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Temporo-spatial distribution of stem cell markers CD146 and p75NTR during odontogenesis in mice

Abstract

Mesenchymal and epithelial stem cells were identified in dental tissues; however, knowledge about the odontogenic stem cells is limited, and there are some questions regarding their temporo-spatial dynamics in tooth development.

Objective

Our study aimed to analyze the expression of the stem cell markers CD146 and p75NTR during the different stages of odontogenesis.

Methodology

The groups consisted of 13.5, 15.5, 17.5 days old embryos, and 14 days postnatal BALB/c mice. The expression of CD146 and p75NTR was evaluated by immunohistochemistry.

Results

Our results showed that positive cells for both markers were present in all stages of tooth development, and the number of positive cells increased with the progression of this process. Cells of epithelial and ectomesenchymal origin were positive for CD146, and the expression of p75NTR was mainly detected in the dental papilla and dental follicle. In the postnatal group, dental pulp cells were positive for CD146, and the reduced enamel epithelium and the oral mucosa epithelium showed immunostaining for p75NTR.

Conclusions

These results suggest that the staining pattern of CD146 and p75NTR underwent temporal and spatial changes during odontogenesis and both markers were expressed by epithelial and mesenchymal cell types, which is relevant due to the significance of the epithelial-ectomesenchymal interactions in tooth development.

Tooth development; Odontogenic stem cells; Odontogenesis; p75 neurotrophin receptor; CD146 antigen

Introduction

Odontogenesis in mammals begins with the migration of neural crest cells derived from the ectoderm. These cells acquire mesenchymal characteristics and are considered ectomesenchymal cells. The epithelial cells proliferate and migrate into the ectomesenchyme, and interactions between these two tissues form a continuous, thickened, so-called primary epithelial band. Dental lamina differentiates from the primary epithelial band and invaginates into the underlying ectomesenchyme to form the epithelial buds. Then, the dental germs undergo subsequent histological stages of tooth development: bud, cap, bell, crown, and root.11 - Alappat S, Zhang ZY, Chen YP. Msx homeobox gene family and craniofacial development. Cell Res. 2003;13(6):429-42. doi: 10.1038/sj.cr.7290185

Epithelial-ectomesenchymal interactions essential for the development of all dental tissues involve a series of molecular and signaling events,22 - Balic A, Thesleff I. Tissue interactions regulating tooth development and renewal. Curr Top Dev Biol. 2015;115:157-86. doi: 10.1016/bs.ctdb.2015.07.006 in which stem cells play a central role, especially the embryonic ones.33 - Yu T, Volponi AA, Babb R, An Z, Sharpe PT. Stem cells in tooth development, growth, repair, and regeneration. Curr Top Dev Biol. 2015;115:187-212. doi: 10.1016/bs.ctdb.2015.07.010 Sources of adult mesenchymal stem cells (MSCs) were already obtained from different dental tissues;44 - Sharpe PT. Dental mesenchymal stem cells. Development. 2016;143(13):2273-80. doi: 10.1242/dev.134189 several studies demonstrated their promising use for tooth repair and regeneration and for cell-based therapies in regenerative medicine.33 - Yu T, Volponi AA, Babb R, An Z, Sharpe PT. Stem cells in tooth development, growth, repair, and regeneration. Curr Top Dev Biol. 2015;115:187-212. doi: 10.1016/bs.ctdb.2015.07.010 On the other hand, the isolation and characterization of stem cells involved in tooth development, i.e., odontogenic stem cells, remains a challenge.

