Secreted Peptidome Profiling of Human Dental Pulp Stem Cells-Conditioned Medium During Odontogenic Differentiation

<h4>Introduction and aims</h4>Odontogenic differentiation of human dental pulp stem cells (hDPSCs) is critical for pulp repair. This study aims to characterise the secretory peptidome of human dental pulp stem cells (hDPSCs) during odontogenic differentiation, thereby providing insights into potential dental pulp repair strategies.<h4>Methods</h4>We employed tandem mass tag labelling coupled with liquid chromatography-tandem mass spectrometry to compare the peptide profiles of undifferentiated and differentiated hDPSCs-conditioned medium (CM), followed by in-depth bioinformatics analysis. A Cell Counting Kit-8 assay was conducted to screen for functional peptides present in the hDPSCs-CM. 5-Ethynyl-2'-deoxyuridine staining, flow cytometry, transwell migration, alkaline phosphatase activity, and alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot analyses were performed to preliminarily investigate the functions of the selected peptide.<h4>Results</h4>A total of 90 peptides were differentially expressed in differentiated hDPSCs-CM compared with undifferentiated hDPSCs-CM. Gene Ontology analysis suggested that the differentially expressed peptides were closely associated with biological processes, including cell proliferation, growth, and reproduction. Kyoto Encyclopedia of Genes and Genomes enrichment analysis suggested that the precursor proteins of these peptides were significantly enriched in the PI3K-Akt signalling pathway, carbon metabolism, and extracellular matrix-receptor interactions. Furthermore, we identified a secreted peptide that promotes the proliferation, migration, and odontogenic differentiation of hDPSCs and named it P1DC (peptide 1 derived from CFL1 protein).<h4>Conclusions</h4>Our study is the first to identify differentially expressed peptides between undifferentiated and differentiated hDPSCs-CM. Furthermore, we discovered a novel secreted peptide, P1DC, which significantly enhances hDPSC function.