Activin and Nodal Are Not Suitable Alternatives to TGFβ for Chondrogenic Differentiation of Mesenchymal Stem Cells

Objective. Previously, we demonstrated the importance of transforming growth factor-beta (TGF beta)-activated SMAD2/3 signaling in chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). However, TGF beta also signals via the SMAD1/5/9 pathway, which is known to induce terminal differentiation of BMSCs. In this study, we investigated whether other SMAD2/3-activating ligands, Activin and Nodal, can induce chondrogenic differentiation of BMSCs without inducing terminal differentiation. Design. Activation of SMAD2/3 signaling and chondrogenesis were evaluated in human BMSCs (N = 3 donors) stimulated with TGF beta, Activin, or Nodal. SMAD2/3 activation was assessed by determining phosphorylated-SMAD2 (pSMAD2) protein levels and SMAD2/3-target gene expression of SERPINE1. Chondrogenesis was determined by ACAN and COL2A1 transcript analysis and histological examination of proteoglycans and collagen type II. Results. Both Activin and TGF beta enhanced pSMAD2 and SERPINE1 expression compared to the control condition without growth factors, demonstrating activated SMAD2/3 signaling. pSMAD2 and SERPINE1 had a higher level of expression following stimulation with TGF beta than with Activin, while Nodal did not activate SMAD2/3 signaling. Of the 3 ligands tested, only TGF beta induced chondrogenic differentiation as shown by strongly increased transcript levels of ACAN and COL2A1 and positive histological staining of proteoglycans and collagen type II. Conclusions. Even with concentrations up to 25 times higher than that of TGF beta, Activin and Nodal do not induce chondrogenic differentiation of BMSCs; thus, neither of the 2 ligands is an interesting alternative candidate for TGF beta to induce chondrogenesis without terminal differentiation. To obtain stable cartilage formation by BMSCs, future studies should decipher how TGF beta-induced terminal differentiation can be prevented.