Potential of Dental Mesenchymal Cells in Developing Teeth - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Hidetoshi Yamazakia, Motokazu Tsunetob, Miya Yoshinob, Ken-Ichi Yamamurac, Shin-Ichi Hayashib作者单位：aDivision of Genomics and Regenerative Biology, Department of Physiology and Regenerative Medicine, Institute of Medical Science, Mie University Graduate School of Medicine, Tsu, Japan;bDivision of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, % z) O- H. H& r, o3 b2 {( _
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      【摘要】
   The tooth, composed of dentin and enamel, develops through epithelium-mesenchyme interactions. Neural crest (NC) cells contribute to the dental mesenchyme in the developing tooth and differentiate into dentin-secreting odontoblasts. NC cells are known to differentiate into chondrocytes and osteoblasts in the craniofacial region. However, it is not clear whether the dental mesenchymal cells in the developing tooth possess the potential to differentiate into a lineage(s) other than the odontoblast lineage. In this study, we prepared mesenchymal cells from E13.5 tooth germ cells and assessed their potential for differentiation in culture. They differentiated into odontoblasts, chondrocyte-like cells, and osteoblast-like cells. Their derivation was confirmed by tracing NC-derived cells as LacZ  cells using P0-Cre/Rosa26R mice. Using the flow cytometry-fluorescent di-&szlig;-D-galactosidase system, which makes it possible to detect LacZ  cells as living cells, cell surface molecules of dental mesenchymal cells were characterized. Large number of LacZ  NC-derived cells expressed platelet-derived growth factor receptor  and integrins. Taken together, these results suggest that NC-derived cells with the potential to differentiate into chondrocyte-like and osteoblast-like cells are present in the developing tooth, and these cells may contribute to tooth organogenesis.
      【关键词】 Dental mesenchyme Neural crest-derived cells Murine tooth Osteoblast Chondrocyte Odontoblast Tissue-specific stem cells Cre-loxP system
   INTRODUCTION

The tooth organ is a vertebrate-specific structure that is constructed via reciprocal interactions between dental epithelium and mesenchyme .

Neural crest (NC) is thought to contribute to dental mesenchymal cells that differentiate into odontoblasts . Thus, NC cells are critical for craniofacial development, including tooth organogenesis.

It is known that NC cells contribute to odontoblasts and cementblasts of the tooth and osteoblasts and chondrocytes in the craniofacial region . However, it is not clear whether dental mesenchymal cells in the developing tooth have the potential to differentiate into lineages other than the odontoblast lineage.: O! U( Q7 S/ ~9 d1 n5 Q: g" J

The recently generated mice carrying the Cre recombinase gene (Cre) under the control of the P0 promoter (P0-Cre mice) and Rosa26R mice (P0-Cre/Rosa26R mice) enable us to trace not only cranial but also trunk NC-derived cells as LacZ-positive cells .

In this study, to clarify whether NC-derived cells are present in the early developmental stage of the tooth and to assess their potential for differentiation, we looked for NC-derived cells in the developing tooth using P0-Cre/Rosa26R mice. NC-derived cells (indicated by LacZ-expressing cells) were present in tooth buds at E13.5 and were mainly located in the dental mesenchyme under the enamel organ. We cultured the mesenchymal cells from the developing tooth and found that NC-derived cells with the potential to differentiate into odontoblasts, osteoblast-like cells, and chondrocyte-like cells were present. These cells were still retained in the dental pulp of 2.5-day-old mice.
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Mesenchymal cells are derived from NC and/or mesoderm , and approximately half of the PDGFR-expressing cells expressed LacZ. Interestingly, the LacZ-negative as well as the LacZ-positive cells had the potential to differentiate into osteoblast-like cells and chondrocyte-like cells. Here, we show the derivation and potential for differentiation of cells in the dental mesenchyme.2 I7 d) d1 `, w- d1 K- y3 Z! M5 ^; h; H

MATERIALS AND METHODS# h( e  U, X# X
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Mice
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Mice carrying Cre recombinase driven by the protein 0 (P0) promoter were produced as described . C57BL/6 mice were purchased from Clea Japan (Tokyo, http://www.clea-japan.com).
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Determination of Genotypes of Transgenic Mice+ e  D& a- g- `; @9 X- [+ z

Genomic DNA was prepared, and transgenes were detected by use of the polymerase chain reaction (PCR). The respective sense and antisense primers used for PCR were as follows: LacZ, 5'-GGT AGC AGA GCG GGT AAA CT-3', 5'-ATC TGA CGG GCT CCA GGA GT-3' Cre, 5'-GGA CAT GTT CAG GGA TCG CCA GGC G-3', 5'-GCA TAA CCA GTG AAA CAG CAT TGC TG-3'. PCR was performed by incubation at 94¡ãC for 4 minutes followed by 35 cycles of incubation at 93¡ãC for 1 minute, 58¡ãC for 1 minute, and 72¡ãC for 1 minute, and a final extension at 72¡ãC for 7 minutes.

