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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;

⁵ corresponding author, present address, Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka, Japan 565-0871, hide-h{at}dent.osaka-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Dental epithelial progenitor cells differentiate into various cell types during development of tooth germs. To study this mechanism, we produced immortalized dental epithelial progenitor cells derived from the cervical-loop epithelium of a rat lower incisor. The expression patterns of cytokeratin 14, nerve growth factor receptor p75, amelogenin, Notch2, and alkaline phosphatase were examined by immnohistochemistry in both lower and higher cell densities. The patterns of each were compared in the dental epithelium of rat lower incisors. The results demonstrated that these cells could produce ameloblast lineage cells, stratum intermedium cells, stellate reticulum, and outer enamel epithelium. Furthermore, fibroblast growth factor 10 stimulated proliferation of dental progenitor cells and subsequently increased the number of cells expressing alkaline phosphatase. These results suggest that fibroblast growth factor 10 plays a role in coupling mitogenesis of the cervical-loop cells and the production of stratum intermedium cells in rat incisors.

KEY WORDS: progenitor cells • ameloblasts • Fgf10 • stratum intermedium • rat incisor

	INTRODUCTION

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Dental epithelial cells in tooth buds differentiate into various cell types during development of tooth germs—inner enamel epithelium, stratum intermedium, stellate reticulum, and outer enamel epithelium. The inner enamel epithelial cells subsequently differentiate into ameloblasts, which then form enamel. The mechanisms responsible for this cellular differentiation are not completely understood.

In vitro experiments are crucial to our understanding of the mechanism of dental epithelial differentiation. Establishment of ameloblast primary culture systems (Limeback, 1987; Kukita et al., 1992; DenBesten et al., 1998; Matsumura et al., 1998) and cell lines of ameloblast-like cells (Chen et al., 1992; DenBesten et al., 1999) has been previously reported. However, these systems did not allow the process of dental epithelial differentiation to be observed, since these cell cultures were a mixture of various dental epithelial cells and/or contained cells expressing amelogenin at the outset. In the current study, we focused on progenitor cells in the cervical-loop epithelium of continuously growing rodent incisors (Harada et al., 1999, 2002b) and attempted to immortalize these cells.

Genes or proteins expressed by the developing enamel organ can serve as indicators of the progression of dental epithelial cell differentiation. Cytokeratin 14 (CK14) is expressed only in the dental epithelium, and not in the mesenchyme of the tooth germ or any of the surrounding tissues in the mandible (Tabata et al., 1996). Nerve growth factor receptor p75 (p75NGFR) is distributed only in the inner enamel epithelium and dental mesenchymal cells (Mitsiadis et al., 1992, 1993; Luukko et al., 1996). Stratum intermedium cells show exceptionally high activity of the enzyme alkaline phosphotase (ALP) (Wise and Fan, 1989). Amelogenin is the major enamel protein produced by ameloblasts (Termine et al., 1980) but is not a specific marker for ameloblasts. Recent studies show that odontoblasts also express amelogenin in a highly restricted developmental-dependent pattern associated with early mantle dentin formation (Oida et al., 2002; Papagerakis et al., 2003). Notch2 mRNA is expressed in the stellate reticulum and outer enamel epithelium (Harada et al., 1999). Here, we characterized the dental epithelial progenitor cells by the combination of these markers.

	MATERIALS & METHODS

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Cell Culture and Immortalization of Dental Epithelial Progenitor Cells
All cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C, and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 units/mL). To culture dental epithelial cells, we used lower incisor tooth germs dissected from the mandibles of six-day-old Sprague-Dawley rats. Both the labial and lingual epithelia were mechanically stripped from the incisor tooth germs, leaving only cervical-loop cells in contact with dental papilla. The cervical-loop epithelium was dissected from the neighboring mesenchyme with 2% collagenase, and seeded onto culture dishes. Initially, small epithelial colonies were seen among the spreading mesenchymal cells. We maintained this primary culture without passage for about 3 mos, changing the culture medium every 3 days. During this time, the majority of epithelial and mesenchymal cells underwent cell death. A month later, a small number of surviving epithelial cells showed slow growth and formed colonies. This cycle of events was repeated once during a one-year period. Subsequently, the culture was enriched for continuously dividing and small polygonal epithelial cells. In the absence of media selective for epithelial cells, the majority of the mesenchymal cells disappeared. After more than 20 passages over a six-month period, single-cell cloning was carried out by limited dilution. A thousand cells were seeded onto a 10-mm culture dish. Two wks later, a colony of small polygonal cells, grown from a single cell, was selected by means of a cloning cylinder and was subcultured. This procedure was performed three times. This was maintained for 200 population doublings and more than 30 passages (more than 3 yrs). The passages were carried out before the culture became confluent, and the cells were maintained as undifferentiated cells in the condition of lower cell density. All animals were treated according to acceptable ethical standards, in compliance with the regulations for the use of experimental animals as established by the Animal Care and Use Committee of Kyushu Dental College.

