A bilaminar stem cell core governs tooth establishment.

Establishment of human teeth relies on coordinated formation of dentin-pulp complex for nurturing and periodontal tissues for anchoring in alveolar bone. The exact cell origin of dentin-pulp complex and periodontal tissues, and the integrative developmental process remain elusive. Here, we identified a bilaminar core of Cd24a+ and Pax9+ stem cells that governs the tooth establishment and persists into adulthood. Specifically, Cd24a+ stem cells gave rise to the dentin-pulp complex while Pax9+ stem cells mainly generated periodontal tissues as well as partial dental pulp. DTA-mediated cell ablation of the Cd24a+/Pax9+ stem cells significantly compromised tooth establishment. Moreover, during development, the Cd24a+/Pax9+ bilaminar core concentrated on the apical region, collectively migrated and contributed to the newly formed dental root, potentially guided by PDGF-B derived from the alveolar bone. Integrated multi-omic analysis and spatial mapping further revealed lineage-associated key signaling pathways in Cd24a+/Pax9+ stem cells and the unique organization of different cell compositions. Finally, the CD24+/PAX9+ bilaminar core was also detected in human teeth at different stages, suggesting it a conserved developmental mechanism. Together, our work identified a unique bilaminar core of bona fide dental stem cells governing tooth establishment and might guide the future regenerative therapy to treat pulpitis, pulp necrosis and periodontal diseases. This study analyzed different types of dental stem cells using single-cell RNA sequencing (scRNA-seq) and H3K4me3 CUT&Tag epigenetic profiling.