Histological Analysis

For the detection of LacZ activity, whole embryos, tissues, and cultured teeth were fixed in phosphate-buffered saline (PBS) solution (pH 7.4) containing 2% formaldehyde (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english), 0.2% glutaraldehyde (Wako), and 0.02% NP-40 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). After washing, samples were stained with a solution containing Bluo-Gal (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) in N,N'-dimethylformamide (Wako) until the desired color intensity had been obtained. For preparation of tissue sections, thymi were embedded in polyester wax (BDH Laboratory Supplies, Poole, U.K.). Sections were prepared at 7 µm thickness and stained with nuclear fast red (Trevigen Inc., Gaithersburg, MD, http://www.trevigen.com) or hematoxylin and eosin.
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Fluorescein Di-ß-D-Galactopyranoside Loading and Flow Cytometric Analysis2 W9 K* ]* ?: a) D* k( t
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Single-cell suspensions from the dental mesenchyme of E13.5 embryos or the cervical loop of the lower incisors of 2.5-day-old mice were prepared by digestion with collagenase D (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com), dispase II (Roche), and trypsin/EDTA (Gibco-BRL). Fluorescein di-ß-D-galactopyranoside (FDG) (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) staining was carried out essentially as described /PBS) containing 1 mM chloroquine for 30 minutes at 37¡ãC in an atmosphere of 5% CO2. The cells were then loaded with FDG by osmotic shock. Briefly, after the cells had been allowed to equilibrate in a water bath at 37¡ãC for 10 minutes, an equal volume of prewarmed 2 mM FDG in sterile water was mixed rapidly with the cell suspension. After precisely 2 minutes of incubation at 37¡ãC, the FDG loading was stopped, and the cells were suspended in ice-cold staining medium containing 10 µg/ml propidium iodide for 5 minutes at 4¡ãC. Then, the cells were blocked with rabbit serum and stained with biotin-conjugated rat anti-mouse monoclonal antibody against PDGFR (Apa5; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), 5 (5H10-27; BD Pharmingen), V (RMV-7; BD Pharmingen), or ß3 (2C9.G2; BD Pharmingen). The stained cells were further incubated with R-phycoerythrin-labeled streptavidin (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com). The stained cells were analyzed using an EPICS-XL flow cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com).

Tooth Organ Culture

Molar tooth germs from the mandibles of E13.5-day-old mice were prepared as described above . The tooth germs were cultured in -minimum essential medium (-MEM; Gibco-BRL) containing 1% FBS, supplemented with the following reagents: 100 µg/ml ascorbic acid (Sigma-Aldrich) and 1 mM ß-glycerophosphate (Peptide Institute, Osaka, Japan, http://www.peptide.co.jp). The tooth germs were washed four times every other day by replacing the medium with 0.5 ml of fresh medium each time and were fed by replacing the medium with 0.3 ml of fresh medium. After 2 weeks, the tooth germs were fixed with 4% paraformaldehyde for histological analysis, or total RNA was isolated from the tooth germs.

Induction of Osteoblast-Like Cells from E13.5 Tooth Mesenchyme and Cervical Loop of Lower Incisors/ O2 |; D' N0 p" p

Cell suspensions from the dental mesenchymal layer of E13.5 embryos or the cervical loop of the lower incisors of 2.5-day-old mice were prepared as described above. Then, 1 x 105 cells were inoculated into 24-well plates coated with 0.1% gelatin (Corning Costar, Acton, MA, http://www.corning.com/lifesciences)  and cultured in Dulbecco's modified Eagle's medium (Gibco-BRL) supplemented with 10% FBS, 10¨C7 M dexamethasone (DEX), 40 nM human ascorbic acid 2 phosphatase (Sigma-Aldrich), 1 nM bone morphogenic protein 4 (BMP4) (Phoenix Pharmaceuticals, Inc., Belmont, CA, http://www.phoenixpeptide.com), and 1 mM ß-glycerophosphate (Sigma) for the induction of osteoblasts. Cultures were fed every other day by replacing half the medium with 0.5 ml of fresh medium. After the cells had been cultured for 21 days, either alizarin red (ALZ) staining was performed to detect calcification, or alkaline phosphatase (ALP) staining was performed to detect osteoblasts. In addition, the cells were stained with LacZ to confirm the presence of NC-derived cells. For detection of LacZ activity, the cells were fixed in PBS solution (pH 7.4) containing 0.25% glutaraldehyde. After having been washed, the cells were stained with a solution containing Bluo-Gal in N,N'-dimethylformamide for 6 hours.
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Induction of Chondrocyte-Like Cells from E13.5 Tooth Mesenchyme and Cervical Loop of Lower Incisors, N0 J! ~% T1 Z3 K
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Cell suspensions were prepared as described above. Then, 1 x 105 cells (1 x 106/ml) were incubated into 24-well plates for 30 minutes (Corning Costar) .
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Induction of Odontoblasts from Cervical Loop of Lower Incisors

Cell suspensions were prepared as described above. Then, 5 x 103 cells were cultured in -MEM supplemented with 20% FBS in a 10-cm culture dish for 2 weeks (Corning Costar). Then, the cells were recovered with 0.25% trypsin/EDTA, and 1 x 105 cells (1 x 106 cells per milliliter) were incubated in each well of a 24-well plate for 30 minutes (Corning Costar) and cultured in -MEM supplemented with 10% FBS, 40 nM ascorbic acid 2 phosphatase, 200 pM basic fibroblast growth factor (bFGF) (Neomarker, Inc.), and 1 mM ß-glycerophosphate for the induction of odontoblasts. The cultures were fed every other day by replacing the medium with 1 ml of fresh medium. After the cells had been cultured for 14 days, total RNA was isolated from them.

