A single-cell transcriptomic atlas of human stem cells from apical papilla during the committed differentiation

Human stem cells derived from the apical papilla (SCAPs) are recognized for their multilineage differentiation potential and their capacity for functional tooth root regeneration. However, the molecular mechanisms underlying odonto/osteogenic differentiation remain largely unexplored. In this study, we utilized single-cell RNA sequencing (scRNA-seq) to conduct an in-depth analysis of the transcriptional changes associated with chemically induced osteogenesis in SCAPs.

scRNA-seq identified SCAPs as distinct subpopulations. Differentially expressed genes (DEGs) and Gene Ontology (GO) analyses were conducted to evaluate the potential function of each cluster. Pseudotime trajectory analysis was employed to elucidate the potential differentiation processes of the identified SCAP populations. To investigate the osteo/odontogenic potential of Deiodinase Iodothyronine Type 2 (DIO2) on SCAPs, we performed alkaline phosphatase staining, western blot analysis, Alizarin Red S staining and immunofluorescence staining. Additionally, SCAP components were transplanted into mouse calvarial defects to evaluate osteogenesis in vivo.

The analysis of cell clusters derived from our scRNA-seq data revealed a significant shift in cellular composition when cells were cultured in a mineralization induction medium compared to those cultured in a complete medium. Both groups exhibited heterogeneity, with some cells intrinsically predisposed to osteogenesis and others appearing to be primed for proliferative functions. Notably, we identified a subpopulation characterized by high expression of DIO2, which exhibited pronounced osteogenic activity during differentiation.

Our study is the first to reveal a shift in the cellular composition of SCAPs when cultured in a mineralization induction medium compared to a complete medium. Following both in vitro and in vivo validation, the DIO2⁺ subpopulation exhibited the highest transcriptional similarity to osteogenic function, suggesting its potential utility in tissue regeneration applications.