Wnt antagonism without TGFβ induces rapid MSC chondrogenesis via increasing AJ interactions and restricting lineage...

Human mesenchymal stem cells (MSCs) remain one of the best cell sources for cartilage, a tissue without regenerative capacity. However, MSC chondrogenesis is commonly induced through TGFβ, a pleomorphic growth factor without specificity for this lineage. Using tissue- and induced pluripotent stem cell-derived MSCs, we demonstrate an efficient and precise approach to induce chondrogenesis through Wnt/β-catenin antagonism alone without TGFβ. Compared to TGFβ, Wnt/β-catenin antagonism more rapidly induced MSC chondrogenesis without eliciting off-target lineage specification toward smooth muscle or hypertrophy; this was mediated through increasing N-cadherin levels and β-catenin interactions-key components of the adherens junctions (AJ)-and increasing cytoskeleton-mediated condensation. Validation with transcriptomic analysis of human chondrocytes compared to MSCs and osteoblasts showed significant downregulation of Wnt/β-catenin and TGFβ signaling along with upregulation of α-catenin as an upstream regulator. Our findings underscore the importance of understanding developmental pathways and structural modifications in achieving efficient MSC chondrogenesis for translational application.