, Calif., USA), and incubated at 37 ° C in 5% CO2. The medium was changed every 3 days. Single cell-derived colony cultures were obtained by limiting dilution technique, and different colonies were gathered as passage (P)0 cells. To avoid changes in cell behaviors caused by prolonged culture, only cells from P3 to P5 were used in this study.
Isolation of ihPDLSCs The inflammatory granulation tissues were isolated as previously described [Hung et al., 2011; Park et al., 2011] from 5 systemically healthy patients with moderate-to-severe chronic periodontitis (40–50 years of age) at the Department of Periodontology and Oral Medicine, Stomatological Hospital of the Fourth Military Medical University, Xi’an, China. All patients provided their informed consent. They had received periodontal nonsurgical treatments prior to a periodontal probing depth of >6 mm with bleeding on probing and radiographic evidence of intrabony pockets. All surgical procedures were performed for the purpose of treatment. After removing the unattached necrotic granulation tis-
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periodontal tissue regeneration by applying PDLSCs [Liu et al., 2008; Ding et al., 2010]. The hPDLSCs are mostly isolated from impacted third molars or extracted teeth during orthodontic therapy, but these are not always available. Excitingly, our lab and others have successfully isolated a type of stromal stem cell from periodontal granulation tissue [Hung et al., 2011; Park et al., 2011], which is usually discarded as medical waste. These cells, namely inflamed human (ih)PDLSCs, exhibited similar tissue regeneration functions as healthy human (hh)PDLSCs in an in vivo transplantation model [Park et al., 2011], and also improved new bone formation when transplanted into calvarial-defect mice [Hung et al., 2011]. A unique feature of mesenchymal stem cells (MSCs) is their ability to mediate potent immunosuppressive and immunoregulatory effects on nearly all kinds of immune cells – the so-called immunomodulatory properties [Marigo and Dazzi, 2011]. These properties are important for MSCs to exert their therapeutic effects and are the foundation for most of the clinical trials focusing on MSC application [Golemovic et al., 2012; Yi and Song, 2012]. Even with low immunogenicity, MSCs can still stimulate the immune responses of the recipient under certain circumstances [Chan et al., 2006; Nauta et al., 2006; Sbano et al., 2008]. Thus, MSCs with normal immunomodulatory properties would be necessary for allogeneic application. Although the mechanism is still largely unknown, only bone marrow MSCs with normal immunomodulatory properties showed therapeutic effects on systemic sclerosis and experimental colitis in mice [Akiyama et al.,
Flow Cytometric Analysis To identify the phenotype of hPDLSCs, approximately 5 × 105 hhPDLSCs or ihPDLSCs (5 lines for each type) were incubated with PE- or FITC-conjugated monoclonal antibodies for human CD31, CD29, CD44, CD90, CD105 (eBioscience, San Diego, Calif., USA), Alexa Fluor 647-conjugated STRO-1, FITC-conjugated CD34 and PE-conjugated CD146 (Biolegend, San Diego, Calif., USA) or isotype-matched control IgGs for 1 h on ice. After washing, cells were subjected to flow cytometric analysis using a Beckman Coulter Epics XL (Beckman Coulter, Fullerton, Calif., USA). PBMNC Proliferation Assay Using Mitogenic Stimulation Concanavalin A (Concanavalin A-Stimulated PBMNC Assay) and Mixed Lymphocyte Reaction Assay To investigate the immunomodulatory effects of hhPDLSCs or ihPDLSCs on lymphocyte proliferation, we established a PBMNC proliferation assay following mitogenic concanavalin A (Con A) stimulation (Con A-stimulated PBMNC assay) and allogeneic mixed lymphocyte reaction (MLR) in vitro. PBMNCs from healthy donors were obtained by the gradient centrifugation separation method. In brief, 20 ml of fresh heparinized peripheral blood was diluted with an equal volume of phosphate-buffered saline (PBS). Diluted blood (5 ml) was carefully layered on 5 ml Ficoll (1.077 g/ ml; Dingguo, Beijing, China) prior to centrifugation at 900 g for 20 min. The PBMNC layer was separated and washed with five volumes of PBS three times, and the precipitated cells were resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Eugene, Oreg., USA). For the Con A-stimulated PBMNC assay, different numbers of γ-irradiated (30 Gy) hhPDLSCs or ihPDLSCs were cocultured with Con A-stimulated PBMNCs (10 μg/ml; Sigma-Aldrich), and the proliferation of PBMNCs was assessed after 3 days. To visualize the cell division cycles by flow cytometry (Epics XL flow cytometer; Beckman Coulter), PBMNCs were labelled with 2 mM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) for 