Establishment and characterization of dental epithelial cell lines from human HERS/ERM and dental pulp

Abstract

Stem cells can differentiate into various cell types and develop into any tissue. Adult stem cells are present in very small populations in each tissue of the body and exist as undifferentiated cells. When tissue damage occurs, stem cells rapidly differentiate and regenerate the tissue. Despite their limited differentiation ability, adult stem cells have the advantages of being easier to obtain and having fewer associated ethical issues than embryonic stem cells. Recently, research on stem cells derived from oral tissues, including periodontal ligaments, has been reported. Although the interaction between ectodermal mesenchymal stem cells and epithelial stem cells is very important throughout the process of tooth development, there are difficulties in researching the interaction between the epithelium and mesenchyme for dental regeneration since epithelial stem cells are relatively difficult to obtain and maintain. Therefore, in this study, I established cell lines of Hertwigs epithelial root sheath cells/epithelial rests of Malassez cells (HERS/ERM cells) and dental pulp epithelial stem cells (DPESCs), and investigated the effect of conditioned medium derived from these dental epithelial stem cell lines on the hard tissue forming ability of Stem cells from human exfoliated deciduous teeth (SHEDs). SHEDs and DPESCs were isolated and cultured from deciduous teeth, and HERS/ERM cells were isolated and cultured from permanent teeth. The obtained HERS/ERM cells and DPESCs were immortalized by introducing the SV40 large T antigen. In addition, a more stable cell line was established by introducing the human telomerase reverse transcriptase (hTERT) gene into the HERS/ERM cell line established with the SV40 large T antigen. To confirm whether the established cell line maintains its dental epithelial stem cell characteristics, FACS analysis of markers of embryonic stem cells and epithelial stem cells was performed to evaluate changes in cell morphology and gene expression when the epithelial-mesenchymal transition (EMT) was induced by TGF-β1. To confirm the interactions between dental epithelial stem cells and mesenchymal stem cells in hard tissue formation, the effects of conditioned medium obtained from primary dental epithelial cells and the cell lines on the hard tissue-forming ability of mesenchymal cells were examined. The immortalized dental epithelial cell lines had an extended life-span, and their morphology was maintained throughout subculture. RT-PCR data showed that the immortalized cell lines had typical epithelial stem cell-like gene expression patterns identical to those of primary dental epithelial cells. In addition, primary dental epithelial cells and the immortalized cell lines exhibited similar characteristics when the epithelial-mesenchymal transition was induced by TGF-β1. Alizarin red S staining indicated that calcium accumulation in SHED cells was equally promoted by conditioned medium derived from primary dental epithelial cells and the immortalized cell lines. Taken together, I established dental epithelial stem cell lines and confirmed that both the primary dental epithelial stem cells and the cell lines showed the same morphological characteristics and gene expression profiles. Furthermore, it was also proven that conditioned medium from the established cell lines affects the acceleration of the hard tissue formation of primary cultured mesenchymal stem cells, SHEDs. These data suggested that HERS-SV40, HERS-SV40 / hTERT and DPESC-SV40, which were established from primary dental epithelial cells, could be expected to contribute to the study of dental stem cell functions and tooth regeneration