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Characterization of Dental Epithelial Progenitor Cells Derived from Cervical-loop Epithelium in a Rat Lower Incisor

¹ Department of Oral Anatomy and Cell Biology, Kyushu Dental College, 2-6-1, Manazuru, Kokurakita-ku, Kitakyushu, Japan, 803-8580;
² Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan, 812-8582;
³ Department of Operative Dentistry and Endodontics, Kanagawa Dental College, 82, Inaoka-cho, Yokosuka, Japan, 238-8580; and
⁴ Department of Oral Biology, Hiroshima University, Graduate School of Biomedical Science, Kasumi 1-2-3, Minami-ku, Hiroshima, Japan, 734-8553;

View larger version (43K): [in this window] [in a new window]   Figure 1. Immunohistochemical classification of dental epithelial cells in the rat lower incisor. (A) Expression patterns of CK14, ALP, amelogenin, p75NGFR, and Notch2 in rat lower incisors. (a) Hemotoxylin/eosin staining. (b) Expression of CK14 was observed in the entire dental epithelium, but not in the mesenchymal cells. (c) ALP activity was expressed in the stratum intermedium (arrowheads) and odontoblasts (arrows). (d) Expression of amelogenin was detected in pre-ameloblasts and ameloblasts. (e) p75NGFR was strongly expressed in inner enamel epithelium (arrowheads). (f) Notch2 was expressed in the stellate reticulum and outer enamel epithelium. The region surrounded by white dots shows lingual epithelium. Asterisks show the location of the cervical loop. Bars = 200 µm. (B) Summary of the distribution of antigens in the rat incisor as shown by immunohistochemical analysis. The intensity of immunostaining is represented as follows: +weakly, ++moderately, +++strongly, and -negative.

View larger version (76K): [in this window] [in a new window]   Figure 2. The appearance of CK14, ALP, p75NGFR, amelogenin, and Notch2 in dental epithelial progenitor cells as revealed by immunostaining. (A) (a-c) CK14 and ALP double-staining. CK14 was continuously expressed in all cells, and ALP-positive cells appeared in the superficial layer (arrows). (d-f) p75NGFR and amelogenin double-staining. Some cells expressing p75NGFR also expressed amelogenin (arrows). (g-i) p75NGFR and ALP double-staining. Cells expressing p75NGFR were not identical to ALP-positive cells. (j-k) Notch2 and ALP double-staining. Many Notch2-positive cells did not express ALP, but some weak ALP-positive cells expressed Notch2 receptors. (l-m) FGFR1 and FGFR2 double-staining. FGFR1 and FGFR2 were continuously expressed in all cells. Bars = 50 µm. (B) Observation of ALP-positive cells by confocal microscope. ALP-positive cells (green) were seen as larger squamous cells in the superficial layer. Lower and light panels show the vertical pictures of the a-b line and c-d line, respevtively. Bar = 25 µm.

View larger version (32K): [in this window] [in a new window]   Figure 3. Expression of amelogenin in the differentiation of dental epithelial progenitor cells as determined at the molecular level. (A)(a) RT-PCR. (b) Immunoblot analysis. Anti-amelogenin antibody reacted with protein bands having a molecular weight of 25 kDa. Lane 1: lower cell-density. Lane 2: higher cell-density. Lane 3: enamel organ of a rat lower incisor. (B) Classification of cells based on CK14, p75NGFR, amelogenin, ALP, Notch2, FGFR1, and FGFR2 expression patterns in lower and higher cell densities. Marker protein expression was as follows: +positive and -negative.

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of Fgf10 on the proliferation and differentiation of dental epithelial progenitor cells. (A) Effect of Fgf10 on cell proliferation. In the presence of Fgf10, the growth rates of dental epithelial progenitor cells increased in a dose-dependent manner. (B) Effect of Fgf10 on the appearance of ALP-positive cells. In the presence of Fgf10, the number of ALP-positive cells increased more rapidly than in the controls. Data were expressed as the mean ± SD (n = 4). Asteriks indicate significant differences from the control value (*P < 0.05). (C) The tracings of the ALP-staining pattern time-dependently in the absence and presence of 100 ng/mL Fgf10 are shown. ALP-positive cells appeared at random in different positions, in both the presence and the absence of Fgf10.

Journal of Dental Research, Vol. 83, No. 2, 129-133 (2004)
DOI: 10.1177/154405910408300209


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