Modulation of iPSC-derived NCMSC cell state by TD-198946 enhances scaffold-free cartilage biofabrication

Abstract Scaffold-free biofabrication has emerged as a promising strategy for cartilage repair, which may facilitate improved tissue integration while avoiding exogenous biomaterials. However, reproducible scaffold-free 3D bioprinting is strongly influenced by the robustness of the expanded cell population, particularly when induced pluripotent stem cell (iPSC)-derived neural crest mesenchymal stem cells (iNCMSCs) undergo repeated monolayer expansion. In this study, we tested whether TD-198946 priming during expansion could stabilize cell population quality and improve fabrication outcomes. TD-198946 priming supported iNCMSC expansion, as evidenced by increased cell number and MTT signal, accompanied by reduced G1 arrest and improved cell-cycle progression. These effects were reversed by the NOTCH3 signaling inhibitor DAPT, supporting the involvement of NOTCH3 as a mediator of TD-198946 activity. In parallel, TD-198946 priming increased N-cadherin expression in expanded iNCMSCs, a cell-cell adhesion molecule associated with spheroid cohesion in scaffold-free biofabrication systems. Applied to scaffold-free 3D bioprinting, TD-198946 priming led to dose-dependent increases in spheroid size, glycosaminoglycan deposition, and mechanical strength of the resulting constructs, with optimal construct quality observed at 50 nM. In contrast, excessive TD priming (100 nM) disrupted extracellular matrix production and resulted in inferior mechanical properties, highlighting the importance of dose optimization. This approach improved the robustness of the expanded iNCMSC population, thereby enhancing the consistency of scaffold-free biofabrication and construct maturation.