Characterization of Putative Amelogenic Cells Isolated from Human Dental Follicle

Objective: Human amelogenic cells are not well characterized, because they disappear after tooth eruption. This study aimed to isolate putative amelogenic cells from human dental follicle tissue, and characterize them from a point of epithelial-mesenchymal interaction. Methods: Human impacted third molars were collected under approved guidelines and informed consent. Dental follicle tissue was detached from extracted teeth, and cells were isolated under a preferential growth condition for epithelial cells. Proliferative capacity was analyzed by staining BrdU-incorporated cells, and calculating a cumulative population doubling (PD). Gene expression profile of dental follicle epithelial (DFE) cells was examined by RT-PCR. Quantitative RT-PCR was used to evaluate the differentiation of DFE cells when cultured with dental mesenchymal (DM) cells. Gingival epithelial (GE) cells were used as a control. Results: Isolated epithelial cells formed colonies and showed a typical polygonal shape. BrdU positive cells were observed at the outer edge of each colony. DFE cells proliferated until 16 PD, which is much higher than GE cells (8 PD). Gene expression of epithelial markers, including cytokeratin 14 and E-cadherin, was positive in both DFE and GE cells. DFE cells were positive for amelogenin, while GE cells not. The expression level of amelogenin in DFE cells cultured for 5 days was increased to 2.1 fold compared to that cultured for 1 day. Very interestingly, amelogenin expression in DFE cells was further increased to 5.3 fold when co-cultured in a direct contact with DM cells for 5 days. However, this increase was not observed when DFE and DM cells were co-cultured without a direct contact. Conclusion: These results suggest that amelogenin-positive putative amelogenic cells can be isolated from the dental follicle tissue, and these cells can significantly differentiate through a direct contact with dental mesenchymal cells. Supported in part by JSPS Grant (#17209062 and 19689038).