Genetic profile of Lepr⁺ skeletal stem/progenitor cells in the periodontal ligament and functional compensation during aging and bone repair.

Abstract

Skeletal stem/progenitor cells (SSPCs) play a fundamental role in maintaining skeletal homeostasis throughout life. Leptin receptor (Lepr)-positive cells were initially identified as SSPCs in the long bone marrow and later in the craniofacial skeleton, where they localize within the periodontal ligament (PDL), particularly among fibroblastic cells (PDLCs). However, their hierarchical organization, maintenance of stemness, and relationships with other SSPC populations remain poorly understood. Through lineage tracing and single-cell RNA sequencing facilitated by Runx2-GFP expression, we identified a Lepr⁺ subpopulation characterized by Thy1 expression but lacking Runx2, which is hierarchically positioned at the apex of the Lepr⁺ PDLC lineage and gives rise to downstream osteogenic progeny. However, their SSPC capacity to contribute to osteocytes and cementocytes is quantitatively limited and declines with age, and this contribution may exhibit sexual dimorphism. At the single-cell transcriptome level, Lepr⁺ PDLCs shared an expression profile with long bone-derived Lepr⁺ SSPCs; however, the expression of Kit ligand (Kitl) was uniquely restricted to Lepr⁺ cells within the PDLCs. In contrast, they were not enriched for other PDLC-associated SSPC markers, such as Axin2, Gli1, and Acta2. Although Lepr⁺ PDLCs contributed to bone regeneration by differentiating into osteocytes after tooth extraction, their contribution was limited. Moreover, genetic ablation of Lepr⁺ PDLCs did not reduce the regenerated bone volume, suggesting compensatory contributions from other SSPC populations. Collectively, these findings reveal a previously unrecognized hierarchical and functional organization of Lepr⁺ PDLCs and demonstrate that their contribution to skeletal homeostasis progressively declines with age, with potential compensation by other SSPC populations.