Role of activin A in MPC multipotency. BM-MPCs transiently transfected either with control siRNA or with activin A siRNA were analyzed. (a) Activin A secretion level in the supernatant of transfected BM-MPCs quantified by ELISA 3 days (D3) after transfection showed the efficiency of knockdown by using activin A siRNA. (b, c) Effect of activin A knockdown on BM-MPC and T-MPC chondrogenic potential. Chondrogenic differentiation was evaluated after 21 days in micropellet culture. (b) Immunohistochemical analysis of cartilage markers (COL2 and AGN) in pellet cultures of BM-MPCs and T-MPCs as well as alcian blue (AB) and picrosirius red (PS) staining revealed that, compared with the untransfected and Control siRNA, MPCs transfected with activin A siRNA exhibited less chondrogenic potential. (c) Quantitative RT-PCR also showed decreased expression of COL2 in MPCs transfected with activin A siRNA, compared with the cells transfected with control siRNA (c). (b, c) Effect of activin A knockdown on BM-MPC osteogenic potential. Both the expression levels of the osteogenic markers (OC and ALP), determined by quantitative RT-PCR, and alizarin red staining, revealed a decrease in the osteogenic activity of BM-MPCs with activin A knockdown. (b, c) Effect of activin A knockdown on BM-MPC adipogenic potential. Both expression levels of the adipogenic markers (PPAR-γ and LPL), determined by quantitative RT-PCR and oil red O staining, revealed an increase in the adipogenic potential of BM-MPCs with activin A knockdown, compared with BM-MPCs transfected with the irrelevant Control siRNA. RT-PCR data represent the mean ± SD of three independent experiments. *P ≤ 0.05, versus Control siRNA.