It is now established that MSC can be isolated from the synovial membrane [7
] as well as from BM or other tissues (for a review, see [1
]). The culture conditions, based in part on adherence to plastic, used to isolate MSC from the synovial membrane are similar to those used to obtain fibroblastic synoviocytes, suggesting that only a subset of cells in this synovium-derived cell population are stem cells, like MSC from BM-derived cultures. As no specific marker for MSC is presently available, their characterization relies essentially on their functional properties, but to date no quantitative data allow comparison between MSC isolated from various tissues. In the present study we have shown that BM-derived and synovium-derived adherent cell populations can be induced to differentiate towards chondrocytes and osteoblasts, but a significant fivefold to 10-fold reduction in the expression levels of the osteogenic markers was observed with the adherent cells from the synovium. Furthermore, we show that the transcriptional profiles permit one to discriminate between the cell populations isolated from BM or the synovium, and that activin A might be a useful marker since it is highly secreted by MSC from BM.
In agreement with other studies [8
], we confirm that, together with skin fibroblasts, synovium-derived adherent cells express various markers known to be present on BM-MSC, such as CD44, CD90 and CD105. We now show that the cells isolated from the synovial membrane also express the CD73 marker, an ecto-5'-nucleotidase recognized by the SH3 antibody, and express prolyl-4-hydroxylase. Prolyl-4-hydroxylase was shown to be expressed by synoviocytes [18
] but to be absent on BM-MSC using FACS analysis [18
]. In the present study we were able to detect the protein in both cell populations by immunocytochemistry, whereas it was negative by FACS analysis, suggesting that this marker was not specific for synoviocytes. Expression of MHC class I molecules on BM-MSC and synovial fibroblasts has been reported, as well as the induction of MHC class II molecules upon treatment with IFN-γ [25
]. However, although IFN-β has been shown to induce the expression of MHC class I and class II molecules on various cell types [27
], no data were available on the potential role of IFN-α and IFN-β on synovial fibroblasts or BM-MSC. To our knowledge, this is the first report to show that IFN-α and IFN-β upregulate the MHC class I molecules and the 6–16 gene, which is one of the early responsive genes induced by IFN, but fail to induce the MHC class II molecules on both cell populations. Altogether, these data illustrate the high similarity between adherent cell populations isolated from BM and the synovium, based on the expression of various phenotypic markers known to be currently tested with MSC. This suggests that these markers correspond to molecules expressed on cells of mesenchymal origin as they are also detected on primary skin fibroblasts (data not shown) and points out the lack of specific markers.
The multilineage potential of MSC from the synovium has already been described [6
]. These studies only described qualitative results based on histological and immunohistological staining or semiquantitative RT-PCR. In our study, we performed quantitative RT-PCR to indicate quantitative differences in the expression level of the markers specific for the differentiated states. Variability in gene expression was observed between samples, independently of gender, age or the status of the patient (normal, OA or RA) (data not shown). In previous studies, the multilineage potential of synovium-derived MSC was reported to be independent of donor age, passaging or cryopreservation [7
]. We now report that synovium-derived cells expressed significantly lower levels of osteogenic marker mRNA after in-vitro
-induced osteogenesis, whereas these cells tended to secrete higher amounts of chondrogenic marker mRNA after chondrogenesis induction, although the differences with BM-derived MSC were not statistically significant. These data may reflect a different cellular phenotype or a different amount of MSC inside the two cell populations. Indeed, in the cells isolated from the synovium tissue we measured 1.8 ± 1.4 colony-forming units in 104
plated cells, which is in the same range obtained with cells from BM (estimated to be 1 in 104
mononuclear cells) [29
]. Alternatively, they may reflect a commitment of stem cells under the influence of the environmental parameters. The presence of progenitors already committed to the chondrogenic lineage may be in higher amounts in the synovial membrane, where they contribute to the homeostasis of the cartilage tissue that is in close contact. Conversely, the higher capacity of differentiation toward osteoblasts observed with cells isolated from BM may suggest a higher numbers of cells committed to the osteogenic lineage inside the BM. Availability of markers specific for the MSC or different stages of differentiation would help to answer this question.