15 min at 37 ° C in PBS and washed three times with RPMI-1640 with 10% FBS. The γ-irradiated hhPDLSCs or ihPDLSCs (1 × 105) were cocultured with the CFSE-labelled PBMNCs (1 × 105) from different donors following Con A stimulation (10 μg/ml). The CFSE-unlabeled and CFSE-labeled PBMNCs were applied as negative and positive controls, respectively. After 3 days of culture, the PBMNCs were analyzed by flow cytometry to detect green fluorescence (CFSE). To study the effects of hhPDLSCs or ihPDLSCs on a two-way MLR, hhPDLSCs or ihPDLSCs were added at the beginning of the MLR. PBMNCs (1 × 105) from two individuals were mixed before coculturing with hhPDLSCs or ihPDLSCs (1 × 105) from a third party. The proliferation of PBMNCs was assessed 5 days later. The proliferation of PBMNCs was measured by a method based on the tetrazolium salt WST-8 according to the manufacturer’s instructions (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). Briefly, at
The Immunomodulatory Properties of ihPDLSCs
the end of each time point (3 days for Con A-stimulated PBMNC assay and 5 days for MLR), the suspended PBMNCs were transferred to a new 96-well plate and 20 μl of WST-8 were added to each well. The plates were then incubated for an additional 4 h at 37 ° C. The absorbance, which represents a direct correlation with the cell number, of each plate was measured at 450 nm. Both assays were performed in 200 μl RPMI-1640 with 10% FBS in 96-well round-bottom plates (Corning Inc., Corning, N.Y., USA), using media alone as the blank control. All the 5-cell lines of hhPDLSCs or ihPDLSCs were used in these tests and all experiments were run in triplicate.
Cytokine Detection For the Con A-stimulated PBMNC assay, the cell-free supernatants were harvested and stored at –80 ° C for cytokine assays. The concentration of IFN-γ in the culture supernatants of PBMNCs with or without hPDLSCs (5 lines for each type) was measured by ELISA (Keyingmei, Beijing, China) according to the manufacturer’s instructions.
Transwell Cocultures hhPDLSCs or ihPDLSCs were added to Con A-stimulated PBMNC assay in a separated coculture system using Transwell plates (Nunc, Roskilde, Denmark). PBMNCs (2 × 105) were placed in the insert with 0.4-mm-pore-sized membrane at its bottom. hhPDLSCs or ihPDLSCs (2 × 105, 5 lines for each type), irradiated with 30 Gy, were seeded in the chamber of the 24-well flat-bottom plate. Cells were cultured in 500 μl RPMI-1640 with 10 μg/ml Con A for 3 days. The proliferation of PBMNCs was then assessed. Quantitative RT-PCR Analysis Total cellular RNA was extracted from hhPDLSCs or ihPDLSCs before or after coculture with the activated PBMNCs using Trizol (Invitrogen) according to manufacturer’s instructions. Isolated total RNA was then subjected to reverse transcription using Oligo dT primer and PrimeScript® RTase (Takara, Dalian, China) according to manufacturer’s instructions. Real-time PCR was performed with SYBR® Premix Ex TaqTM II (Takara, Dalian, China) using a C1000TM Thermal Cycler (Bio-Rad, Hercules, Calif., USA). The primers used in this study were as follows: IL-10: forward, 5′-GTGATGCCCCAAGCTGAGA-3′, and reverse, 5′-CACGGCCTTGCTCTTGTTTT-3′; inducible nitric oxide synthases (iNOS): forward, 5′-AAAGACCAGGCTGTCGTTGA-3′, and reverse, 5′-ACGGGACCGGTATTCATTCT-3′; TNF-α-induced protein 6 (TNFAIP6): forward, 5′-TCATGTCTGTGCTGCTGGATG-3′, and reverse, 5′-GGGCCCTGGCTTCACAA-3′; indoleamine 2,3 dioxygenase (IDO): forward, 5′-AGAGTCAAATCCCTCAGTCC-3′, and reverse, 5′-AAATCAGTGCCTCCAGTTCC3′; cyclooxygenase-2 (COX-2): forward, 5′-TGAAACCCACTCCAAACACAG-3′, and reverse, 5′-AGAGAAGGCTTCCCAGCTTTT-3′; β-actin: forward, 5′-CTCCACCCTGGCCTCGCTGT-3′, and reverse, 5′-GCTGTCACCTTCACCGTTCC-3′. The expression levels of the target genes were normalized to that of the housekeeping gene β-actin. Statistical Analysis All values are expressed as mean ± SD. To test the statistically significant differences between paired observations, the Student t test for paired data was used. A p value 0.05). We further investigated the cell division cycles of PBMNCs cocultured with or without hPDLSCs in Con A-stimulated PBMNC assay. Con Astimulated PBMNCs in the coculture groups showed that cell division peaked at the undivided phase (similar to that of PBMNCs without stimulation). However, the cell
The Immunomodulatory Properties of ihPDLSCs
Cells Tissues Organs DOI: 10.1159/000367986
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Fig. 3. The inhibitory effects of hhPDLSCs and ihPDLSCs on PBMNC IFN-γ secretion. Supernatant of Con A-stimulated PBMNCs cocultured with or without hPDLSCs was collected and detected using an ELISA kit for IFN-γ. * p < 0.05 (n = 5).