Antibodies and Immunohistochemistry0 [) E* A9 @3 Y% T9 w# x2 C
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On serial days after induction of the differentiation of dental mesenchyme cells, the cultured cells were stained separately with the monoclonal antibodies: mouse anti-mouse type II collagen (6B3; Neomarker, Inc.) or mouse anti-human human leukocyte antigen (HLA)-DQ (Neomarker, Inc.) as follows. The endogenous peroxidase activity was blocked, and the cells were fixed with 0.3% hydrogen peroxide in 100% methanol. The cells were next blocked with rabbit serum. Then, the cells were incubated with the first antibody. The bound antibody was visualized by subsequent incubation with horseradish peroxidase (HRP)-conjugated goat antibody against mouse IgG (HistoMark streptavidin-HRP Kit). After being washed, the cells bearing immunocomplexes were visualized using a diaminobenzidine Reagent Set (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD, http://www.kpi.com).

Reverse Transcription-PCR Analysis of Osteoblast-, Chondrocyte-, Odontoblast-, and Ameloblast-Specific Gene Expression; E9 J2 ?) T7 a5 F" N* o/ @
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Total tissue RNAs were prepared using Isogen (Nippon Gene, Tokyo, http://www.nippongene.com). After treatment of the RNA fraction with DNase I (Amersham Pharmacia Biotech, Piscataway, NJ, http://www.amershambioscience.com), cDNA synthesis was carried out using reverse transcriptase (ReverTraAce; Toyobo, Osaka, Japan, http://www.toyobo.co.jp) with oligo(dT) primer (Gibco-BRL). PCR using the cDNA mixture as template was performed with Taq polymerase (Toyobo). The oligonucleotide primers used for PCR were as follows: Runx2/Cbfa1, 5'-GGACCGGCCCCGACTGTAATC-3', 5'-GTAGGGCAACGCAAAGGACTCAT-3'; Bglap1(Osteocalcin), 5'-CCATGGAGAAGGCTGGGG-3', 5'-CAAAGTTGTCATGGATGACC-3'; Col2a1, 5'-GTTGCGTCTCTAAGATCCTG-3', 5'-AGCGTCCTTGGCAATTTGAC-3'; Col10a1, 5'-ACCCCTGGATCTCCCGTATCCGAGT-3', 5'-GGACAGGGAAACGGCTTTCGATCTG-3'; Sox9, 5'-CACTACACCGACCAGCCGTCCACTT-3', 5'-GATAGAGTCGTATATACTGGCTGCT-3'; Dspp, 5'-AGACATCCACTACTGTGCG-3', 5'-TCCCGGACATAAATCCTCGTGCAGA-3'; Amelx (Amelogenin), 5'-CTGGGCA CTGTAGGTCAATCTC-3', 5'-GGACGTCCTCGATGGCGTGAGG-3'; Hprt, 5'-CTTTATCTGTCCTTGACATCAACC-3', 5'-TCGCCGCTGCAGGTGCTTCACTTA-3'.% W7 ]' {4 k1 `) ~' v
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PCR was performed by incubation at 94¡ãC for 4 minutes, followed by 35 cycles of incubation at 93¡ãC for 1 minute, 58¡ãC (60¡ãC for col10a1) for 1 minute, and 72¡ãC for 1 minute, and a final extension at 72¡ãC for 7 minutes. No PCR products were amplified from templates when cDNA synthesis was performed without reverse transcriptase.
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Statistical Analysis; S6 A9 _) I( y) ?% V( T7 F
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Data are presented as mean ¡À SD. Statistical significance was assessed by using Student's t test.. ?! h$ p/ y1 X( \: X% u$ L
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RESULTS+ }0 f7 v. W3 z* u, @" h

Detection of NC-Derived Cells in the Developing Tooth
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As we recently reported, P0-cre/Rosa26R mice enabled us to detect NC-derived cells as LacZ-positive (LacZ ) cells . Approximately, 60% of the total cells from E13.5 tooth germs of P0-Cre/Rosa26R mice were LacZ  cells (Fig. 1E). In addition, 60% of the cells prepared from the mesenchyme in the tooth germ expressed LacZ (Fig. 1E). In contrast, 14% of cells expressed LacZ in the fraction of epithelial layers.

Figure 1. Contribution of neural crest (NC)-derived cells to cranium/face and dental mesenchyme. LacZ  cells are present in the craniofacial region, including the maxilla and mandible, and in the dental mesenchyme (B) of E13 P0-Cre/Rosa26R but not Rosa26R mice. A section with LacZ staining (A, B), and with both LacZ and nuclear fast red staining (C, D). LacZ  cells are observed in the dental mesenchyme under the EO (D) (arrowheads). (E): Flow cytometric analysis of NC-derived cells in the E13 tooth germ using FDG. E13.5 tooth germs of P0-Cre/Rosa26R mice or Rosa26R mice were divided into mesenchyme and epithelium, and then cells from the mesenchyme, epithelium, and both were dissociated into single cells and subjected to flow cytometric analysis. (F¨CH): Mesenchymal cells from E13 tooth germs of Rosa26R mice (F) and P0-Cre/Rosa26R mice (G) were cultured for 1 week, and the cells were stained for LacZ. Cells recovered from the culture dishes were analyzed by flow cytometry using FDG. FDG  cells were rarely observed in cells from E13 tooth germ of Rosa26R mice (H). Abbreviations: EO, enamel organ; epi, epithelium; FDG, fluorescent di-ß-D-galactosidase; mesen, mesenchyme.' u+ l; d# p  M

