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TISSUE-SPECIFIC STEM CELLS |
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, Tottori University, Yonago, Japan;
cDivision of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
Key Words. Dental mesenchyme • Neural crest-derived cells • Murine tooth • Osteoblast • Chondrocyte • Odontoblast Tissue-specific stem cells • Cre-loxP system
Correspondence: Hidetoshi Yamazaki, D.D.S., Ph.D., Department of Physiology and Regenerative Medicine, Division of Genomics and Regenerative Biology, Institute of Medical Science, Mie University Graduate School of Medicine, Tsu 514-8507, Japan. Telephone: 81-59-231-5498; Fax: 81-59-231-5004; e-mail: yamazaki{at}doc.medic.mie-u.ac.jp
Received June 12, 2006;
accepted for publication August 23, 2006.
First published online in STEM CELLS EXPRESS August 31, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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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.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Neural crest (NC) is thought to contribute to dental mesenchymal cells that differentiate into odontoblasts [7, 8]. Alterations of the migration and differentiation of cranial NC cells by targeted mutations of a number of growth and transcriptional factor genes result in craniofacial malformations, including missing teeth [9–12]. 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 [13–15]. 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.
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 [16, 17]. Almost all NC-derived cells are thought to express the P0-Cre gene, and therefore the majority of NC-derived cells in P0-Cre/Rosa26R mice express the LacZ gene. These mice are useful for assessing the contribution of NC-derived cells to cardiovascular development and thymic development [18, 19]. Using these mice, we previously demonstrated that NC-derived melanocytes, glia cells, and neurons are LacZ-expressing cells both in vivo and in vitro [17].
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.
Mesenchymal cells are derived from NC and/or mesoderm [20, 21]. In general, the formation of the cranioface, including the mandible and maxilla, is mainly contributed by NC cells; therefore, it is likely that NC-derived mesenchymal cells differentiate into chondrocytes and osteoblasts in the craniofacial region [13, 22–26], whereas the skeleton outside of craniofacial regions is mainly derived from mesodermal mesenchymal cells [21, 23, 27]. However, using the Wnt-1-Cre and Rosa26R mouse system, Chai et al. have suggested that dental mesenchyme consists of both NC and non-NC cells [13]. Recently, the neck and shoulder were reported to consist of mesenchymal cells derived from both mesoderm and NC cells [28]. Therefore, to characterize the dental mesenchymal cells, we assessed their platelet-derived growth factor receptor (PDGFR) expression using P0-Cre/Rosa26R mice. The majority of cells prepared from the dental mesenchyme expressed PDGFR, which is expressed in mesenchymal cells [29, 30], 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.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Determination of Genotypes of Transgenic Mice
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°C for 4 minutes followed by 35 cycles of incubation at 93°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute, and a final extension at 72°C 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.
Fluorescein Di-ß-D-Galactopyranoside Loading and Flow Cytometric Analysis
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 [32, 33]. To reduce background fluorescence, we incubated the cells in FDG staining medium (4% fetal bovine serum [FBS] [JRH, Lenexa, KS, http://www.sigmaaldrich.com/SAFC/Biosciences.html]/10 mM HEPES [pH 7.3]/PBS) containing 1 mM chloroquine for 30 minutes at 37°C 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°C 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°C, 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°C. 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 [3, 25, 34]. 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
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) [35] and cultured in Dulbecco's modified Eagle's medium (Gibco-BRL) supplemented with 10% FBS, 10–7 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.
Induction of Chondrocyte-Like Cells from E13.5 Tooth Mesenchyme and Cervical Loop of Lower Incisors
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) [35, 36] and cultured in -MEM supplemented with 10% FBS, 10–7 M DEX, 40 nM ascorbic acid 2 phosphatase (Sigma-Aldrich), 1 nM transforming growth factor (TGF)-ß3 or BMP2 (Neomarker, Inc., Freemont, CA, http://www.labvision.com), and 1 mM ß-glycerophosphate for the induction of chondrocyte. Cultures were fed every other day by replacing the medium with 1 ml of fresh medium. After the cells had been cultured for 21 days, staining with Alcian Blue or with mouse anti-type II collagen antibody (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) was performed to detect the chondrocytes produced. Furthermore, the cells were stained with LacZ to confirm the presence of NC-derived cells. ATDC5 cells that are known as chondrogenic cell line were used as the positive control of chondrogenesis [37].
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 Immunohistochemistry
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
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'.
PCR was performed by incubation at 94°C for 4 minutes, followed by 35 cycles of incubation at 93°C for 1 minute, 58°C (60°C for col10a1) for 1 minute, and 72°C for 1 minute, and a final extension at 72°C for 7 minutes. No PCR products were amplified from templates when cDNA synthesis was performed without reverse transcriptase.
Statistical Analysis
Data are presented as mean ± SD. Statistical significance was assessed by using Student's t test.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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-MEM containing 10% fetal calf serum (FCS). The ratio of LacZ+ cells in the cells recovered from the culture was enriched (Fig. 1F–1H) before culturing (Fig. 1E).