division peak of Con A-stimulated PBMNCs shifted greatly to the left (fig. 2c). While the results showed that Con A led to extensive division in PBMNCs alone, coculture with PDLSCs inhibited this effect of Con A in PBMNC division. Cytokine Detection To assess whether ihPDLSCs can suppress the secretion of proinflammatory cytokine in Con A-stimulated PBMNCs, we detected the concentration of IFN-γ in the supernatant of PBMNCs cocultured with or without ihPDLSCs. In PBMNCs alone without Con A stimulation, the concentration of IFN-γ was 389 ± 50 pg/ml while, in the PBMNCs with Con A stimulation, the concentration of IFN-γ was increased to 767 ± 35 pg/ml (p < 0.05). With the existence of ihPDLSCs or hhPDLSCs in the culture, the concentration of IFN-γ in PBMNCs significantly declined to 480 ± 42 and 450 ± 60 pg/ml, respectively (p < 0.05, compared with the PBMNCs with Con A group). However, there was no statistical significance between the hhPDLSC and ihPDLSC groups (p > 0.05; fig. 3). Mechanisms Mediating Inhibitory Effects of hhPDLSCs and ihPDLSCs on PBMNC Proliferation To determine whether ihPDLSCs exert their immunosuppressive effects on activated PBMNCs by cell-cell contact, hhPDLSCs or ihPDLSCs were cocultured with Con A-stimulated PBMNCs using Transwell culture. In parallel studies, conditioned media of cultured hhPDLSCs or 6
Cells Tissues Organs DOI: 10.1159/000367986
ihPDLSCs was applied to Con A-stimulated PBMNCs to examine whether the immunomodulatory properties of ihPDLSCs were mediated via the secretion of inhibitory factors. The results showed that PBMNC proliferation was still significantly inhibited (p < 0.05) in the Transwell experiments, but to a somewhat lesser extent (fig. 4a). The expressions of iNOS, IDO, COX-2, TNFAIP6 and IL-10 by hhPDLSCs and ihPDLSCs before or after coculturing with Con A-stimulated PBMNCs for 3 days in Transwell culture were investigated. There were no detectable levels of iNOS, IDO, COX-2, TNFAIP6 and IL-10 expression in ihPDLSCs or hhPDLSCs prior to coculturing with Con A-activated PBMNCs (fig. 4b–f). However, expressions of these mediators were greatly upregulated following coculture (fig. 4b–f; p < 0.05). Moreover, hhPDLSCs illustrated statistically higher expressions of both iNOS and IDO than ihPDLSCs (p < 0.05). In addition, hhPDLSCs showed a distinctly lower level of TNFAIP6 expression than ihPDLSCs (p < 0.05). However, there was no statistical significance between hhPDLSCs and ihPDLSCs for iNOS and IL-10 expressions following coculture (p > 0.05; fig. 4b–f).
