Establishment and Characterization of Human Ameloblast Lineage Cell Lines

Dental enamel is formed by ameloblasts, which are derived from the epithelial cells of the tooth organ. Cultured ameloblast lineage cells are difficult to grow and can only be passaged a few times. Objectives: To immortalize and characterize human ameloblast lineage cell lines at different stages of differentiation. Methods: Cells from enamel organ epithelia isolated from human fetal teeth, were selected in serum-free keratinocyte growth media. Passage 1 cells were transfected with vectors containing either human telomerase transcriptase (hTERT) or SV40 genome, and plated at low density on a 96-well plate for single cell isolation. The cells were screened for their morphological characteristics, and two types of cells, one containing primarily cobblestone-shaped cells, and the other containing cells with a ruffled border were selected. Cells transfected with SV40 were characterized by PCR and immunohistochemistry for expression of cell markers and matrix proteins present in the various stages of ameloblast differentiation. Results: Transfection with both SV40 and hTERT prolonged the life span of ameloblast lineage cells. Whereas non-transfected cells could be passaged only 3 times, hTERT transfection prolonged the cell life span for 8 passages. SV40 transfected cell lines were both passaged over 20 times, and both cell lines expressed amelogenin, AE2, DSPP, KLK4, LAMP1, MMP20, and WISP1. Amelotin, a recently identified protein detected in late stage ameloblast differentiation, was dramatically up-regulated in ruffle -bordered cells as compared to cobblestone-shaped cells. Conclusions: SV40 transfection of ameloblast lineage cells was most effective in prolonging the cell life span. The two SV40 transfected ameloblast lineage cells lines investigated in this study differed in their expression of amelotin, a marker of ameloblast differentiation. These cell lines will be useful models to better understand enamel formation.

Supported by NIH grant# R21-DE017910