Cells from E13.5 P0-Cre/Rosa26R dental mesenchyme were cultured for 1 week in -MEM containing 10% fetal calf serum (FCS). The ratio of LacZ  cells in the cells recovered from the culture was enriched (Fig. 1F¨C1H) before culturing (Fig. 1E).
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Cell Surface Molecules on Dental Mesenchymal Cells
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To characterize the surface molecules on NC-derived cells from dental mesenchymal layers, we stained the cells prepared from P0-Cre/Rosa26R mice with antibodies against integrins-v, -5, and -ß3, and PDGFR. It is known that the majority of mesenchymal cells express PDGFR and several integrins . Representative histograms from three independent experiments are shown in Figure 2. The dental mesenchymal cell fraction in control Rosa26R mice contained 82.4 ¡À 9.2% PDGFR-expressing cells (Fig. 2, upper panel). In P0-Cre/Rosa26R mice, 81.4 ¡À 9.2% of mesenchymal cells expressed PDGFR, and 55.0 ¡À 18.3% of PDGFR-expressing cells were LacZ . These results suggest that LacZ  NC-derived cells may be present in dental mesenchymal layers. The majority of LacZ  cells expressed integrin-v as well as PDGFR. Integrins-5 and -ß3 were expressed in 54% and 59% of LacZ  cells, respectively (Fig. 2, lower panel). In the LacZ-negative (LacZ¨C) cells of the P0-Cre/Rosa26R dental mesenchymal fraction, 88%, 44%, and 50% of cells expressed integrins-v, -5, and -ß3, respectively. We assessed the possibilities of nonspecific and low-level expression of the LacZ gene; however, LacZ  cells were rarely detected in the E13 Rosa26R mice or hematopoietic cells derived from mesoderm in the P0-Cre/Rosa26R fetal liver (data not shown).$ ]3 x  {) S3 |; x! q

Figure 2. Expression of PDGFR and integrins on neural crest (NC)-derived cells of E13 dental mesenchyme. Shown is the expression of adhesion molecules such as PDGFR and V, 5, and ß3 integrins on the dental mesenchymal cells of E13.5 Rosa26R mice (upper panels) and P0-Cre/Rosa26R mice (lower panels). First, cells were stained FDG to detect NC-derived cells, and then they were stained with antibodies against PDGFR and integrins. Abbreviations: FDG, fluorescent di-ß-D-galactosidase; PDGFR, platelet-derived growth factor receptor .
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Induction of Odontoblasts from the E13.5 Dental Mesenchyme
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To clarify the potential of dental mesenchymal cells for differentiation into odontoblasts, E13.5 tooth germs of C57BL/6 mice were cultured with ascorbic acid (AA) for 2 weeks using standard tooth organ culture methods. As shown in Figure 3A, enamel-secreting ameloblasts (red arrowhead) and dentin-secreting odontoblasts (blue arrowheads) were present in the tooth germ tissue sections. Neither type of cell was observed in the E13.5 tooth germs at the initiation of the cultures. The gene expression of odontoblast-specific dentin sialophosphoprotein (Dspp) and ameloblast-specific amelogenin X chromosome (Amelx) (Amel/Amg) was detected at 2 weeks in the tooth germ cultures (Fig. 3B) .

Figure 3. Presence of cells with the potential to differentiate into Od and melanocytes in the E13.5 tooth germ. (A): E13.5 tooth organ culture was performed in the presence of ascorbic acid and L-glutamate. Two weeks later, the cultured teeth were fixed and examined histologically. Normal cusp formation (open arrowheads) with large amounts of dentin matrix and enamel matrix produced by differentiated Am and Od (A) (arrowheads) were observed in the cultured teeth, although no cusp formation with Am and Od was observed in E13.5 tooth germ before culture. (B): Expression of Amelx and Dspp in E13.5 cultured teeth after 2 weeks and E13.5 tooth germ. Dental mesenchyme of A0.5-day-old lower incisor was used as a positive control. ST2 cells, known to be hematopoietic supporting cells, or a preadipocytic cell line were used as negative controls. Total RNAs were prepared from E13.5 or A0.5 tooth germ, cultured teeth, and ST2 cells, and reverse transcription-polymerase chain reaction was performed to analyze the Amelx and Dspp gene expression. (C, D): To assess whether the cells with the potential to differentiate into Od were derived from neural crest cells, the cells were stained for LacZ and hematoxylin and eosin. Rectangular Od (D) (black arrow) were stained with LacZ. (D'): High magnification of (D). Am (red arrow) next to dentin (D) were not stained with LacZ. (E): Pigmented melanocytes were sometimes observed in the dental papilla of the cultured teeth (E, E') (arrowheads). (E''): High magnification of (E'). Abbreviations: Am, ameloblasts; Amelx, amelogenin; Dspp, dentin sialophosphoprotein; Hprt, hypoxanthine guanidine phosphoribosyl transferase; Od, odontoblasts; T, teeth.8 v% b3 F- r2 C8 H' t

Using P0-Cre/Rosa26R mice, the derivation of odontoblasts in culture was assessed by staining for LacZ and hematoxylin and eosin. As shown in Figure 3C and 3D, rectangular-shaped odontoblasts (black arrow) located in the upper layer of the dental papilla were stained with LacZ. Ameloblasts next to dentin (red arrow) were not stained with LacZ. These results indicated that odontoblasts were derived from NC.