Discussion
While PDLSCs offer promising potential for periodontal regeneration with their unique abilities in forming cementum/PDL-like structures [Seo et al., 2004] and immunomodulatory properties [Ding et al., 2010], there are limited sources readily available. Consistent with others [Hung et al., 2011; Park et al., 2011], this study demonstrated the feasibility of isolating a type of stromal cell from granulation tissue, namely ihPDLSCs, which possess a similar phenotype to hhPDLSCs while also having the capacity to differentiate into osteogenetic or adipogenetic cells under special differentiation conditions (data not shown). It is also reported that these cells can form cementum/PDL-like structures after in vivo transplantation [Park et al., 2011]. Although ihPDLSCs exhibited decreased mineralized potential compared to hhPDLSCs [Park et al., 2011], we still consider them as potential alternative cells to hhPDLSCs owing to their easier availability. The differentiation and replacement into damaged cells is only a rare event in injuries [Quarto et al., 2001; Togel et al., 2005]. This strongly suggests that MSCs do not mainly contribute to tissue regeneration by differentiating into the appropriate cell type, but rather by promoting the regenerative potentials of residual host parenchymal stem cells. The immunomodulaLi /Wang /Tan /Wang /Wang
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Fig. 4. Mechanisms mediating the inhibitory effects of hPDLSCs. a PBMNCs (2 × 105) were placed in inserts with 0.4-mmpore-sized membrane at its bottom, and hhPDLSCs or ihPDLSCs (2 × 105) irradiated with 30 Gy were seeded in the chamber of a 24-well flat-bottom plate. Cells were cultured in 500 μl of RPMI-1640 with 10 μg/ml Con A for 3 days. The proliferation of PBMNCs was then assessed. * p < 0.05 (n = 5). The gene expression of iNOS (b), IDO (c), COX-2 (d), TNFAIP6 (e) and IL-10 (f) by hhPDLSCs and ihPDLSCs before and after coculture was investigated by quantitative RT-PCR analysis. * p < 0.05 (n = 5).
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tory property of MSCs is considered to play a very important role during this therapeutic process [Shi et al., 2010]. Periodontitis is associated with abnormal or imbalanced immune reaction. Anti-inflammatory mediators can counteract and attenuate disease progression [Gemmell et al., 2002; Bascones et al., 2005; Garlet, 2010]. Similar to bone marrow MSCs, hhPDLSCs were also reported to have immunomodulatory properties [Wada et
al., 2009; Ding et al., 2010]. Nonetheless, this is still unclear for ihPDLSCs. CD146, a 113- to 119-kDa transmembrane glycoprotein, was initially identified as a marker of melanoma progression and invasion, and is involved in cell-to-cell junctions [Ouhtit et al., 2009]. It is widely used as an important surface marker in PDLSC research [Seo et al., 2004]. CD146 expression differs significantly between
The Immunomodulatory Properties of ihPDLSCs
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the effects of the former on suppressing the proliferation of PBMNCs were weakened, though not eliminated. NO is involved in the MSC-mediated immune suppression and can only function locally due to its high instability [Sato et al., 2007; Ren et al., 2008]. NO production is catalyzed by NOS, of which there are three types in human: iNOS, inducible primarily in macrophages, nNOS, in neuronsand, and eNOS, in endothelial cells. iNOS expression is inducible and plays a major role in immune regulation. We found that after hhPDLSCs or ihPDLSCs were cocultured with PBMNCs for 3 days, the mRNA expression of iNOS was dramatically elevated, which indicates that NO may also take part in the immunosuppression of hPDLSCs. This can partly explain why the immunosuppression effects of hPDLSCs in Transwell culture were decreased; the separation by Transwell prohibits NO from fully exerting its immunosuppressive effects. Soluble mediators secreted by MSCs are necessary for their immunomodulatory properties and proinflammatory cytokine stimulation is a prerequisite for the secretion of these mediators. Conditioned media derived from either hhPDLSCs or ihPDLSCs showed no suppression on PBMNC proliferation at all concentrations examined (10, 20 and 50% of total media; data not shown), suggesting hPDLSCs require stimulating factors from activated PBMNC to exert their suppressive effects. In agreement with our results, IDO and PGE2, two important mediators of MSC immunosuppression [Meisel et al., 2004; Aggarwal and Pittenger, 2005], have also been reported to be associated with the immunomodulation of PDLSCs [Wada et al., 2009; Ding et al., 2010]. Following coculture, the mRNA expressions of IDO and COX-2 (the enzyme for PGE2 production) were