Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by chronic inflammation of the joints. Previously, several independent groups have explored the therapeutic effects of MSCs in a collagen-induced arthritis (CIA) model, and generated conflicting results [19–22]. Augello et al. reported that MSC treatment decreased the serum concentration of tumour necrosis factor (TNF)-α and alleviated CIA by educating

regulatory T cells (Tregs) PF-562271 manufacturer [19], but Djouad et al. found that the addition of TNF-α to in vitro co-culture of MSCs and lymphocytes reversed the proliferation-suppressive properties of MSCs, and proposed that the presence of TNF-α in CIA animals led to aggravation of the disease after MSC treatment [20]. Indeed, there is some evidence showing that MSCs may up-regulate the immune response [23–25]. The underlying reasons for the

discrepancy are currently unknown. MSCs are heterogeneous cells without a defined phenotype and are always cultured using different check details modified methods by different laboratories. The difference in cells may account at least partly for the conflicting results in animal studies. Moreover, the circumstances in CIA animals are much more complicated than in vitro culture: the phenomena observed in cultured cells may not happen exactly as in animal models. To clarify this issue, it is important to investigate the therapeutic effect with a defined MSC population and explore the underlying mechanisms in vivo. We have been engaged in the studies of Flk-1+ MSCs for a long time. They are a MSC subpopulation

with a defined phenotype. We have completed Phase I clinical trials and have shown that Flk-1+ MSCs are safe and effective in the treatment of GVHD [26]; Phase II clinical trials for GVHD are on the way. In this study, we investigated the therapeutic effect of Flk-1+ MSCs on CIA mice. Considering the present application of Flk-1+ MSCs in clinical trials, this study is of great importance for the establishment of inclusion criteria in enrolling potential candidates. Flk-1+ MSCs were isolated from bone marrow of dilute brown non-Agouti (DBA-1) mice and cultured as we have described previously [1,3]. Briefly, mononuclear cells were obtained Acesulfame Potassium by Ficoll-Paque density gradient centrifugation from bone marrow flushes, depleted of haematopoietic cells, and cultured in Dulbecco modified Eagle medium and Ham F12 medium (DF12) culture medium containing 40% MCDB-201 medium complete with trace elements (MCDB) medium (Sigma, St Louis, MO, USA), 2% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 10 ng/ml platelet-derived growth factor BB, insulin transferring selenium, linoleic acid and bovine serum albumin (BSA) at 37°C and 5% CO2. The non-adherent cell population was removed after 24–48 h and the adherent layer was cultured for approximately 1 week. When cells reached 90% confluence they were harvested by trypsinization (0·25% trypsin).