In Vitro Single-Cell Transcriptomic Profiling of Cultured Stem Cells From Apical Papilla and Dental Pulp Stem Cells: Unveiling Cellular Heterogeneity

Dental-derived mesenchymal stem cells show considerable variability in their differentiation potential due to the use of nonspecific surface markers and technical limitations in isolation protocols. This study aimed to employ single-cell RNA sequencing to compare the cellular composition of cultured stem cells from apical papilla (SCAPs) and dental pulp (DPSCs), with the goal of detecting subpopulations underlying their divergent regenerative behaviour and identifying markers that can facilitate future isolation and functional targeting. SCAP and DPSC tissues were obtained from 3 human donors and cultured to passage 2. Single-cell suspensions were sequenced to generate gene expression profiles. Dimensionality reduction and clustering were performed using the Seurat package and visualised using uniform manifold approximation and projection. Cluster-specific differential gene expression was computed as log2 fold change, followed by gene set enrichment analysis. Pseudotime trajectory analysis was used to map lineage progression based on transcriptional gene expression. Transcriptomic analysis identified 12 distinct clusters shared across DPSC and SCAP cultures. While both cell types contributed comparably to overall biological processes, key differences emerged within specific clusters. Clusters 3, 4, 5, 6, and 9 expressed high levels of proliferative markers ( MKI67, TOP2A, and TYMS ), suggesting active proliferating populations. Cluster 9 was notable for the coexpression of pericyte-associated markers (NOTCH3, PDGFRB ) alongside canonical mesenchymal stromal cell markers ( MCAM, THY1, DCN ), identifying a previously uncharacterised progenitor-like subset. NOTCH3 and PDGFRB were also present in a more mature fibroblast-enriched population in cluster 7, dominated by collagen-related genes. IGFBP3 and IGFBP5 were selectively enriched in SCAP-derived clusters 7 and 9, whereas IGFBP4, 6 , and 7 were expressed across both DPSC and SCAP populations. Clusters 10 and 11, primarily derived from DPSCs, were enriched in stress-response, heat shock, and apoptotic genes, which may reflect culture-induced adaptations. Pseudotime trajectory inference positioned cluster 9 at a putative progenitor-like node; however, this represents a hypothesis-generating model based on transcriptional similarity in cultured cells rather than validated lineage relationships. This study provides a high-resolution single-cell transcriptomic comparison of cultured SCAPs and DPSCs, revealing distinct transcriptional profiles and cellular heterogeneity. SCAPs may harbour a broader spectrum of proliferative progenitors under these culture conditions, particularly in clusters 7 and 9, enriched in IGF-, PDGFRB-, and NOTCH-associated genes. These findings generate testable hypotheses regarding subpopulation-specific regenerative roles. Future studies using prospective isolation based on NOTCH3 + /PDGFRB + and functional in vitro and in vivo validation are needed to establish their regenerative potential and translational relevance.