Presence of Cells with the Potential to Differentiate into Melanocytes in the Developing Tooth) o5 D  t& w& l* `6 W  G
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It was not clear whether there were cells with the potential to differentiate into cell types other than odontoblasts among the dental mesenchymal cells. We sometimes observed pigmented melanocytes, one type of NC progeny, in cultures of E13.5 tooth germs of C57BL/6 mice after 2 weeks (Fig. 3E, 3E'). We could not rule out the possibility that the progenitor cells for melanocytes in the dermis spuriously invaded the tooth germs; however, melanocytes were only detected inside dental papillae (Fig. 3E', arrows). These results imply the presence of NC-derived cells with the potential to differentiate into not only odontoblasts but also melanocytes in the developing tooth.6 d" C: _, s' H0 v$ a+ }; \4 J1 i( |

LacZ  osteoblast-like cells were induced from the E13.5 dental mesenchyme The above observations prompted us to clarify whether cells with the potential to differentiate into a variety of cell lineages were present in the E13.5 dental mesenchyme. We prepared the dental mesenchymal cells from E13.5 tooth germs of C57BL/6 mice and cultured the cells in the presence of AA, ß-glycerophosphate (ß-gly), and BMP4 for the induction of osteoblastogenesis. Three weeks later, the cultured cells from E13.5 dental mesenchyme were stained with ALZ to detect calcification. Large numbers of ALZ-positive cells were observed in the presence of AA/ß-gly/BMP4 (Fig. 4A, lower panel) but not in the absence of AA/ß-gly/BMP4 (Fig. 4A, upper panel). We also assessed whether these cells had ALP activity, which is known to be one of the specific features of osteoblasts. Cells cultured in the presence but not absence of AA/ß-gly/BMP4 showed significant ALP activity (Fig. 4B, bottom). In this culture, as a positive control, we used ST2 cells, a preadipocytic cell line that has the potential to differentiate into osteoblasts. ST2 cells showed weak ALP activity in the absence of AA/ß-gly/BMP4 (data not shown) , whereas they showed strong ALP and ALZ activity in the presence of these three reagents (data not shown).- O7 ]' q+ D; ^' f
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Figure 4. Presence of neural crest (NC)-derived cells with the potential to differentiate into osteoblast-like cells in the E13.5 dental mesenchyme. Mesenchymal cells from E13.5 tooth germ of C57BL/6 mice were cultured in the presence of AA, ß-gly, and BMP4. Three weeks later, the cells were stained with alizarin red (ALZ) (A) or alkaline phosphatase (ALP) (B). ALZ-positive (ALZ ) and ALP-positive (ALP ) cells were observed in the presence of these three reagents. No ALZ and only weakly ALP  cells were observed in the absence of these three reagents (medium). (C): Expression of osteogenic or odontogenic- or chondrogenic-specific genes in mesenchymal cells from E13.5 tooth germ and these cultured cells in the presence or absence of these reagents. Spleen cells consisting of hematopoietic cells were used as a negative control. (D, E): To assess whether the cells with the potential to differentiate into osteoblast-like cells were derived from NC cells, the cells were stained for both LacZ and ALP. Both LacZ  cells (blue) and ALP  osteoblast-like cells (red, red arrowhead) (E) were observed. ALP  cells without LacZ (black arrowhead), only LacZ  (black arrow), and both LacZ¨C and ALP¨C cells were also observed. Abbreviations: AA, ascorbic acid; ß-gly, ß-glycerophosphate; BMP4, bone morphogenic protein 4.: V3 G3 C  k3 F  i+ u
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Subsequently, we assessed the expression of representative genes for osteogenic, chondrogenic, and odontogenic cells (i.e., the osteocalcin/bone -carboxyglutamate protein 1 (Ocn/Bglap1) .