The odontogenic epithelial stem cells (OEpSCs) are present in the dental lamina, epithelial cell rests of Malassez, reduced enamel epithelium, and junctional epithelium.55 - Padma Priya S, Higuchi A, Abu Fanas S, Pooi Ling M, Kumari Neela V, Sunil PM, et al. Odontogenic epithelial stem cells: hidden sources. Lab Invest. 2015;95(12):1344-52. doi: 10.1038/labinvest.2015.108 At the apical end of the murine incisors, there is an epithelial stem cell niche with cells that migrate and differentiate into ameloblasts.66 - Krivanek J, Soldatov RA, Kastriti ME, Chontorotzea T, Herdina AN, Petersen J, et al. Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth. Nat Commun. 2020;11(1):4816. doi: 10.1038/s41467-020-18512-7 Moreover, ectomesenchymal stem cells (EMSCs) are present in the dental papilla and in the dental follicle that surrounds the tooth germ. These cells are essential to both the epithelium-ectomesenchymal interaction and the tooth morphogenesis.77 - Yang K, Wang Y, Ju Y, Li G, Liu C, Liu J, et al. p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells. Cell Prolif. 2017;50(1):e12290. doi: 10.1111/cpr.12290,88 - Chai Y, Jiang X, Ito Y, Bringas P Jr., Han J, Rowitch DH, et al. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127(8):1671-9.

Mesenchymal and epithelial stem cell are identified by using cell surface markers;99 - Mo M, Wang S, Zhou Y, Li H, Wu Y. Mesenchymal stem cell subpopulations: phenotype, property and therapeutic potential. Cell Mol Life Sci. 2016;73(17):3311-21. doi: 10.1007/s00018-016-2229-7,1010 - Jones KB, Klein OD. Oral epithelial stem cells in tissue maintenance and disease: the first steps in a long journey. Int J Oral Sci. 2013;5(3):121-9. doi: 10.1038/ijos.2013.46 however, the odontogenic stem cell markers are still not well established. CD146 (MCAM/MUC18) is a transmembrane glycoprotein expressed mainly at the endothelial intercellular junction. It is involved in angiogenesis and may play a role in cell signaling, migration, and differentiation.1111 - Wang Z, Xu Q, Zhang N, Du X, Xu G, Yan X. CD146, from a melanoma cell adhesion molecule to a signaling receptor. Signal Transduct Target Ther. 2020;5(1):148. doi: 10.1038/s41392-020-00259-8 This adhesion molecule was initially used as a selective melanoma biomarker,1111 - Wang Z, Xu Q, Zhang N, Du X, Xu G, Yan X. CD146, from a melanoma cell adhesion molecule to a signaling receptor. Signal Transduct Target Ther. 2020;5(1):148. doi: 10.1038/s41392-020-00259-8,1212 - Lei X, Guan CW, Song Y, Wang H. The multifaceted role of CD146/MCAM in the promotion of melanoma progression. Cancer Cell Int. 2015;15(1):3. doi: 10.1186/s12935-014-0147-z but, currently, it is considered an important marker of MSCs and pericytes.99 - Mo M, Wang S, Zhou Y, Li H, Wu Y. Mesenchymal stem cell subpopulations: phenotype, property and therapeutic potential. Cell Mol Life Sci. 2016;73(17):3311-21. doi: 10.1007/s00018-016-2229-7,1313 - Crisan M, Corselli M, Chen WC, Péault B. Perivascular cells for regenerative medicine. J Cell Mol Med. 2012;16(12):2851-60. doi: 10.1111/j.1582-4934.2012.01617.x In different tissues such as dental pulp, periodontal ligament and apical papilla, the CD146+/STRO-1+ profile proved to be the most efficient to isolate MSCs.1414 - Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002;81(8):531-5. doi: 10.1177/154405910208100806

15 - Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364(9429):149-55. doi: 10.1016/s0140-6736(04)16627-0
-1616 - Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod. 2008;34(2):166-71. doi: 10.1016/j.joen.2007.11.021 One of the first studies that identified the perivascular niche of dental pulp stem cells (DPSCs) used CD146 in situ expression to locate these cells.1717 - Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res. 2003;18(4):696-704. doi: 10.1359/jbmr.2003.18.4.696 In addition, this surface marker selects cells that are more clonogenic and that show better multi-differentiation properties; it can also separate DPSCs from dental pulp fibroblasts.1818 - Tavangar MS, Attar A, Razmkhah M, Hosseini SM, Hosseini A, Monabati A, et al. Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts. Clin Exp Dent Res. 2020;6(4):448-56. doi: 10.1002/cre2.297

