Spatiotemporal expression dynamics of CD73, CD90, and CD105 based on scRNA-seq and immunofluorescence analysis of dental pulp toward establishing a triple-positive population.

Dental pulp stem cells (DPSCs), a subset of mesenchymal stem/progenitor cells, exhibit remarkable self-renewal and multipotent differentiation capacity, making them promising candidates for regenerative applications. While their in vitro characteristics have been extensively studied, the in vivo dynamics and molecular regulation during development are much less known. This study examined the embryonic/prenatal (E16.5) and early postnatal (P3.5 and P7.5) stages of mouse molar development to trace the temporospatial expression and overlap of five key molecules associated with DPSC: a trio of positive markers (CD73/Nt5e, CD90/Thy1, and CD105/Eng) and two negative markers (CD34/Cd34 and CD45/Ptprc). Reanalysis of single-cell RNA sequencing data revealed a triple-positive and double-negative population overlapping in the dental mesenchyme by P7.5. The results were confirmed by multiple immunofluorescence-based in situ labeling. These findings contribute to the understanding of the spatiotemporal in vivo expression landscape and dynamics of DPSC markers, thus bridging the gap between in vitro characterization and the complex developmental reality of the dental pulp.