Low and high osteogenic potential of periodontal ligament-derived mesenchymal stem cells correlates with RUNX2, SP7, EZH2 and KDM6B expression

Periodontal ligament-derived mesenchymal stem cells (PDLCs) possess selfrenewal and multilineage differentiation capacities, supporting their application in regenerative therapies. However, PDLCs are regulated by epigenetic mechanisms that influence chromatin accessibility and gene expression, and may exhibit distinct phenotypes under osteogenic conditions. This study aimed to investigate whether differences in osteogenic potential of PDLCs (h-PDLCs and l-PDLCs, high and low, respectively) correlate with the expression of key transcription factors and epigenetic regulators. We performed qPCR for mRNA expression analysis, immunofluorescence, and Western blotting for protein levels, as well as chromatin immunoprecipitation followed by qRT-PCR to investigate H3K27me3 enrichment at the RUNX2 and SP7 promoter regions in PDLCs cultured under basal conditions (DMEM, T0) and after 3 and 10 days of osteogenic induction (T3 and T10). At baseline, h-PDLCs and l-PDLCs exhibited distinct transcriptional and protein profiles. During osteogenic stimulation, EZH2 expression significantly increased in l-PDLCs but decreased in h-PDLCs (p < 0.001), while KDM6B expression decreased in l-PDLCs and increased in hPDLCs (p < 0.0001). Analysis of RUNX2, RUNX2p57, and SP7 revealed dynamic transcriptional modulation: RUNX2p57 and SP7 were downregulated in l-PDLCs (p < 0,001), whereas RUNX2 was upregulated in h-PDLCs (T3 and T10, p < 0,0001). Furthermore, l-PDLCs exhibited greater accumulation of the repressive histone mark H3K27me3, with significant enrichment at the RUNX2 and SP7 promoter regions compared to h-PDLCs during osteogenic induction (p < 0.05). Together, these findings demonstrate that low and high osteogenic potential of PDLCs correlate with coordinated differences in the expression of the transcription factors RUNX2 and SP7 and the epigenetic regulators EZH2 and KDM6B, as well as with H3K27me3 enrichment patterns.