The p75 neurotrophin receptor (p75NTR), also known as NGFR/CD271, is a component of the tumor necrosis factor receptor superfamily that commonly binds to neurotrophins, such as neurotrophin-3 and -4/5, nerve growth factor, and brain-derived neurotrophic factor.1919 - Tomellini E, Lagadec C, Polakowska R, Le Bourhis X. Role of p75 neurotrophin receptor in stem cell biology: more than just a marker. Cell Mol Life Sci. 2014;71(13):2467-81. doi: 10.1007/s00018-014-1564-9 p75NTR mediates cellular events such as cell apoptosis, adhesion, differentiation, and invasion, and may be involved in intracellular signaling.1919 - Tomellini E, Lagadec C, Polakowska R, Le Bourhis X. Role of p75 neurotrophin receptor in stem cell biology: more than just a marker. Cell Mol Life Sci. 2014;71(13):2467-81. doi: 10.1007/s00018-014-1564-9,2020 - Pan W, Kremer KL, Kaidonis X, Ludlow VE, Rogers ML, Xie J, et al. Characterization of p75 neurotrophin receptor expression in human dental pulp stem cells. Int J Dev Neurosci. 2016;53:90-8. doi: 10.1016/j.ijdevneu.2016.07.007 Besides being a tumor marker, especially in the gastrointestinal tract,2121 - Blondy S, Christou N, David V, Verdier M, Jauberteau MO, Mathonnet M, et al. Neurotrophins and their involvement in digestive cancers. Cell Death Dis. 2019;10(2):123. doi: 10.1038/s41419-019-1385-8 p75NTR is regarded as a marker of mesenchymal and epithelial stem cells. In populations of embryonic stem cells, p75NTR is a key marker for purifying neural crest cells.2222 - Lee G, Kim H, Elkabetz Y, Al Shamy G, Panagiotakos G, Barberi T, et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol. 2007;25(12):1468-75. doi: 10.1038/nbt1365 In the pulp of permanent teeth, p75NTR is expressed in a small subpopulation of undifferentiated neural stem cells, which coexpress CD146.2020 - Pan W, Kremer KL, Kaidonis X, Ludlow VE, Rogers ML, Xie J, et al. Characterization of p75 neurotrophin receptor expression in human dental pulp stem cells. Int J Dev Neurosci. 2016;53:90-8. doi: 10.1016/j.ijdevneu.2016.07.007 This population has been referred to as ectomesenchymal stem cells.2323 - Alvarez R, Lee HL, Hong C, Wang CY. Single CD271 marker isolates mesenchymal stem cells from human dental pulp. Int J Oral Sci. 2015;7(4):205-12. doi: 10.1038/ijos.2015.29,2424 - Martens W, Wolfs E, Struys T, Politis C, Bronckaers A, Lambrichts I. Expression pattern of basal markers in human dental pulp stem cells and tissue. Cells Tissues Organs. 2012;196(6):490-500. doi: 10.1159/000338654 The presence of p75NTR-positive cells in the dental pulp and in the periodontal ligament indicates that neural crest stem cells, with potential for neurodifferentiation, are present in adult tissues.2525 - Alvarez R, Lee HL, Wang CY, Hong C. Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers. Int J Oral Sci. 2015;7(4):213-9. doi: 10.1038/ijos.2015.42

There are still many questions regarding stem cells and tooth development. The temporo-spatial dynamics among the odontogenic stem cells is unknown, and it is unclear if they proportionally decrease in number or remain constant in the different tissues during development. The murine models give important contributions to the study of cellular and molecular events in odontogenesis; they are relevant for reproducing these mechanisms in vitro,2626 - Jernvall J, Thesleff I. Tooth shape formation and tooth renewal: evolving with the same signals. Development. 2012;139(19):3487-97. doi: 10.1242/dev.085084 and also for the research in stem cell-based dental regenerative therapies and tooth bioengineering. Therefore, this study aimed to evaluate the immunoexpression of CD146 and p75NTR during the different stages of molar tooth development in mice.

Methodology

Animals and tissue preparation

This study was approved by the Ethics Committee on Animal Research of our Institution.

Four male and four female BALB/c mice (Mus musculus; 4-week-old; initial body weight: 18-21 g) were housed in individual cages with free access to food and water. They were maintained in a room under a 12-hour light/dark cycle, with a controlled temperature of 23ºC and were allowed to acclimate for 7 days before any procedure.