Cell Surface Molecules on Dental Mesenchymal Cells
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 [29, 30, 38]. These gene-disrupted mice or injected mice of inhibitory antibodies against integrins show abnormalities of NC-derived cells and tissues [39–44]. Representative histograms from three independent experiments are shown in Figure 2. The dental mesenchymal cell fraction in control Rosa26R mice contained 82.4 ± 9.2% PDGFR-expressing cells (Fig. 2, upper panel). In P0-Cre/Rosa26R mice, 81.4 ± 9.2% of mesenchymal cells expressed PDGFR, and 55.0 ± 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–) 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).
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Presence of Cells with the Potential to Differentiate into Melanocytes in the Developing Tooth
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.
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) [35, 48], whereas they showed strong ALP and ALZ activity in the presence of these three reagents (data not shown).
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-carboxyglutamate protein 1 (Ocn/Bglap1) [49], dentin matrix protein 1 (Dmp1) [50], runt-related transcription factor 2 (Runx2/Cbfa1) [51, 52], type II-collagen 1 (Col2a1) [53], and Dspp genes) in these cells in the presence (osteoblast differentiation medium) or absence (medium only) of osteogenic reagents. Both E13.5 freshly prepared and cultured dental mesenchymal cells expressed osteogenic genes Bglap1, Dmp1, and Runx2 (Fig. 4C). However, these cells did not express the Dspp gene specific for odontoblasts (Fig. 4C). Both cells cultured with and without AA/ß-gly/BMP4 expressed Col2a1, a gene specific for differentiated chondrocytes [35, 36, 54]. 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.
Presence of Cells with the Potential to Differentiate into Chondrocyte-Like Cells in the E13.5 Tooth Mesenchyme
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.
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 [37].
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1 (Col10a1) [55], 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).
Presence of Cells with the Potential to Differentiate into Osteoblast-Like Cells and Chondrocyte-Like Cells in the Mesenchyme Surrounding Tooth Germ
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 [13, 23]. 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).
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-MEM with 10% FCS were LacZ+ (Fig. 7A).
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-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 [57]. As shown in Figure 7B, the expression of the Dspp gene was detected at 2 weeks in the presence of AA/ß-gly/bFGF. Next, cells prepared from the cervical loop or the tips of the pulps of incisors in 2.5-day-old C57BL/6 mice were cultured in the presence of AA/ß-gly/BMP4 as a substitute for AA/ß-gly/bFGF for 3 weeks. They were calcified and were stained with ALZ (Fig. 7C). However, we did not detect Dspp-expressing odontoblasts under these culture conditions (Fig. 7D). Furthermore, when these cells were cultured under chondrogenic conditions (AA/TGF-ß3/BMP2), cells from the cervical loop (chondrocyte differentiation medium) and the tips of pulps expressed the Col10a1 and Col2a1 genes, respectively (Fig. 7E) [55]. 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. |
DISCUSSION |
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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 [16–19]. It was also reported that P0-Cre mice showed same expression pattern even if the mouse background was changed [58]. Thus, the P0-Cre reporter system seems to be relatively adequate for genetic marking of the NC-derived cells. Genetic marking using Cre-recombinase has been applied to the long-term tracing of NC-derived cells in not only P0-Cre but also Wnt1-Cre mice. We recently got the comparable results of LacZ-expression in dental mesenchyme of the Wnt1-Cre/Rosa26R mouse system (data not shown). Very recently, Kanakubo et al. reported that the specificity of P0-Cre as a genetic marker for NC-derived cells through direct DiI labeling in P0-Cre/Gfp (green fluorescent protein) transgenic embryos, and the expression pattern was comparable to our P0-Cre/Rosa26R system [58]. 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.
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– cells express PDGFR and integrins in dental mesenchymal fractions [29, 30]. Cell surface molecules useful for identifying and isolating NC or mesoderm have not been reported yet. Recently, neural crest cell-specific deletion of PDGFR or ß1-integrin gene under the control of NC-specific promoters showed the function in NC-derived cells [42, 43]. The findings of studies using that approach support the notion that mesenchyme might be composed of NC-derived and non-NC-derived mesoderm [20, 21]. Previously, Saga et al. reported that Mesp1-Cre mouse enabled us to detect mesoderm-derived cells [59]. 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 [60–62]. On the other hand, it has been reported that multipotent NC stem cells are present in the gut and sciatic nerve of fetal rats [63, 64]. Recently, we reported that multipotent NC-derived cells were present in the developing mouse thymus [17]. Sieber-Blum et al. also reported that multipotent NC-derived cells were present in the hair follicles of adult mice [65]. 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.
Recently, multipotent cells have been reported to be present in several tissues [66]. Dental pulp stem cells (DPSCs) were established from permanent teeth [67], and stem cell-like multipotent cells were observed in human exfoliated deciduous teeth (SHED) [68]. DPSCs differentiate into dentin-secreting odontoblasts but not osteoblasts or preadipocytes that support hematopoiesis [67]. SHEDs give rise to odontoblasts, adipocytes, and neuronal cells in culture [68]. Furthermore, it was reported that odontoblasts could be induced from immature NC-derived cells or very early progenitors prior to commitment to the NC lineage, such as bone marrow mesenchymal stem cells and embryonic stem cells in case of cotransplantation with dental epithelia [69–71]. The differentiation of stem cells established from periodontal ligament into cementoblasts and collagen-producing Sharpey-like cells was also reported [72]. 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.
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 [73]. 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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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