highly increased, while ihPDLSCs showed less IDO expression than hhPDLSCs. TNFAIP6 is a 30-kDa glycoprotein. It can produce antiinflammatory effects through different mechanisms. TNFAIP6 expression in MSCs is highly upregulated following intravenous administration. After myocardial infarction, intravenous administration of MSCs, but not MSCs transduced with TNFAIP6 siRNA, exhibited therapeutic effects in mice [Lee et al., 2009]. Interestingly, we have found that TNFAIP6 may also play a role in PDLSCs immunosuppression, as shown by dramatically elevated gene expression after coculture. The gene expression of IL-10 in PDLSCs was also greatly upregulated after coculture with activated PBMNCs. IL-10 is a well-established immune suppressor and is also one of the reported soluble cytokines that take part in the immunomodulatory properties of MSCs [Jui et al., 2012]. However, we also discovered its possible involvement with PDLSCs. Li /Wang /Tan /Wang /Wang
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hhPDLSCs and ihPDLSCs in our results (58.4% for hhPDLSCs and 9.2% for ihPDLSCs). Consistently, Xu et al. [2009] also reported that the positive rate of CD146 varies in periodontal ligament, fluctuating from 9.33 to 60.42%. Since this study is limited by the small number of samples analyzed (five samples for each cell type), further investigation is needed to validate whether CD146 expression is lower in ihPDLSCs in comparison with hhPDLSCs. The inflammation environment can affect the cytokine profile of MSCs and their immunomodulatory properties. The proinflammatory cytokine IFN-γ upregulates HGF and TGF-β1 expressed by MSCs and thus promotes the immunosuppressive capacity of adult human MSCs [Ryan et al., 2007]. Meanwhile, COX-2 and prostaglandin E2 (PGE2) expression by MSCs can also be upregulated by both IFN-γ and TNF-α in murine MSCs [English et al., 2007]. It appears, therefore, that a proinflammatory environment will enhance the immunosuppressive effects elicited by MSCs. In order to better understand the immunomodulatory property changes in ihPDLSCs, we performed a PBMNC proliferation assay as previously described [Bartholomew et al., 2002; Jarvinen et al., 2008; Wada et al., 2009; Sun et al., 2011]. Our results indicated that the immunomodulatory effects of ihPDLSCs were comparable to those observed in hhPDLSCs. They can not only suppress the proliferation of PBMNCs in a dosedependent manner, but also suppress the secretion of IFN-γ, which is the main proinflammatory cytokine secreted by activated PBMNCs. CFSE staining suggested that the suppression of PBMNC proliferation is mainly due to the inhibition of the cell cycle. It is unlikely that the reduction in PBMNC proliferation was caused by the exhaustion of culture medium. Firstly, the color of the RPMI-1640 medium in coculture groups was similar to that of control groups without PDLSCs. Secondly, we have also tried to replace half the amount of the culture medium every day or using culture medium containing higher levels of FBS (20% instead of 10% v/v) with similar results (data not shown). The mechanism underlying the immunomodulatory properties of MSCs is complex and still not fully understood. Some reports have shown that soluble factors are the major mediators of suppression [Di Nicola et al., 2002; Meisel et al., 2004; Aggarwal and Pittenger, 2005], whereas others have demonstrated that T cell-MSC contact is required for this suppression [Djouad et al., 2003; Ren et al., 2008; Akiyama et al., 2012]. We have found that both cell-cell contact and soluble factors contribute to the immunomodulatory properties of hPDLSCs. When using Transwell culture to separate the hPDLSCs and PBMNCs,
The mechanism of MSC immunosuppression is complex and there are many mediators involved in this process. Immunosuppression of PDLSCs is more likely a combination effect of all the molecules. Though there are some significant differences between hhPDLSCs and ihPDLSCs in terms of the gene expressions of immune mediators, such as iNOS, IDO and TNFAIP6, their potency in suppressing PBMNC proliferation and secreting IFN-γ shows no apparent differences. However, because of a lack of blocking experiments, our research indicates a possible involvement of the mediators when tested during the immunosuppression of hhPDLSCs or ihPDLSCs. It therefore merits further investigation and clarification. In conclusion, ihPDLSCs have similar immunomodulatory properties as hhPDLSCs. Although different in
dose, the gene expression of some immune mediators in ihPDLSCs displayed a similar pattern when compared with hhPDLSCs. Along with their easier accessibility, our findings suggest that ihPDLSCs offer considerable potential as a valuable alternative to hhPDLSCs.
