Epigenetic regulation of the osteogenic potential in mesenchymal stem cells derived from periodontal ligament

Author(s)

Rahyza Inacio Freire de Assis

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2021-12-07

Examining board members

Denise Carleto Andia; Ricardo Della Coletta; Rodrigo Augusto da Silva; Márcio Mateus Beloti; Renato Assis Machado

• Advisor: Denise Carleto Andia

Abstract

Human mesenchymal stem cells from periodontal ligament (PDLCs) are candidates for cellular therapy and tissue regeneration. However, the individual epigenetic profile might influence the osteogenic potential acquisition, causing heterogeneity during cell differentiation. Therefore, PDLCs collected from different donors may present distinct impairments to the osteoblastic phenotype, impacting on clinical outcomes. Transcription factors (TFs), chromatin accessibility and epigenetic regulation are associated with osteogenic regulation; however, epigenetic regulation of PDLCs remains unknow. Genome wide methodologies, such as DNA (hydroxy)methylome and assay for transposase accessible chromatin followed by sequencing (ATAC-seq) are tools for epigenetic analysis to identify types and functional regulatory regions of genome, indicating which TFs are associated to regions with open and accessible chromatin. On the other hand, the transcriptome provides a map of gene expression, informing how, what and whether epigenetic mechanisms modulate the gene expression. Epigenetic modulators can influence patterns of gene expression and cell fate commitment, increasing the capacity of mineral nodules deposition, in vitro. Initially, we used genome wide methodologies to identify how epigenetic and transcriptional regulations affect the osteoblast phenotype acquisition, comparing PDLCs with low and high osteogenic potential (l-PDLCs and h-PDLCs, respectively), at basal levels (DMEM – without induction) and during osteogenic differentiation (OM – 10 days), in vitro. Results showed distinct genomic accessibility and transcriptional maps between l-PDLCs and h-PDLCs at both time points. While h-PDLCs expressed more TFs related to osteoblast commitment, facilitating the expression of osteoblastic genes and deposition of mineral nodules, l-PDLCs showed DNA hypermethylation in genes that can delay the osteoblast differentiation, confirmed by late activation of genes and biological processes showed by h-PDLCs at basal levels already. Moreover, a unique set of TFs was observed in each PDLCs could distinctly contribute to the osteoblast phenotype commitment. In sequence, RG108, an inhibitor of enzymes responsible for methylation of DNA, was used to modulate the phenotype commitment and enhance the osteogenic potential in PDLCs. Analysis of DNA methylation, gene expression and protein levels were performed at basal (DMEM – without induction) and at osteogenic differentiation (OM – 3 days), in vitro. Results showed l-PDLCS and h-PDLCs have distinct levels of gene expression of epigenetic enzymes, multipotential and osteogenic markers, at basal levels. After treatment with RG108 and osteogenic induction, the RUNX2 promoter was demethylated, increasing its gene expression and protein at cell nucleus. RG108 triggered changes in gene expression of enzymes that control DNA methylation, multipotential and osteogenic markers, leading to an increase in the mineralization capacity of l-PDLCs, in vitro. In conclusion, we contributed to the advance of knowledge of epigenetic regulation in PDLCs, highlighting the role of DNA methylation, chromatin accessibility, genes transcription and protein levels in supporting the development of osteogenic potential of PDLCs, opening a new avenue for therapeutic modulation in future studies and application of PDLCs in regenerative procedures (AU)

• FAPESP's process: 17/12158-9 - Epigenetic regulation of osteogenic potential in mesenchymal stem cells derived from periodontal ligament
• Grantee: Rahyza Inacio Freire de Assis
• Support Opportunities: Scholarships in Brazil - Doctorate