# Epigenetic Signatures of Dental Stem Cells: Insights into DNA Methylation and Noncoding RNAs
## 3 Results
### Gene Ontology and Pathway Analyses  
Gene Ontology (GO) and KEGG pathway analyses enabled the identification of different biological processes associated with the different clusters, highlighting functional differences among the 3 cell lines, reflecting their embryological origins and differentiation potentials. Cluster 1 (Table S1) displays hypomethyllation in DPSCs, intermediate methylation in DFPCs, and hypermethylation in PDLSCs. This cluster contains genes involved in neurogenesis and neural development, multiorgan embryonic morphogenesis, cell adhesion and cytoskeleton organization, cell signaling (Wnt, PI3K-Akt, and TGF-\(\beta\)), mesenchymal, osteogenic, and chondrogenic differentiation. Key pathways include cell signaling pathways (as MAPK, Wnt, and TGF-\(\beta\) signaling pathways) that regulate critical cellular functions such as growth, differentiation, survival, and migration, cancer-related pathways, as well as  
Figure 1: **Heatmap of methylation beta values for top elbow-defined probes, grouped in six clusters. ** Each row represents a CpG probe, and each column corresponds to a sample. Rows are clustered hierarchically based on methylation patterns. Beta values were row-scaled (z-score normalization), and colors represent relative methylation levels: blue indicates hypomethylation, white indicates intermediate levels, and red indicates hypermethylation. DPSCs = dental pulp stem cells, DFPCs = dental follicle progenitor cells, PDLSCs = periodontal ligament stem cells. cytoskeletal and adhesion mechanisms, regulating cellular shape, motility, and interactions with the extracellular matrix, all crucial for morphogenesis and differentiation, and already known or suggested to play a role in odontogenesis. Cluster 2 (Table S2) shows intermediate methylation in DPSCs, hypomethyllation in DPPCs, and hypermethylation in PDLSCs. The most representative biological processes highlight a strong convergence around embryonic morphogenesis, including Wnt signaling regulation, renal and neural development, and mesenchymal differentiation. Of note, several processes directly pertain to odontogenesis. The pathway analysis disclosed the occurrence of developmental and morphogenetic pathways (e.g., Wnt, Hippo, Adherens junctions), regulating tissue patterning, growth control, and morphogenetic processes in multicellular organisms; signal transduction and hormonal pathways (e.g., cAMP, hormonal pathways), which mediate hormone-driven cellular responses and metabolic regulation; and cancer-associated pathways, which reflect the dysregulation of core signaling networks such as Wnt, MAPK, PI3K-Akt, and TGF-\(\beta\), common in both oncogenesis and development. Cluster 3 (Table S3) is characterized by hypermethylation in DPSCs, intermediate methylation in DEFPCs, and hypomethyllation in PDLSCs, disclosing an opposite pattern compared to Cluster 1. The main biological processes include cytoskeletal dynamics and cell morphogenesis, developmental processes and organogenesis, signal transduction and cellular communication, as well as tissue repair. Pathways span crucial domains of development, metabolism, immunity, and disease. They include signal transduction pathways (e.g., PI3K-Akt, MAPK, mTOR, Wnt, Rap1, Ras, Hippo), which regulate cell proliferation, survival, metabolism, stress response, and plasticity; pathways involved in cell architecture and adhesion, which regulate structural organization and interactions with the cell environment; morphogenesis, hormonal, and endocrine pathways, which are involved in hormonal control and systemic homeostasis; and pathways regulating the pluripotency of stem cells. Cluster 4 (Table S4) shows hypermethylation in DPSCs, slight hypomethyllation in DEFPCs, and intermediate levels in PDLSCs. The biological processes span several functional categories, including embryonic development and morphogenesis, Wnt and BMP signaling regulation, cell migration and adhesion, neurodevelopmental processes, and growth and differentiation.