Newly established cell lines from mouse oral epithelium regenerate teeth when combined with dental mesenchyme

Abstract

The present study attempted to examine whether clonal cell lines of the oral epithelium can differentiate into ameloblasts and regenerate tooth when combined with dental germ mesenchyme. Clonal cell lines with a distinct morphology were established from the oral epithelium of p53-deficient fetal mice at embryonic day 18 (E18). The strain of mouse is shown to be a useful source for establishing clonal and immortalized cell lines from various tissues and at various stages of development. Tooth morphogenesis is almost completed and the oral epithelium is segregated from the dental epithelium at E18. In RT-PCR analysis of cell lines, mucosal epithelial markers (cytokeratin 14) were detected, but ameloblast markers such as amelogenin and ameloblastin were not detected when cells were cultured on plastic dish. They formed stratified epithelia and expressed a specific differentiation marker (CK13) in the upper layer when cultured on feeder layer or on collagen gel for 1-3 wk, demonstrating that they are of oral mucosa origin. Next, bioengineered tooth germs were prepared with cell lines and fetal molar mesenchymal tissues and implanted under kidney capsule for 2-3 wk. Five among six cell lines regenerated calcified structures as seen in natural tooth. Our results indicate that some oral epithelial cells at E18 possess the capability to differentiate into ameloblasts. Furthermore, cell lines established in the present study are useful models to study processes in tooth organogenesis and tooth regeneration.

Figure 1

Morphology of clonal cell lines. foec-1 cells (a), foec-2 cells (b), foec-3 cells (c), foec-4 cells (d), foec-5 cells (e), foec-6 cells (f), foec-7 cells (g), and foec-8 cells (h). They had a distinct morphology when cultured on plastic dish in 10%FCS supplemented with transferrin, insulin, and cholera toxin. Inserts were at high density. Bar, 50 µm.

Figure 2

Immunochemical analyses of foec lines. Immunocytochemistry of epithelial and mesenchymal marker proteins in foec-2 line (A). Vimentin (1), CK10 (2), CK13 (3), and CK18 (5) were not detected. CK14 (4) and p63 (6) were detected. Nuclei were stained with DAPI in (6). Bar, 100 µm. Western blotting (B) detected CK14 in all lines. CK18 was weakly detected only in foec-7 line. Vimentin was not detected in any lines. Tongue was used as control.

Figure 3

RT-PCR analyses of gene expression. Expressions of undifferentiated epithelial marker genes (A) were observed in all lines examined. emtg-3 line is a dental epithelial line. Oral epithelium (E18) was used as control. Expression of tissue-specific epithelial marker genes was examined (B). mBD1, a mucosal epithelium-specific gene, was detected in foec-2, -3, -6, -7, and -8 lines, but not in foec-5 and ameloblasts prepared from incisors. Ameloblast-specific genes (amelogenin and ameloblastin) were undetected in any of oral cell lines. Plexin expression was examined (C). Plxna1 was detected in all cell lines. Plxna2 and plxna3 were undetected in emtg-3. Plxna4 was undetected in any cell lines. Control was prepared from fetal and adult brains.

Figure 4

Immunohistochemistry of fetal oral mucosa (E18.5). The basal layer of stratified epithelia was positive for CK14 (1). CK13 was detected in stratified epithelia except the basal layer (2). p63 was strongly detected at the basal layer and immunoreactivity became weaker toward to the surface (3). β1-integrin was detected at the basement membrane along the dotted line (4). (5) was H-E staining. OC oral cavity, OE oral epithelium, t tongue. Bar, 50 μm.

Figure 5

Reconstructed oral epithelia on feeder layer. foec-5 line formed epithelia with 2-3 cell layers (a, H–E staining). CK13 was detected only in cells at the surface (b). CK14 was detected in all cells (c). p63 protein was detected in cuboidal cells at the basal layer (d). Nuclei were stained with DAPI. Bar, 20 µm.

Figure 6

Reconstructed oral epithelia on collagen gel. Top panel is of foec-8 line and bottom panel is of foec-6 line. Cells were cuboidal at the basal layer, and cell shape became flat toward the surface (a, e H–E staining). CK13 was strongly detected in surface layers of foec-8 (b) and foec-6 lines (f). CK14 was detected in all cells in layers of foec-8 (c) and foec-6 (g) lines. β1-integrin was strongly expressed in basal cells of foec-8 (d) and foec-6 (h) lines. Nuclei were stained with DAPI. Bar, 50 µm.

Figure 7

Regenerated teeth with oral epithelial cell lines and dental mesenchyme. Calcified teeth were regenerated from germs prepared with dental mesenchyme and foec-7 (1) and foec-5 cells (2). A rectangle in (2) was enlarged in (3). Ameloblasts regularly lined up along with calcified tissue (3). Amelogenin was detected in differentiated ameloblasts and residues of enamel in a section near (1) (4). A rectangle in (4) was enlarged in (5). Keratinized tissues developed from germs prepared with dental mesenchyme and foec-2 cells (6). Osteogenic tissues developed from germs prepared with dental mesenchyme and foec-6 cells (7). a ameloblast, d dentin, e enamel, k kidney, o odontoblast, p dental pulp. Black bar, 200 µm; white bar, 50 µm.

Figure 8

Ameloblasts were differentiated from implanted oral epithelial cells. Tooth germs were prepared with mesenchyme and GFP-expressing foec-5 cells. GFP-expressing foec-5 cells were found around the calcified tissues and differentiated into amelogenin positive ameloblasts. (1) was a whole image of a regenerated tooth. (2) and (3) were immunohistochemistry of amelogenin at high magnification of areas in rectangles in (1). a ameloblast, d dentin, e enamel, k kidney, p dental pulp. green GFP, red amelogenin. Bar, 100 µm.