Osteogenic Commitment of Human Periodontal Ligament Cells Is Predetermined by Methylation, Chromatin Accessibility and Expression of Key Transcription Factors

Periodontal ligament stem cells (PDLCs) can be used as a valuable source in cell therapies to regenerate bone tissue. However, the potential therapeutic outcomes are unpredictable due to PDLCs' heterogeneity regarding the capacity for osteoblast differentiation and mineral nodules production. Here, we identify epigenetic (DNA (hydroxy)methylation), chromatin (ATAC-seq) and transcriptional (RNA-seq) differences between PDLCs presenting with low (l) and high (h) osteogenic potential. The primary cell populations were investigated at basal state (cultured in DMEM) and after 10 days of osteogenic stimulation (OM). At a basal state, the expression of transcription factors (TFs) and the presence of gene regulatory regions related to osteogenesis were detected in h-PDLCs in contrast to neuronal differentiation prevalent in l-PDLCs. These differences were also observed under stimulated conditions, with genes and biological processes associated with osteoblast phenotype activated more in h-PDLCs. Importantly, even after the induction, l-PDLCs showed hypermethylation and low expression of genes related to bone development. Furthermore, the analysis of TFs motifs combined with TFs expression suggested the relevance of SP1, SP7 and DLX4 regulation in h-PDLCs, while motifs for SIX and OLIG2 TFs were uniquely enriched in l-PDLCs. Additional analysis including a second l-PDLC population indicated that the high expression of OCT4, SIX3 and PPARG TFs could be predictive of low osteogenic commitment. In summary, several biological processes related to osteoblast commitment were activated in h-PDLCs from the onset, while l-PDLCs showed delay in the activation of the osteoblastic program, restricted by the persistent methylation of gene related to bone development. These processes are pre-determined by distinguishable epigenetic and transcriptional patterns, the recognition of which could help in selection of PDLCs with pre-osteoblastic phenotype.