Single-Cell Atlas Of Human Periodontal Ligament Stem Cells

The biological characteristics of periodontal ligament stem cells (PDLSCs) vary at different developmental stages, yet the cellular heterogeneity and regulatory mechanisms are still lacking. To obtain the single-cell atlas of PDL and PDLSCs at different developmental stages, we applied single-cell RNA sequencing (scRNA-seq) on PDL of 20 immature and 20 mature human teeth. All donors signed informed consent forms, and the study was approved by the Ethics Committee. Primary analysis was performed using cell clustering to find PDLSCs cluster, with downstream analysis via sub-clustering and functional enrichment to define osteogenic PDLSCs. The SCENIC analysis provided insights into transcription factor (TF) regulatory networks across different PDLSCs subgroups. We used small interfering RNA (siRNA) to knockdown TF, the effect of TF on osteogenesis was detected by ALP staining, alizarin red staining, RT-PCR and western blot. By scRNA-seq we found the heterogeneity in cell populations of PDL and in subclusters of PDLSCs at different developmental stages, with osteogenic PDLSCs overexpressing TF RelB. After down-regulated RelB, the osteogenic differentiation potential of PDLSCs of immature and mature teeth was significantly decreased. This study provided a single-cell atlas of PDL and PDLSCs at different developmental stages, which found that PDLSCs had varying degrees of osteogenic differentiation ability at different developmental stages and RelB played a regulatory role in this process. These findings not only deepen our understanding of PDL biology, but also provide potential targets for periodontal bone regeneration.