Immunohistochemistry
The lower incisors were carefully dissected from the mandibles of day-old rats and immediately frozen. Sections (8 µm) were cut and fixed with 4% PFA. The primary antibodies used were anti-CK14 monoclonal antibody (1:400, NOVOCASTRA, Newcastle, UK), anti-p75NGFR (1:200, Boehringer Mannheim, Mannheim, Germany), anti-amelogenin polyclonal antibody (1:5000) (Uchida et al., 1991), anti-Notch2 polyclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-fibroblast growth factor receptor 1(FGFR1) polyclonal antibody (1:200, Santa Cruz Biotechnology), and anti-FGFR2 monoclonal antibody (1:300, Santa Cruz Biotechnology). As secondary antibodies, Alexa fluor^TM 488 (1:200, Molecular Probes, Eugene, OR, USA) and Alexa fluor^TM 546 (1:200, Molecular Probes) were used. We determined the specificity of immunoreactivity by substituting buffer for the primary antibody.

Cell Proliferation Assay
Five hundred cells were inoculated onto 96-well microtiter plates containing 100 µL medium (DMEM/F12, 10% FBS) per well, and recombinant human Fgf10 protein (R&D, Minneapolis, MN, USA) was added to the medium at various concentrations (10 ng/mL, 50 ng/mL, and 100 ng/mL). Bovine serum albumin (BSA) was used as a control. The cells were counted by means of a WST-1 Cell Counting Kit (Dojin, Japan), following the manufacturer’s instructions.

Detection of Alkaline Phosphatase Activity
Samples were fixed with 4% PFA. ALP activity was detected with the use of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitroblue-tetrazolium chloride (NBT) substrate (DAKO, Glostrup, Denmark) or the 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate (HNPP) Fluorescent Detection Set (Roche, Mannheim, Germany), according to the manufacturer’s instructions. To examine effects on cell differentiation, we added 10 ng/mL, 50 ng/mL, and 100 ng/mL of recombinant Fgf10 protein (R&D, USA) to the medium and used BSA as a control. Furthermore, tracings of ALP-staining patterns time-dependently in the absence and presence of 100 ng/mL Fgf10 were shown.

Cells expressing ALP were also observed by a confocal microscope (Radiance 2000 Rainbow, Bio-Rad, Hercules, CA, USA). We used 4',6-diamidino-2-phenylindole (DAPI) for the counterstaining.

Polyacrylamide Gel Electrophoresis and Immunoblotting
After the cells were washed twice with PBS, they were suspended in sodium dodecyl sulfate (SDS) sample buffer and concentrated 20 times by centrifugation at 3000 x g for 30 min in an Ultrafree-15 centrifugal filter (Millipore, Bedford, MA, USA). Equal amounts (50 µg) of the samples were subjected to 15% (w/v) SDS-PAGE, and the separated proteins were electrophoretically transblotted onto nitrocellulose membrane. Non-specific binding sites were blocked by incubation in 5% skim milk. The membrane was then incubated with antibody against amelogenin at a dilution of 1:5000. After being washed, the membrane was incubated with ALP-conjugated anti-rabbit IgG at a dilution of 1:4000, and the bands were visualized with the use of BCIP/NBT.

RNA Extraction and RT-PCR Analysis
mRNA was isolated from a cell pellet (containing 10⁶ cells) with the use of ISOGEN (Nippon Gene, Tokyo, Japan). Reverse transcription (RT) was performed by means of the Omniscript^TM RT Kit (QIAGEN, Hilden, Germany) and Oligo-dT primer according to the manufacturer’s instructions. The first strand of cDNA was then used as a template for polymerase chain-reaction (PCR) with rat amelogenin primer: (upstream) 5'-CCCCAGCAACCAATGATGCCAGT-3', (downstream) 5'-GAAGCTTGGCCAGCGACAGACAAGACCAAGCGGGAAGAAG-TGGAT-3', and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer (upstream, 5'-TGAAGGTCGGTGTGAACGGA; downstream, 5'-GTACATCC-GGTACTCCAGGT). The enamel organ from the incisor tooth germ was used as a positive control. PCR amplification with LA Taq DNA polymerase (Takara, Otsu, Japan) was performed under the following conditions: 40 cycles of 94°C for 1.5 min, 54°C for 1.5 min, and 72°C for 1.5 min, followed by a 10-minute extension at 72°C. The amplified products were analyzed by electrophoresis through a 2% agarose gel.

