Transcriptome alteration of cultured dental mesenchymal cells

Dental mesenchymal cells (dMCs) differentiate into dentin-secreting odontoblasts and are utilized in the majority of tooth regeneration studies to produce an entire tooth organ. An in vitro culture platform of dMCs is useful in generating an adequate quantity of cells for experiments and regenerative therapy. However, culturing techniques may induce functional modification in dMCs. Here, we examined the transcriptomic alterations induced by in vitro culture and investigated the biological properties of cultured dMCs. Hierarchical clustering analysis and principal components analysis reveal an overall transcriptional similarity between the first and second passages that differed significantly from freshly obtained cells. Following in vitro culture, the biological processes of cell adhesion, chromatin organization, cell differentiation and development are significantly altered. Components of signaling pathways, including p53, transforming group factor ß/bone morphogenetic protein (TGFß/BMP), Hedgehog, and Wnt, as well epigenetic regulators are involved. Furthermore, the osteogenic potential of cultured dMCs is also impaired. Our study provides valuable insights for the modification of the current culturing methods to maintain the characteristics of in vivo dMCs. Overall design: To determine the transcriptional regulation following in vitro culture, dMCs from developing molar germs in embryonic day 14.5 (E14.5) mouse embryos were isolated and designated as P0. The cells were then subcultured in standard medium and passaged once they reached 90% confluence, with the first-passage culture designated as P1 and the second-passage as P2. Illumina-based high-throughput sequencing (Illumina, San Diego, CA, USA) was used to obtain the transcriptome profiles of P0, P1 and P2 cells