Acknowledgements This work was supported by grants from the Nature Science Foundation of China (81170963 and 81271137).
Disclosure Statement There are no conflicts of interest.
References
The Immunomodulatory Properties of ihPDLSCs
Ding, G., Y. Liu, W. Wang, F. Wei, D. Liu, Z. Fan, Y. An, C. Zhang, S. Wang (2010) Allogeneic periodontal ligament stem cell therapy for periodontitis in swine. Stem Cells 28: 1829– 1838. Djouad, F., P. Plence, C. Bony, P. Tropel, F. Apparailly, J. Sany, D. Noel, C. Jorgensen (2003) Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 102: 3837–3844. English, K., F.P. Barry, C.P. Field-Corbett, B.P. Mahon (2007) IFN-γ and TNF-α differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 110: 91–100. Garlet, G.P. (2010) Destructive and protective roles of cytokines in periodontitis: a re-appraisal from host defense and tissue destruction viewpoints. J Dent Res 89: 1349–1363. Gemmell, E., K. Yamazaki, G.J. Seymour (2002) Destructive periodontitis lesions are determined by the nature of the lymphocytic response. Crit Rev Oral Biol Med 13: 17–34. Golemovic, M., M. Skific, B.G. Cepulic (2012) Mesenchymal stem cells: immunomodulatory properties and clinical application (in Croatian). Lijec Vjesn 134: 42–49. Hung, T.Y., H.C. Lin, Y.J. Chan, K. Yuan (2011) Isolating stromal stem cells from periodontal granulation tissues. Clin Oral Investig 16: 1171–1180. Jarvinen, L., L. Badri, S. Wettlaufer, T. Ohtsuka, T.J. Standiford, G.B. Toews, D.J. Pinsky, M. Peters-Golden, V.N. Lama (2008) Lung resident mesenchymal stem cells isolated from human lung allografts inhibit T cell proliferation via a soluble mediator. J Immunol 181: 4389–4396.
Cells Tissues Organs DOI: 10.1159/000367986
Jui, H.Y., C.H. Lin, W.T. Hsu, Y.R. Liu, R.B. Hsu, B.L. Chiang, W.Y. Tseng, M.F. Chen, K.K. Wu, C.M. Lee (2012) Autologous mesenchymal stem cells prevent transplant arteriosclerosis by enhancing local expression of interleukin-10, interferon-γ, and indoleamine 2,3-dioxygenase. Cell Transplant 21: 971– 984. Kinane, D.F., G.J. Marshall (2001) Periodontal manifestations of systemic disease. Aust Dent J 46: 2–12. Lee, R.H., A.A. Pulin, M.J. Seo, D.J. Kota, J. Ylostalo, B.L. Larson, L. Semprun-Prieto, P. Delafontaine, D.J. Prockop (2009) Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5: 54–63. Liu, Y., Y. Zheng, G. Ding, D. Fang, C. Zhang, P.M. Bartold, S. Gronthos, S. Shi, S. Wang (2008) Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells 26: 1065–1073. Marigo, I., F. Dazzi (2011) The immunomodulatory properties of mesenchymal stem cells. Semin Immunopathol 33: 593–602. Meisel, R., A. Zibert, M. Laryea, U. Gobel, W. Daubener, D. Dilloo (2004) Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenasemediated tryptophan degradation. Blood 103: 4619–4621. Nauta, A.J., G. Westerhuis, A.B. Kruisselbrink, E.G. Lurvink, R. Willemze, W.E. Fibbe (2006) Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 108: 2114–2120.
9
Downloaded by: Selçuk Universitesi 193.255.248.150 - 1/12/2015 12:09:16 PM
Aggarwal, S., M.F. Pittenger (2005) Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105: 1815–1822. Akiyama, K., C. Chen, D. Wang, X. Xu, C. Qu, T. Yamaza, T. Cai, W. Chen, L. Sun, S. Shi (2012) Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell 10: 544– 555. Bartholomew, A., C. Sturgeon, M. Siatskas, K. Ferrer, K. McIntosh, S. Patil, W. Hardy, S. Devine, D. Ucker, R. Deans, A. Moseley, R. Hoffman (2002) Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30: 42–48. Bascones, A., S. Noronha, M. Gomez, P. Mota, M.A. Gonzalez Moles, M. Villarroel Dorrego (2005) Tissue destruction in periodontitis: bacteria or cytokines fault? Quintessence Int 36: 299–306. Chan, J.L., K.C. Tang, A.P. Patel, L.M. Bonilla, N. Pierobon, N.M. Ponzio, P. Rameshwar (2006) Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-γ. Blood 107: 4817–4824. Chen, F.M., H.H. Sun, H. Lu, Q. Yu (2012) Stem cell-delivery therapeutics for periodontal tissue regeneration. Biomaterials 33: 6320– 6344. Di Nicola, M., C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, S. Grisanti, A.M. Gianni (2002) Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99: 3838–3843.
