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. Overall design: This study analyzed different types of dental stem cells using single-cell RNA sequencing (scRNA-seq) and H3K4me3 CUT&Tag epigenetic profiling.