3confirmed the immunohistochemistry staining

3confirmed the immunohistochemistry staining. cells is not yet compromised. Only Herbacetin at much higher concentrations is TGF- signaling also inhibited in vitro, confirming the compounds preference for BMP type I receptor inhibition (Fig. 1 and and in hMSCs, excluding binding to ALK1 as a mechanism involved in this model (Fig. S1and and and and 0.05) in expression compared with the control. Results obtained at the microscale level were also confirmed in 3D macropellet culture (Fig. S3 and and expression and refer to basal expression in day 0 hMSCs; all fold-changes in transcript levels are shown in logarithmic scale (= 3, * 0.05, ** 0.005). ALK2 and ALK3 Inhibition on hMSCs-Derived Constructs Is Sufficient to Maintain a Stable Cartilaginous Phenotype in Vitro. We investigated how stable the effect of hypertrophy silencing was elicited by ALK2 and ALK3 inhibition in adult hMSC-derived constructs. Three-dimensional hMSC-derived macropellets were incubated in standard chondrogenic medium for 28 d with either compound supplemented continuously (Fig. S3 expression compared with compound B and control (Fig. 3and expression compared with control (Fig. 3 and expression was also reduced, even if not statistically significantly (Fig. 3confirmed the immunohistochemistry staining. All Ct values are normalized relative to expression and refer to basal expression in day 0 hMSCs; all fold-changes in transcript levels are shown in logarithmic scale (= 3, * 0.005, *** 0.001). Ctrl, control. Simultaneous Inhibition of ALK2 and ALK3 Down-Regulates the BMP Pathway and Triggers the Activation of Genes Characteristic for Articular Cartilage in Adult Human MSCs. The modulation of key players of the BMP pathway was monitored upon treatment with BMP type I receptor inhibitors. Among endogenous BMP antagonists, was up-regulated by both compounds, with compound A inducing the most pronounced increase. hMSCs cultured in standard chondrogenic medium Herbacetin instead down-regulated during the whole culture period (Fig. 4uniformly increased and no significant differences were detected among the different conditions, suggesting that Noggin is not modulated by inhibiting ALK2 and ALK3 (Fig. 4was strongly up-regulated in standard chondrogenic conditions compared with treatment with compound A (Fig. 4and (and of (expression and refer to basal expression in day 0 hMSCs; all fold-changes in transcript levels are shown in logarithmic scale (= 3; * Herbacetin 0.05, ** 0.005). Ctrl, control. To characterize the nature of the hMSC-derived cartilaginous tissues obtained by treatment with the different compounds, gene-expression changes of a panel of articular or transient cartilage signature genes were assessed by quantitative RT-PCR. Expression of Lubricin (and expression was down-regulated in the control conditions compared with basal levels during the whole culture period. expression was up-regulated in all conditions compared with basal levels, with compound A-treated constructs showing the least increase of (Fig. 4exhibited instead a variable trend (Fig. 4and Fig. S6and Fig. S6and Fig. S6and Fig. S6= 3 donors). (Scale bar, 300 m.) Simultaneous ALK2 and ALK3 Inhibition Prevents hMSCs from Bone Remodeling by Activating a Protective Mechanism Against Vascularization. Immunofluorescence staining for vessel invasion (CD31, SMA and DAPI) (Fig. Herbacetin 6 and and and in all conditions. All Ct values are normalized relative to expression and refer to basal expression in day 0 hMSCs; all fold-changes in transcript levels are shown in logarithmic scale (= IL18R antibody 3, * 0.05, ** 0.005). ( em J /em ) VEGF secreted by hMSCs was measured by ELISA at day 2, day 7 and day 14 of culture. Ctrl, control. Discussion Following the concept of developmental engineering (1), we investigated in this study the role Herbacetin of BMP signaling on the differentiation of adult mesenchymal progenitor cells, namely hMSCs, toward articular cartilage. The combination of ( em i /em ) a microfluidic system for time- and dose-dependent screening of soluble factors on 3D microaggregates and of ( em ii /em ) synthetic compounds selectively and specifically silencing the BMP pathway by targeting ALK2 and ALK3 receptors allowed us to successfully design a strategy effective in guiding hMSCs toward stable articular-like cartilage, both in vitro and in vivo. We also postulate the role of ALK2 and ALK3 inhibition in triggering a protective mechanism against vascularization, eventually favoring the maintenance of stable and avascular cartilage tissue. Recently, the achievement of an organized cartilage template based on hMSCs was reported by recreating in vitro the native articular cartilage spatial organization. However, the only partial inhibition of hypertrophic markers achieved in this model suggests that key players.