10
Cells Tissues Organs DOI: 10.1159/000367986
Sato, K., K. Ozaki, I. Oh, A. Meguro, K. Hatanaka, T. Nagai, K. Muroi, K. Ozawa (2007) Nitric oxide plays a critical role in suppression of Tcell proliferation by mesenchymal stem cells. Blood 109: 228–234. Sbano, P., A. Cuccia, B. Mazzanti, S. Urbani, B. Giusti, I. Lapini, L. Rossi, R. Abbate, G. Marseglia, G. Nannetti, F. Torricelli, C. Miracco, A. Bosi, M. Fimiani, R. Saccardi (2008) Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model. Arch Dermatol Res 300: 115–124. Seo, B.M., M. Miura, S. Gronthos, P.M. Bartold, S. Batouli, J. Brahim, M. Young, P.G. Robey, C.Y. Wang, S. Shi (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364: 149–155. Shi, Y., G. Hu, J. Su, W. Li, Q. Chen, P. Shou, C. Xu, X. Chen, Y. Huang, Z. Zhu, X. Huang, X. Han, N. Xie, G. Ren (2010) Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Res 20: 510– 518. Sun, Z., Q. Han, Y. Zhu, Z. Li, B. Chen, L. Liao, C. Bian, J. Li, C. Shao, R.C. Zhao (2011) NANOG has a role in mesenchymal stem cells’ immunomodulatory effect. Stem Cells Dev 20: 1521–1528.
Togel, F., Z. Hu, K. Weiss, J. Isaac, C. Lange, C. Westenfelder (2005) Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol 289: F31–F42. Wada, N., D. Menicanin, S. Shi, P.M. Bartold, S. Gronthos (2009) Immunomodulatory properties of human periodontal ligament stem cells. J Cell Physiol 219: 667–676. Wang, L., H. Shen, W. Zheng, L. Tang, Z. Yang, Y. Gao, Q. Yang, C. Wang, Y. Duan, Y. Jin (2010) Characterization of stem cells from alveolar periodontal ligament. Tissue Eng Part A 17: 1015–1026. Xu, J., W. Wang, Y. Kapila, J. Lotz, S. Kapila (2009) Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells. Stem Cells Dev 18: 487–496. Yi, T., S.U. Song (2012) Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications. Arch Pharm Res 35: 213–221. Zhang, J., Y. An, L.N. Gao, Y.J. Zhang, Y. Jin, F.M. Chen (2012) The effect of aging on the pluripotential capacity and regenerative potential of human periodontal ligament stem cells. Biomaterials 33: 6974–6986.
Li /Wang /Tan /Wang /Wang
Downloaded by: Selçuk Universitesi 193.255.248.150 - 1/12/2015 12:09:16 PM
Ouhtit, A., R.L. Gaur, Z.Y. Abd Elmageed, A. Fernando, R. Thouta, A.K. Trappey, M.E. Abdraboh, H.I. El-Sayyad, P. Rao, M.G. Raj (2009) Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146. Biochim Biophys Acta 1795: 130– 136. Park, J.C., J.M. Kim, I.H. Jung, J.C. Kim, S.H. Choi, K.S. Cho, C.S. Kim (2011) Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations. J Clin Periodontol 38: 721–731. Quarto, R., M. Mastrogiacomo, R. Cancedda, S.M. Kutepov, V. Mukhachev, A. Lavroukov, E. Kon, M. Marcacci (2001) Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344: 385– 386. Ren, G., L. Zhang, X. Zhao, G. Xu, Y. Zhang, A.I. Roberts, R.C. Zhao, Y. Shi (2008) Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2: 141–150. Ryan, J.M., F. Barry, J.M. Murphy, B.P. Mahon (2007) Interferon-γ does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol 149: 353–363.
