Regenerative teeth induced by in vitro mesenchymal cells in mice via repressing BMP4 and activating retinoic acid/osteopontin.

Maintaining the odontogenic potential of dental mesenchymal cells (DMCs) in vitro remains a critical challenge in tooth regeneration research. Current culture systems fail to sustain DMC functionality beyond short-term periods, limiting their utility for tissue engineering applications. Here, we developed an optimized N2B27-based culture medium that preserves the odontogenic capacity of mouse DMCs (mDMCs) for up to 14 days with passaging-a significant improvement over conventional methods (≤ 24 h). Single-cell RNA sequencing (scRNA-seq) revealed distinct transcriptional profiles and cellular trajectories between traditionally cultured (FBS-based) and N2B27-cultured DMCs. Mechanistically, excessive BMP4 signaling in standard media suppressed odontogenesis, whereas elevated Spp1 (osteopontin, OPN) expression in the N2B27 system enhanced regenerative potential. We demonstrate that optimal maintenance of DMC functionality requires balanced BMP4 activity and is enhanced by high OPN levels. Notably, supplementation with recombinant OPN or all-trans retinoic acid (ATRA) partially restored tooth-forming capacity in suboptimal cultures. Our findings establish a robust in vitro platform for DMC expansion while preserving odontogenic competence, advancing both mechanistic studies of tooth development and the generation of clinically relevant cell sources for bioengineered dental tissues. This work provides key insights on the features of a regenerative tooth germ and its odontogenic microenvironment for future translational applications in tooth regeneration.