In the present study, we have isolated and characterized a new population of precursor cells from human gingival tissues, termed GMSC, which exhibit several unique stem cell-like properties as MSCs derived from bone marrow and other postnatal tissues (8
). These characteristics include in vitro proliferation as plastic-adherent cells with fibroblast-like morphology, colony-forming ability, multipotent differentiation into different cell lineages, including mesodermal (adipocytes, osteocytes), endodermal, and neuroectodermal progenies, and expression of mesenchymal cell surface markers and stem cell-specific genes (1
). More importantly, we have demonstrated that single colony-derived GMSCs possess in vivo self-renewal and differentiation capacities, further supporting their stem cell-like properties. In addition, compared with MSCs derived from several other adult dental tissues such as DPSCs (13
) and PDLSCs (16
), GMSCs express a similar profile of cell surface molecules, a high proliferative rate, and an increased population doubling, and thus can be easily expanded ex vivo for several cell-based clinical applications. However, s.c. transplantation of GMSCs could form connective tissue-like structures, whereas transplantation of DPSCs and PDLSCs could generate dentin-like and cementum/PDL-like structures (13
). These findings have provided evidence that human gingiva, an easily accessible tissue from the oral cavity or a discarded tissue sample following some dental procedures, might serve as a unique source of postnatal stem cells with potential therapeutic functions in tissue regeneration and repair (1
In recent years, a major breakthrough was the discovery that MSCs are immune privileged and, more importantly, possess profound immunosuppressive and anti-inflammatory effects both in vitro and in vivo via inhibiting the proliferation and function of several major types of innate and adaptive immune cells such as NK cells, dendritic cells, and T and B lymphocytes (25
). However, to date, the underlying mechanisms of MSC-mediated suppression of lymphocyte proliferation remain largely unknown (28
). In one study, the suppressive activity of human bone marrow MSCs was shown to be independent of cell-cell contact (31
); however, several other studies have reported that cell-cell contact contributed, at least in part, to the immunosuppression mediated by MSCs derived from human bone marrow, adipose, or umbilical cord blood (32
). In this study, we showed that GMSCs when cocultured with PBMCs under cell-cell contact conditions exhibited a slightly stronger inhibition on PBMC proliferation than GMSCs when cocultured with PBMCs separately in Transwells, thus supporting the notion that the cell-cell contact mechanism may partly contribute to GMSC-mediated suppression of PBMC proliferation.
Various studies have indicated that soluble factors such as TGF-β
1, HGF, IL-10, HLA-G5, PGE2
, NO, and IDO play an important role in MSC-mediated immunosuppression (27
). However, it is noteworthy that the relative contribution of these soluble factors to the immunosuppressive effects of MSCs varies under different experimental conditions, and neutralizing these soluble factors does not completely abrogate the immunosuppressive activity of MSCs (32
). For example, IL-10, HGF, and TGF-β
1 have been shown to contribute to BMSC-mediated immunosuppression (33
), but in other studies, these three factors appeared not to be related to immunosuppression mediated by BMSCs and human adipose-derived stem cells (hASCs) (30
). In addition, controversies about the role of PGE2
in MSC-mediated immunosuppression have also been reported. In some studies, blocking PGE2
production by COX-2 resulted in partial abrogation of immunosuppression by BMSCs and hASCs (29
); however, Cui et al. (67
) have recently reported that PGE2
is the major soluble factor in the in vitro inhibition of allogeneic lymphocyte reaction. In the present study, we observed that blocking TGF-β
, or NO by using specific neutralizing Abs or antagonists for synthetic enzymes showed no obvious effects on GMSC-mediated suppression of PBMC proliferation. However, blocking IL-10 led to moderate abrogation of GMSC-mediated suppression of PBMC proliferation, albeit to a greater extent than in BMSCs. These findings suggest that IL-10 might partially contribute to GMSC-mediated immunosuppression.
IDO is an enzyme that catabolizes tryptophan, an essential amino acid. A growing body of evidence has indicated that IDO plays a critical role in immunosuppression mediated by MSCs of various tissue origins, whereas 1-MT, a specific antagonist of IDO, can abrogate the immunosuppressive effects (30
). The immunomodulatory effects of IDO are attributed to tryptophan depletion and/or accumulation of the downstream metabolites such as kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid (30
). Most recently, studies have shown that IDO activity is involved in PDLSCs and gingival fibroblast-mediated immunosuppression (70
). Consistently, we have demonstrated that the addition of 1-MT also significantly ablated GMSC-mediated suppression of PBMC proliferation in response to mitogen stimulation under both cell-cell contact and Transwell conditions, suggesting that IDO might play a major role in GMSC-mediated immunosuppression.
