Proinflammatory cytokines reduced
significantly the expression of 13 of a total of 45 types of collagens (Fig. 2j). Culture of ASC with MLR reduced expression of collagen type 15α1 only (threefold). ASC may also induce fibrosis via the secretion of factors such as connective tissue growth factor, TGF-β and platelet-derived growth factor that act on other cell types. The expression of these factors by ASC, however, did not change in response to inflammatory conditions. Furthermore, except from small increases in actin α1 (0·2-fold) and actin γ2 (2·0-fold) after culture with MLR, no significant changes in gene expression of cytoskeletal proteins such as actins or intermediate filaments were observed in ASC after exposure to proinflammatory conditions. Next, functional analysis of ASC mTOR inhibitor cultured under inflammatory conditions was performed. ASC cultured under inflammatory conditions showed morphological changes compared to ASC cultured under control conditions (Fig. 3a). ASC cultured under control conditions grew in a monolayer and were distributed equally on the surface of the culture flask, while ASC cultured with alloactivated PBMC clustered in star-shaped formations. The number of ASC cultured
Selleckchem CP 690550 for 7 days with MLR increased compared to control ASC cultures (Fig. 3b). In contrast, the number of ASC treated with proinflammatory cytokines was reduced significantly. Culture of ASC with MLR or proinflammatory cytokines increased Nintedanib (BIBF 1120) significantly the diameter of ASC (Fig. 3c). ASC cultured under control conditions had a diameter of
21 (interquartile range 19–25) µm. After culture with MLR, ASC had a diameter of 24 (22–28) µm and treatment of ASC with inflammatory cytokines led to an increase in cell diameter to 29 (25–32) µm. To investigate whether the immunophenotype of ASC changed after culture with inflammatory factors, flow cytometric analysis was performed (Fig. 3d). ASC expressed the characteristic cell surface markers CD90, CD105 and CD166 and the expression of these markers was unaffected by culture of ASC with MLR or proinflammatory cytokines. Levels of HLA class I expression by ASC were independent of inflammatory culture conditions. Control ASC were slightly positive for HLA class II (6%), while culture of ASC with MLR or proinflammatory cytokines resulted in an increase in HLA class II-positive cells of 62% and 86%, respectively. Independently of culture conditions, ASC stained positive for the co-stimulatory molecule CD80 and were weakly positive for CD86. CD40 was not expressed on control or MLR-cultured ASC, but culture of ASC with proinflammatory cytokines induced expression of CD40. ASC, cultured previously for 7 days under inflammatory conditions, were cultured under adipogenic and osteogenic conditions for 3 weeks (Fig. 4). Independent of previous culture conditions, ASC were able to differentiate in adipogenic and osteogenic lineages.