	RESULTS

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Characterization of Dental Epithelial Progenitor Cells
The expression of CK14, p75NGFR, amelogenin, and ALP in the rat lower incisor tooth germ was determined immunohistochemically, so that we could assess dental epithelial cell differentiation. Immunoreactivity for CK14 was observed in the entire dental epithelium, and was especially intense in differentiated ameloblasts, outer enamel epithelium, and lingual epithelium. There was, however, no reactivity observed in mesenchymal tissues of the incisor tooth germ (Fig. 1A-b). ALP activity was detected in the stratum intermedium of the dental epithelium, odontoblasts, and subodontoblast layers (Fig. 1A-c). Expression of p75NGFR was observed in the inner enamel epithelium, the dental papilla cells, and the dental follicle (Fig. 1A-e). In addition, immunoreactivity to amelogenin was detected in the coronal area of the inner enamel epithelium, pre-ameloblasts, and ameloblasts (Fig. 1A-d). Weak staining was also observed in odontoblasts. Notch2 protein was detected in the stellate reticulum and outer enamel epithelium, and was identical to the expression of Notch2 mRNA (Harada et al., 1999). Immunostaining of FGFR1 and FGFR2 was observed in both dental epithelial and mesenchymal tissues (data not shown). Based on the results of immunostaining in vivo, the combined immunostaining with CK14, p75NGFR, ALP, amelogenin, and Notch2 was indicative of dental epithelial cell differentiation (Fig. 1B).

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 (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.

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.

Effect on Fgf10 in Dental Epithelial Progenitor Cells
Immunoreactivity for anti-FGFR1 and -2 antibodies was intense and continuous in almost all cells (Fig. 2A-m-o). To examine whether Fgf10 also functions as a proliferative signal for these cells, we added 10 ng/mL, 50 ng/mL, and 100 ng/mL of recombinant Fgf10 protein to the medium and used BSA as a control. In the presence of Fgf10 protein, the cell-growth rate increased in a dose-dependent manner (Fig. 4A). Furthermore, we examined Fgf10 effects on the appearance of ALP-positive cells (G5) by adding 100 ng/mL recombinant Fgf10 protein to the medium and counting the ALP-positive cells (G5). ALP-positive cells (G5) were observed, and their number increased rapidly in the presence of Fgf10 during 3–6 days (Fig. 4B). A few dots of ALP-staining started to appear beyond three-day culture, in both the presence and the absence of Fgf10 (Fig. 4C). The number of dots increased rapidly in the presence of Fgf10, but the size did not expand in most of the dots.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we immortalized dental epithelial progenitor cells derived from the cervical loop of the rat lower incisor. This was performed without the use of viral genes, such as SV-40, or carcinogens. The present study is the first to show successful establishment of dental epithelial progenitor cells for the observation of dental epithelial differentiation. Expression of CK14, p75NGFR, and amelogenin can be used to indicate differentiation into inner enamel epithelium and ameloblasts. Recent studies suggested that odontoblasts express markers for ameloblasts (Oida et al., 2002; Papagerakis et al., 2003) and/or that ameloblasts express odontoblast markers. Accordingly, we identified various differentiated cells based on their expression of multiple markers. As stratum intermedium markers, we used both ALP and CK14. Whether ALP is expressed by pre-ameloblasts is controversial, since its transcripts in the pre-ameloblasts is much less than that of stratum intermedium (Hotton et al., 1999), and immunolocalization of ALP is restricted to the proximal portion of pre-ameloblasts (Hoshi et al., 1997). However, in these cells or primary culture at various stages of differentiation, none of the cells expressing amelogenin or p75NGFR was ALP-positive (Fig. 2 and unpublished observation). These results support the use of ALP activity, where available, for use as a marker for stratum intermedium cells. As stellate reticulum and outer enamel epithelium markers, Notch2 was used.

Expression patterns exhibited during changes from lower to higher cell density show the process of dental epithelial-cell differentiation. At lower cell density, cells lack cell-cell interaction through "jostling" each other. At higher cell density, some cells are expelled from the basal layer and situated in the upper layers. In this condition, some cells can differentiate by the pressure or some signals from the surrounding cells. The cell stratification is similar to that reported in previous studies of rat incisor cervical-loop epithelial cell culture (Farges et al., 1991). However, in the present study, the situation of the ALP-positive cells (G5) provides a clue for studying the process in the differentiation of stratum intermedium cells, but not Hertwig’s epithelial sheath. The appearance patterns (Fig. 4C) suggest that the increase of ALP-positive cells (G5) was the result of differentiation of undifferentiated cells (G1), and not the proliferation of G5 cells. Therefore, recombinant Fgf10 proteins stimulated cell proliferation of G1 and subsequently induced the production of cells expressing ALP (G5), without stimulating division of G5 cells. Our previous studies of Fgf10 expression patterns and in vitro application of Fgf10-soaked beads suggested that Fgf10 acts as a regulatory signal from mesenchyme to epithelium through the FGFR1b and FGFR2b receptors during tooth development (Kettunen et al., 1998; Harada et al., 1999, 2002a; Kettunen et al., 2000; Ohuchi et al., 2000). Taken together, these results suggest that Fgf10 plays a role in coupling mitogenesis and induction of stratum intermedium cells expressing ALP in the dental epithelium.

	ACKNOWLEDGMENTS

 
This work was supported by The Kato Memorial Bioscience Foundation and KAKENHI (No. 13470387, No. 13877308 to H.H.) from MEXT, Japan.

Received April 21, 2003; Last revision October 15, 2003; Accepted November 20, 2003

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Kawano, S. Articles by Harada, H. Search for Related Content PubMed PubMed Citation Articles by Kawano, S. Articles by Harada, H.