To determine the derivation of cells differentiating into osteoblasts, double staining of ALP and LacZ was performed to detect NC-derived cells. As shown in Figure 4D and 4E, ALP (red) and LacZ (blue) double-positive cells (red arrowhead) were detected in these cultures. ALP-positive cells without LacZ (black arrowhead) were also present in this culture. These results suggest that cells with the potential to differentiate into osteoblasts might consist of both NC- and non-NC-derived cells. However, it is not clear that 100% of NC-derived cells were traced by using P0-cre/Rosa26R mice.
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Presence of Cells with the Potential to Differentiate into Chondrocyte-Like Cells in the E13.5 Tooth Mesenchyme
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We also examined the presence of cells giving rise to chondrocytes in the E13.5 dental mesenchyme of C57BL/6 mice. For this, cells from dental mesenchyme were cultured in the absence or presence of TGF-ß3 and BMP2. Unlike in the osteogenic cultures, 3 weeks later, calcification was not observed in either culture condition.1 S' N0 [" [# z1 b' ~8 u
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The development of the chondrocyte lineage in these cultures was assessed by Alcian Blue (ALB) staining to detect chondrogenic specific proteoglycans such as aggrecan and by staining with anti-type II collagen (Col II) antibody to detect the production of Col II in chondrocytes but not osteoblasts. In the presence of AA/TGF-ß3/BMP2 in cultures from E13.5 dental mesenchyme, ALB-positive cells (Fig. 5A, bottom, arrowhead) and cells strongly positive for Col II were observed (Fig. 5B, arrows). Unexpectedly, ALB-positive cells and a small but significant number of Col II-positive cells were sometimes detected in cultures even in the absence of AA/TGF-ß3/BMP2 (Fig. 5B, medium, arrowheads). The specificities of ALB-staining and anti-Col II antibody immunostaining were ascertained by culturing of a bone marrow-derived stromal cell, ST2, with AA/TGF-ß3/BMP2 as a negative control, and culturing of a prechondrocytic cell line, ATDC5, with this agent as a positive control ./ ?, I- E6 }6 f6 \( L2 N% Q0 c* ?# q
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Figure 5. Presence of neural crest (NC)-derived cells with the potential to differentiate into chondrocyte-like cells in the E13.5 tooth germ. Mesenchymal cells from E13.5 tooth germ of C57BL/6 mice were cultured in the presence of ascorbic acid, TGF-ß3, and BMP2. Three weeks later, the cells were stained with Alcian Blue (ALB) (A) or anti-Col II antibody (B). Cells reactive with ALB are stained blue (A) (arrowhead), whereas cells reactive with the antibody are stained brown (B) (arrow and arrowheads). ALB-positive (ALB ) cells were observed in the presence of these reagents. Col II-positive (Col II ) cells were observed in the presence of these reagents (B) (arrows), but Col II  cells were still present in the absence of these reagents (B) (arrowheads). No ALB  or Col II  cells were detected in the mesenchymal cells from E13.5 tooth germ before culture (data not shown). No cells reactive with the anti-human leukocyte antigen (HLA) antibody (control) were observed in either the presence or absence of these reagents (B). (C): Expression of osteogenic-, chondrogenic-, or odontogenic-specific genes in mesenchymal cells from E13.5 tooth germ and cells cultured in the presence (Chondro-D) or absence (Chondro-M) of these reagents. Col2a1 (chondrogenic gene) was expressed in cells cultured in the both the presence and absence of these reagents. Spleen cells were used as a negative control. (D): To assess whether cells with the potential to differentiate into chondrocyte-like cells were derived from the NC, cells were stained with both LacZ and anti-Col II antibody. Both LacZ  cells (blue) and Col II  chondrocyte-like cells (brown) were observed (black arrowhead). No LacZ  cells were stained with antibody against HLA (control), and E13.5 NC-derived dental mesenchymal cells positive for LacZ were not stained with Col II. Col II  cells without LacZ were observed in this culture (D) (white arrowheads). Abbreviations: AA, ascorbic acid; BMP2, bone morphogenic protein 2; Ab, antibody; Col II, type II collagen; Chondro-D, culture with ascorbic acids; Chondro-M, culture with medium only; Hrpt, hypoxanthine guanidine phosphoribosyl transferase; TGF-ß3, transforming growth factor-ß3.
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Reverse transcription-PCR for chondrocyte marker genes further confirmed these observations (Fig. 5C). Although E13.5 dental mesenchyme cells rarely express chondrogenic genes Col2a1 and type X-collagen 1 (Col10a1) , these cells cultured both with (chondrocyte differentiation medium) and without (medium only) AA/TGF-ß3/BMP2 expressed these genes (Fig. 5C). Two types of Col2a1 transcripts were detected in chondrocytes using the primers we used. Longer and shorter transcripts were detected in undifferentiated and differentiated chondrocytes, respectively. The shorter transcript was strongly observed in cultures under the chondrogenic conditions (Fig. 5C). These results suggest that cells with the potential to differentiate into chondrocyte-like cells are also present in the E13.5 dental mesenchyme. Using P0-Cre/Rosa26R mice, the derivation of these chondrocyte lineage cells expressing Col II molecules in culture was assessed by double staining for LacZ and Col II. At the initiation of the culture, LacZ  (blue) but not Col II-positive (brown) cells were present (day 0; Fig. 5D, upper left). Three weeks later, Col II and LacZ double-positive cells were observed in the cultures (Fig. 5D, bottom right, arrowhead). Col II-positive cells without LacZ expression were also present (Fig. 5D, bottom left, white arrowheads).; O2 a$ o; m; G# j# M. }
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Presence of Cells with the Potential to Differentiate into Osteoblast-Like Cells and Chondrocyte-Like Cells in the Mesenchyme Surrounding Tooth Germ
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NC-derived cells contribute to the mandible and maxilla, which are composed of cartilage and bone, and it is difficult to distinguish dental mesenchyme from nondental mesenchyme surrounding the tooth germ . Therefore, we cannot exclude the possibility that nondental mesenchymal cells surrounding the tooth germ differentiate into chondrocytes and/or osteoblasts. To examine this possibility, mesenchymal cells surrounding the tooth germ and cells from the dental mesenchyme were isolated to assess their developmental potential. Both types of cells calcified when cultured under osteogenic conditions (AA/ß-gly/BMP4) for 3 weeks (data not shown). Larger numbers of Col II-positive cells (Fig. 6, arrows) were detected among the cells from the dental mesenchyme than among those from the mesenchyme surrounding the tooth germ under the chondrogenic conditions (AA/TGF-ß3/BMP2).

