Transcriptional analysis of osteogenic and adipogenic gene markers in human periodontal ligament cells, presenting high and low osteogenic potential

Abstract

Multipotent periodontal ligament cells (PDLCs) have shown the ability to differentiate in some lineages, being good candidates to regenerative therapies. Recently, our research group have characterized PDLCs presenting distinct osteogenic potentials, showing high (h-PDLCs) or low (l-PDLCs) capacity of mineral nodules formation, in vitro. These h- and l-DLCs were submitted to the Assay for Transposase Accessible Chromatin followed by next generation sequencing and bioinformatic analysis (ATAC-Seq) (FAPESP/University of Birmingham - 2017/07944-5), aiming to identify specific open chromatin regions, which are potentially regulatory regions related to those distinct osteogenic potential observed. Interestingly, even at basal levels (without any differentiation), both h- and l-PDLCs have already demonstrated differences in several parameters analyzed, highlighting the super enhancers analysis and the signaling pathways and biological processes. The initial analysis pointed out to the adipogenic pathway as active in the l-PDLCs and the osteogenic pathways in the h-PDLCs. These results have built the basis for this IC project, aiming to confirm the ATAC-Seq results at transcript levels, i.e., whether the regulatory regions are functionally active, confirming not only the distinct regulatory background but also the distinct transcription background between those PDLCs. The PDLCs will be obtained by third molars from patients aging 18 to 20 and will be characterized as undifferentiated (flow cytometry) and distinct osteogenic (Alizarin red - 21 days) and adipogenic (Oil red O - 25 days) potential. The PDLCs will be collected after 10 days in culture (without any differentiation induction) and the RNA will be extracted and purified by the TRIzol method. The transcript levels of adipogenic (PPAR-gamma, CEBPD/B/A e KLF15) and osteogenic markers (CTNNB1, WNT10a and Collagen type 1), pointed out by the ATAC-Seq analysis, will be evaluated by real time PCR, with GAPDH, B-ACTIN or 16S as reference gene (the more stable one). The calculations will be performed according to the ””CT method.