# Epigenetic Signatures of Dental Stem Cells: Insights into DNA Methylation and Noncoding RNAs
## 4 Discussion  
In this study, a comprehensive epigenomic analysis of three dental stem cell populations--dental pulp stem cells (DPSCs), dental follicle progenitor cells (DFPCs), and periodontal ligament stem cells (PDLSCs)--was conducted, by interrogating publicly available DNA methylation data profiles [8]. Unsupervised hierarchical clustering was based on 17,175 highly variable probes and disclosed the occurrence of six distinct clusters that clearly differentiate the three cell types and identify specific methylation profiles that reflect their developmental origin and functional role. Each cluster was characterized by a specific methylation signature across the three samples. For example, DPSCs displayed hypermethylation in specific clusters (Clusters 3 and 4) and hypomethyllation in others (Clusters 1 and 5), suggesting repression of some developmental pathways while maintaining accessibility in regions potentially relevant to neural or mesenchymal differentiation. DFPCs displayed hypermethylation in two clusters (Clusters 5 and 6), while hypomethyllation in two other different clusters (Cluster 2 and slightly in Cluster 4), indicating distinct regulatory constraints that are potentially linked to their embryonic follicular identity. Conversely, PDLSCs showed hypermethylation in the first two clusters (Clusters 1 and 2) and hypomethyllation in two other clusters (Clusters 3 and 6), which aligns with their periodontal niche and their potential role in structural remodeling and mechanical response. The analysis of the distribution of methylation probes across genomic regions revealed consistent and functionally relevant patterns. Cluster 2, for instance, was highly enriched  
Figure 6: **Gene-concept network. ** Gene-concept network showing protein-coding genes (blue nodes), noncoding RNAs (red nodes), and GO terms (yellow nodes). Edges link noncoding RNAs with protein-coding genes that share promoters, as well as protein-coding genes with enriched BP terms. in CpG islands, typically located in gene promoters, suggesting a role in canonical transcriptional regulation. Other clusters were preferentially distributed in CpG shores, regions known to undergo dynamic methylation during development [30]. These patterns suggest that epigenetic regulation in dental stem cells is region-specific and modulates key loci involved in differentiation and tissue-specific functions. Furthermore, the gene region analysis disclosed a predominant occurrence of methylated CpGs in gene bodies in all clusters, with lower representation in promoters. This finding is consistent with prior evidence showing that gene body methylation may support transcriptional elongation and tissue-specific expression patterns through alternative splicing [31]. This suggests a potential role for epigenetic regulation in shaping isoform diversity during odontogenesis. Gene Ontology and KEGG pathway enrichment analyses provided functional insights into the biological roles of each cluster. The clusters hypomethyllated in DPSCs (e.g., Cluster 1) were enriched in neurogenesis, axon guidance, and Wnt signaling, which reflects the neurogenic potential of pulp-derived cells [32; 33]. The clusters hypomethyllated in PDLSCs (e.g., Cluster 3) instead showed enrichment in processes related to ECM remodeling, actin cytoskeleton, and MAPK signaling, all pathways being relevant to mechanotransduction and periodontal ligament function [34; 35]. DEFPCs' hypomethyllated Cluster 2 was associated with embryonic organ development, canonical Wnt signaling, and odontogenic pathways, consistent with their involvement in tooth root formation and follicular development [1; 36]. Clusters with high hypermethylation in DPSCs, such as Cluster 4, showed suppression of PI3K-Akt and BMP signaling, suggesting the downregulation of pathways that could otherwise favor alternate differentiation trajectories.