Another functional characteristic of MSC is their capacity to inhibit the proliferation of T cells in a MLR [15
]. We show here for the first time that synovium-derived cells not only suppress the proliferative activity of T cells, but also exhibit functional IDO activity upon stimulation with IFN-γ to the same extent as BM-MSC. IDO activity has recently been suggested to contribute to the T-cell suppressive mechanism in human MSC. IDO has been identified as a T-cell inhibitory effector pathway in professional antigen-presenting cells upon induction by IFN-γ and other proinflammatory molecules such as TNF-α. This enzyme catalyzes the conversion from tryptophan to kynurenine; because tryptophan is an essential amino acid, its depletion will impair protein synthesis, leading to inhibition of cell proliferation. Depletion of tryptophan has also been shown to lead to stabilization of IL-6 and IL-8 mRNA, resulting in increased IL-6 and IL-8 responses that were proposed to be implicated in enhanced inflammatory responses to bacterial challenges after a viral infection [30
]. A comparison of patients with RA, OA, psoriatic arthritis and gout recorded the highest levels of IL-1β, IL-6, IL-8 and IDO as well as the lowest levels of tryptophan in RA synovial fluids, indicating stimulated cellular immune responses in RA patients [31
]. Indeed, the possible dual activity of IDO in synoviocytes as well as in BM-MSC still needs to be elucidated. In conditions where TNF-α and IFN-γ are only poorly present, the IDO activity may lead to an immunosuppressive environment inside the joint favoring the inhibition of immune cell proliferation. In the inflammatory context, where TNF-α and IFN-γ are prominent, induction of proinflammatory cytokines may reverse the cytokine balance, leading to a reversion of the immunosuppressive capacity of MSC as we previously showed in the collagen-induced arthritis model of arthritis [33
The presence of MSC in the synovial membrane addresses the question of their origin and function within the joint. MSC in the synovium may be recruited from the blood that enters the synovial tissue, as they are present in normal conditions and even in higher numbers in the case of injury [8
] due to their recruitment from the other tissues where they reside. MSC may also come from the bone marrow, which is connected with the intra-articular space by channels, enlarged in RA [10
]. The role of MSC is possibly related to their potential to repair tissues of mesodermal origin present inside the joint in the case of traumatic or pathologic injuries [10
]. Another postulated role for MSC is their possible involvement in the early phases of osteoarticular diseases and, in particular, in RA [10
]. Although MSC possess immunosuppressive capacities, we have previously shown that they are unable to display a benefit in the collagen-induced arthritis model because they lose this property in the presence of TNF-α [33
]. Moreover, the increase of MHC class II expression on MSC upon IFN-γ stimulation may further contribute to the aggravation of the immune response. We thus may postulate that TNF-α is the key molecule at the onset of RA pathogenesis that induces or contributes to modifying the characteristics of the MSC, which then act to favor the accumulation of immunocompetent cells into the joint.
An important feature revealed in the present study is that cells isolated from BM and the synovial membrane could be distinguished by distinct gene expression profiles. Both populations are thus characterized by the differential expression of various genes, in particular activin A that is upregulated in BM-MSC. Activins and inhibins are members of the transforming growth factor beta super-family that play roles in skeletal development and bone morphogenesis [23
]. Activin A is a multifunctional cytokine that regulates cell growth and differentiation, whose effects are diverse depending on the cell type. In synoviocytes, activin A has been reported to promote proliferation, to be induced by IL-1β and to be upregulated in OA and RA patients [35
]. It is still unclear, however, whether activin A accelerates or inhibits RA autoimmunity and inflammation. The secretion of activin A in BM-MSC is induced by BMP-2, at least in vitro
, and therefore has been suggested to be downstream BMP-2 in the differentiation program that results in skeletal development [38
]. In the bone marrow, where MSC are under the influence of transforming growth factor beta and BMP molecules, the upregulation of activin A may be involved early at the beginning of the cascade of events promoting chondrogenic/osteogenic differentiation [39
]. However, activin A has been recently shown to play a role in the maintenance of the pluripotency of human embryonic stem cells [40
]. The maintenance of pluripotency of embryonic stem cells could involve Wnt signaling and could occur through a crosstalk between the transforming growth factor beta/activin and Wnt pathways [41
]. Indeed, activin A may play a dual role according to the environmental parameters: proliferation or maintenance of pluripotency. The higher amounts of activin A produced by BM-MSC together with similar numbers of colony-forming units further suggest their higher multipotent potential.
In summary, the similarity between adherent cells cultured from BM and from synovial tissue suggests a common origin. The few discrepancies between cells may reflect the impact of the tissue environment on the properties of MSC. Thus, due to pathological conditions, reduced differentiation properties and reversion of immunosuppression of MSC have been reported. It will be of therapeutic interest to determine whether MSC originating from various tissue sources share the same features. In this respect, the demonstration that high levels of activin A are produced by BM-MSC may potentially be of relevance in arthritis and repair since it may be associated with the pluripotency of the cells. MSC isolated from various tissues not involved in the specific pathology may be an alternative and more suitable source of cells with fully functional features for tissue engineering.