Generally, IDO is not constitutively expressed by mesenchymal stromal cells, but can be significantly induced by a variety of inflammatory mediators (30
). Accumulating evidence has shown that IFN-γ
plays a critical role in the cross-talk between MSCs and immune cells. Upon activation, immune cells secrete a high amount of inflammatory cytokines, especially IFN-γ
, which may subsequently stimulate MSCs to express various immunosuppressive molecules, such as IDO, resulting in a negative feedback inhibition of inflammatory cell responses in terms of proliferation and cytokine secretion (11
). In agreement with previous reports (29
), GMSCs do not constitutively express IDO, but in response to IFN-γ
stimulation, harbored a significantly increased level of functional IDO. Coculture with GMSCs led to moderate suppression of mitogen-stimulated PBMC proliferation and IFN-γ
secretion; however, the presence of stimulated PBMCs enhanced IL-10 secretion and IDO expression by GMSCs. Furthermore, the addition of IFN-γ
-neutralizing Ab significantly blocked the secretion of IL-10 and the expression of functional IDO in GMSCs. These findings suggest that the up-regulated inflammatory signals dominated by IFN-γ
in the coculture of GMSCs and stimulated PBMCs can induce GMSC-mediated immunosuppression, mediated in part, via the up-regulation of IL-10 and functional IDO expression. However, further studies are required to determine whether other inflammatory cytokines such as TNF-α
are involved in priming GMSC-mediated immunosuppression.
Recently, several studies have reported that treatment with human bone marrow- or adipose-derived MSCs exhibits early efficacy in attenuating the progression of several experimental inflammatory diseases in murine models, including experimental arthritis (38
), colitis (43
), and autoimmune encephalomyelitis (39
). The apparent lack of graft rejection and positive treatment effects of human MSCs on these murine disease models could be due to their inherent capabilities to harness inflammatory cell infiltration, suppress inflammatory mediator production, and/or regulate immune tolerance by increasing the production of anti-inflammatory cytokines (e.g., IL-10) and inducing the generation/activation of Tregs (38
). Most recently, a study by Gonzalez et al. (38
) suggested that the viability of human adipose-derived MSCs was not required for their long-term immunosuppressive activities since these cells were only detectable in the recipient for ~1 wk after injection. Similar to recent studies using hASCs to treat experimental colitis (43
), the present study has demonstrated that infusion of GMSCs could ameliorate the severity of inflammatory-related colonic tissue injuries in experimental colitis, possibly by reducing colonic infiltrates of inflammatory cells, down-regulating the production of inflammatory cytokines, and by promoting the generation/activation of Tregs. However, it remains unclear why infusion of human MSCs, including GMSCs, into immunocompetent mice in our murine models can attenuate disease progression in the absence of an apparent graft-versus- host disease response. Further studies are warranted to address this important issue.
Despite the potential benefits of MSCs in clinical applications, several questions remain unanswered, especially regarding the identity and biological properties of MSCs as compared with other stromal cells such as fibroblasts (72
). Accumulating evidence has shown that MSCs share many common features with fibroblasts, including a spindle-like cell morphology, plastic adherence, expression profile of certain cell surface markers, multipotent differentiation, and even immunomodulatory functions (72
). Previous analysis of human bone marrow MSC subclones revealed that the lineage commitment was hierarchical in nature (75
) and may differ among MSC subpopulations derived from different tissues (75
). As such, the so-called fibroblast population may represent a more differentiated subpopulation of MSCs (22
). Up to date, there is still a lack of evidence whether such hierarchy exists in relevance to several biological functions, specifically the immunomodulatory properties of MSCs, and should be further addressed.
In conclusion, the unique immunomodulatory and anti-inflammatory properties of GMSCs as well as their ease of isolation, abundant tissue source, and rapid ex vivo expansion render these postnatal stem cells an ideal source for stem cell-based therapeutic approaches in clinical applications, including inflammatory diseases.