Figure 6. Presence of chondrocyte progenitors in the E13.5 dental mesenchyme and surrounding tooth germ. Dental mesenchymal cells and mesenchymal cells surrounding the tooth germ were isolated from the E13.5 tooth germ of C57BL/6 mice and cultured and stained with anti-type II collagen (Col II) antibody as described above (see legend for Fig. 5). Larger numbers of Col II  cells (arrows) were detected among the cells from dental mesenchyme than among the mesenchymal cells surrounding the tooth germ. No cells positive for immunostaining with anti-human leukocyte antigen antibody (control) were observed.) C3 S' A+ K& s. N! ?5 b5 h

Induction of Osteoblast-Like and Chondrocyte-Like Cells from Cells in the Cervical Loop of 2.5-Day-Old Mouse Lower Incisors$ o# d, b6 e6 u: ^. q$ U3 D1 C

Murine incisors are not exchanged, but they elongate in response to occlusion between the upper and lower incisors, indicating that progenitor cells differentiating into ameloblasts and odontoblasts remain in the roots of incisors . Since teeth consist of dentin and enamel, the progenitor cells for both odontoblasts and ameloblasts must be retained in those roots. We assessed whether NC-derived cells were present in the pulps of the lower incisors of 2.5-day-old P0-Cre/Rosa26R or Rosa26R mice using the FDG system. Using P0-Cre/Rosa26R mice, approximately 70% of freshly prepared cells and 80% of cells cultured for 1 week in -MEM with 10% FCS were LacZ  (Fig. 7A).
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Figure 7. Maintenance of neural crest-derived cells with the potential to differentiate into cell types other than odontoblasts in lower incisors of 2.5-day-old mice. (A): Cells from the lower incisors of 2.5-day-old P0-Cre/Rosa26R mice and Rosa26R mice were isolated and cultured for 1 week, and freshly isolated cells and cells cultured for 1 week were recovered and analyzed by flow cytometry using FDG. (B): Expression of Amelx and Dspp in cultured cervical loop cells of 2.5-day-old lower incisor for 2 weeks. Tooth-m or whole tooth of the lower incisors of 2.5-day-old mice were used as a control. Total RNAs were prepared from day 2.5 lower incisors and cultured cells. (C): Cells from the tips of pulps (square) or the cervical loop of lower incisors (dotted square) of 2.5-day-old C57BL/6 mice were cultured in the presence of osteogenic reagents. Three weeks later, cells were stained with alizarin red (ALZ). ALZ  cells were observed in the presence of both osteogenic reagents. (D, E): Expression of osteogenic or chondrogenic or odontogenic specific genes in mesenchymal cells from Tip and Cer of the lower incisors of C57BL/6 mice cultured in the presence of osteogenic (D) or chondrogenic (E) reagents. Chondrogenic genes, including Col2a1 and Col10a1, were expressed in cultured cells of the Tips or Cer in the presence of chondrogenic reagents. ATDC5 cells, which are known to be a chondrogenic cell line, were used as a positive control for chondrogenesis. Abbreviations: A2.5 tooth-m, cells of 2.5-day-old lower incisors before culture; AA, ascorbic acid; Amelx, amelogenin; BMP4, bone morphogenic protein 4; Cer, cervical loops; chondro-D, culture with chondrogenic reagents; Dspp, dentin sialophosphoprotein; FDG, fluorescein di-ß-D-galactopyranoside; ß-gly, ß-glycerophosphate; Od-D, culture with ascorbic acid, ß-glycerophosphate, and basic fibroblast growth factor; Od-M, culture without ascorbic acid, ß-glycerophosphate, or basic fibroblast growth factor; osteo-D, culture with osteogenic reagents; Tip, tips of pulps; Tooth-m, tooth mesenchyme.

To clarify the potential of dental mesenchymal cells for differentiation into odontoblasts, tissues surrounding the cervical loop of lower incisors of 2.5-day-old C57BL/6 mice were isolated and cultured in -MEM with 20% FCS and L-glutamate for 3 weeks. Subsequently, the recovered cells were cultured in -MEM with AA, ß-gly, and bFGF for 2 weeks as described in Materials and Methods . These results suggest that cells with the potential to differentiate into odontoblasts, osteoblast-like cells, and/or chondrocyte-like cells may be still remained in the cervical loops of the incisors in 2.5-day-old mice.7 G3 @- w% ?- ~1 k/ L# m
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DISCUSSION

