Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.
Human bone marrow-derived mesenchymal stem cells (hBMSCs) are important for tissue regeneration but their epigenetic regulation is not well understood. Here we investigate the ability of a non-nucleoside DNA methylation inhibitor, RG108 to induce epigenetic changes at both global and gene-specific levels in order to enhance mesenchymal cell markers, in hBMSCs. hBMSCs were treated with complete culture medium, 50 μM RG108 and DMSO for three days and subjected to viability and apoptosis assays, global and gene-specific methylation/hydroxymethylation, transcript levels’ analysis of epigenetic machinery enzymes and multipotency markers, protein activities of DNMTs and TETs, immunofluorescence staining and western blot analysis for NANOG and OCT4 and flow cytometry for CD105. The RG108, when used at 50 μM, did not affect the viability, apoptosis and proliferation rates of hBMSCs or hydroxymethylation global levels while leading to 75% decrease in DNMTs activity and 42% loss of global DNA methylation levels. In addition, DNMT1 was significantly downregulated while TET1 was upregulated, potentially contributing to the substantial loss of methylation observed. Most importantly, the mesenchymal cell markers CD105, NANOG and OCT4 were upregulated being NANOG and OCT4 epigenetically modulated by RG108, at their gene promoters. We propose that RG108 could be used for epigenetic modulation, promoting epigenetic activation of NANOG and OCT4, without affecting the viability of hBMSCs. DMSO can be considered a modulator of epigenetic machinery enzymes, although with milder effect compared to RG108.
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.
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