In this study, we showed that dental mesenchymal cells with the potential to differentiate into odontoblasts and lineages other than odontoblasts were present in the developing tooth. We obtained results indicating that the dental mesenchyme or dental pulp contained large number of NC-derived (LacZ ) cells using FDG or LacZ staining. Moreover, we cultured cells from E13 dental mesenchyme and found the presence of LacZ  odontoblasts, osteoblast-like cells, and chondrocyte-like cells in cultures. Using P0-Cre/Rosa26R mice and the FDG system, we could only detect LacZ expression in 55% of the E13 dental mesenchymal cells. This probably indicates that not all dental mesenchymal cells consist of NC-derived cells, even taking into account the possibility that NC-derived cells might not be completely traced by using P0-cre/Rosa26R mice. By using the FDG system, we could detect nearly 100% LacZ-expressing cells in Chinese hamster ovary-derived cells carrying CAG-LacZ (data not shown). In contrast, non-NC-derived cells, such as hematopoietic cells, were rarely stained by LacZ staining (data not shown). Therefore, the P0-Cre/Rosa26R mouse system, especially when combined with the FDG system, might enable us to detect almost all LacZ-expressing cells effectively.
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Since the tooth mesenchyme was prepared mechanically and enzymatically, we could not exclude completely the contamination of outside of the tooth mesenchyme. However, we do not think that approximately half of the cells were contaminated out of the tooth mesenchyme. The problem is the penetrance of P0-cre/Rosa26R system in each cell lineage rather than the contamination. The activity of P0 promoter was reported in various tissues, such as dorsal root ganglia, sympathetic ganglia, melanocytes, and craniofacial mesenchyme . It might mean that the Rosa26R system also functioned in the dental mesenchyme. As shown in Figures 4 and 5, NC-derived LacZ  cells in the P0-Cre/Rosa26R tooth germ differentiated into osteoblast- and chondrocyte-like cells. However, we also detected osteoblast- and chondrocyte-like cells without expression of LacZ. These LacZ-negative cells may be mesoderm-derived cells, but the derivation is not quite clear yet. The possibility remains that P0-Cre or Rosa26R gene expression does not penetrate at 100% in the dental mesenchyme.
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It is noteworthy that the cell surface molecules expressed on both the mesenchymal NC-derived cells and non-NC, putative mesoderm-derived cells were quite similar. Both LacZ  and LacZ¨C cells express PDGFR and integrins in dental mesenchymal fractions . Therefore, using both P0-Cre and Mesp-1-Cre mice, we might detect NC- and mesoderm-derived cells, respectively, in the dental mesenchyme.

It is unclear whether multipotent NC-derived cells that differentiate into chondrocytes, osteoblasts, and odontoblasts are present in the developing tooth. The fate of NC-derived cells has been thought to be determined during their migration, resulting in the loss of multipotency of NC-derived cells before they reach the peripheral tissues . Therefore, multipotent NC-derived cells may be present in the developing tooth and dental pulp. Alternatively, these NC-derived cells might be composed of heterogeneous populations with progenitors for odontoblasts, chondrocytes, osteoblasts, and even melanocytes. The expression of osteogenic genes (Bglap1/Ocn and Runx2/Cbfa1) was already detected in preparations of E13.5 dental mesenchymal cells (Fig. 4). The expression of chondrogenic genes was frequently observed in culture conditions not optimized for chondrogenesis, and even under the conditions for osteoblast induction (Figs. 4, 5). These results suggest that the E13.5 dental mesenchyme is composed of a heterogeneous population containing osteogenic cells and chondrogenic cells. Of course, we cannot exclude the possibility that our culture conditions allowed precursors to differentiate into chondrocytes preferentially. Further examination using a clonal culture system is needed to clarify whether NC-derived multipotent cells exist in the E13 dental mesenchyme and pulps.* J, H  ^# y( N9 R8 G2 I
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Recently, multipotent cells have been reported to be present in several tissues . Our findings that cells with the potential to differentiate into chondrocytes, osteoblasts, and odontoblasts are present in the developing tooth and pulp might be in accord with those reports, although it is still not clear whether stem cells in the teeth are derived from mesoderm. It will be necessary to ascertain the differences between the progenitor cells shown here and these multipotent cell populations., a8 n! A; o' a/ O6 u5 A: J- Y6 v
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In this study, we showed that the cell fractions prepared from the dental mesenchyme and dental pulps might have restricted potential that includes the potential to differentiate into odontoblasts, chondrocytes, and osteoblasts. However, neither bone nor cartilage was present in the teeth. In the emergent state, such as defects of the dentine (dental caries) or alveolar bone (periodontitis), dental mesenchymal cells may participate to repair the defects . This study using P0-Cre/Rosa26R mice enabled us to show the novel possibility that these cell populations might consist of both NC-derived and non-NC-derived cells.1 _, `6 ]  s$ f( k. r0 R

DISCLOSURES2 t. e5 t; i& G3 [- V
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS9 P" I" X5 A9 h' l0 ]+ t( E3 y: Y+ q: B
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We thank Dr. S. Niida (National Center for Geriatrics and Gerontology), Dr. T. Yamane (Stanford University), and Drs. T. Era and H. Sakurai (Riken, Center for Developmental Biology, Kobe, Japan) for helpful discussions. We also gratefully acknowledge Dr. N. Suzuki (Mie University) for maintenance of the mice and K. Yoshida for technical assistance. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (H.Y., S.H.); by a grant from Research on Demential and Fracture, Health and Labour Sciences Research Grants, the Japanese Government (S.H.); and by funding from the Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd. M.T. is currently afiliated with Max Plank Institute for Infectionbiology, Berlin, Germany.
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