The four couples were mated overnight, and the day after the mating was designated as “0.5” day of pregnancy if the vaginal plug was present. After mating, the females were individually housed in cages with the same aforementioned conditions, and males were euthanized.

For this qualitative study, each experimental group was composed by the litter of a female. The groups consisted of embryos of 13.5 (E13.5, n=7), 15.5 (E15.5, n=9) and 17.5 (E17.5, n=9) days old, and 14 days postnatal mice (PN14, n=5). In total, 21 embryos (seven mice in each group: E13.5, E15.5 and E17.5) were used to analyze the stages of odontogenesis. In the PN14 group, tooth germs from 5 lower jaws and 5 upper jaws were evaluated.

The mice were euthanized with an intramuscular injection of xylazine (100 mg/mL; Anasedan, Ceva, Paulínia – Brazil) and ketamine (50 mg/mL; Dopalen, Ceva, Paulínia – Brazil). After the euthanasia, the heads of the embryos were collected and processed, whereas, in the postnatal group, the upper and lower jaws were first dissected and then processed. All samples were fixed in 4% formaldehyde for 24 hours at room temperature. The molar regions from E17.5 and PN14 were decalcified in 4.13% EDTA for a period sufficient to achieve complete demineralization. Subsequently, the specimens were dehydrated in ascending concentrations of ethanol, diaphanized and embedded in paraffin.

Morphological evaluation and immunohistochemistry

For each specimen that composed the sample of this study, 1 representative hematoxylin and eosin histological section was selected, in which at least 1 tooth germ of the corresponding odontogenesis stage could be visualized. Afterwards, serial sections were obtained for immunohistochemistry.

Five-μm thick sections were stained with hematoxylin and eosin and examined in a light microscope for morphological analysis.

For the immunohistochemical study, 3 μm thick sections were dewaxed, rehydrated and pretreated for antigen retrieval (citrate buffer, pH 6.0) for 30 min at 95°C in water bath. Then, the slides were incubated for 30 min in a 6% hydrogen peroxide/methanol (1:1) solution to quench endogenous peroxidase activity. After a water rinse, sections were incubated in Tris-buffered saline (TBS, pH 7.4) for 15 min. Primary monoclonal antibodies against CD146 (clone EPR3208, dilution 1:100, Abcam, Cambridge, UK) and p75NTR (clone D4B3, dilution 1:100, Cell Signaling Technology, Danvers, MA) were incubated for 18 h at 4°C. EnVision+ Dual Link System-HRP (Dako, Carpinteria, CA, USA) was used for 30 min at room temperature to detect the reactions, and diaminobenzidine (DAB+, Dako) was used as the chromogen. Samples were counterstained with Mayer’s hematoxylin and analyzed under a light microscope. All reactions were performed with appropriate positive and negative controls. Negative controls were incubated in the absence of the primary antibody. Oral mucosa sections of human specimens and adult mice were used as positive controls.

The analysis of immunohistochemical reactions was semiquantitative. In every section examined, cells were considered positive or negative for CD146 and p75NTR. The intensity of the immunostaining was not analyzed due to its subjectivity, and the scoring for the expression of both markers was considered: negative (-); up to 5% positive cells (+); from 5% to 50% of positive cells (++); more than 50% positive cells (+++).

Results

At the bud stage (E13.5), the dental lamina and the oral epithelium were considered as “dental epithelium” due to the difficulty in differentiating them anatomically. We observed that the ectomesenchymal cells gathered around the invaginated dental epithelium. Positive cells for CD146 were detected on the dental epithelium and ectomesenchyme, whereas p75NTR was expressed only in the ectomesenchymal condensation (Figure 1A–C).

Figure 1
Morphological aspects and CD146 and p75NTR immunoexpression in mice odontogenesis. Rows represent, from top to bottom, the bud (A–C), cap (D–F) and bell (G–I) stages of the tooth germ, and the 14 days postnatal tooth (J–L). (A, D, G, J) show the main morphological characteristics in the different stages. At the bud stage, the immunohistochemical expression of CD146 was detected in the dental epithelium and ectomesenchymal condensation (B). For p75NTR, the immunostaining was restricted to the ectomesenchymal cells (C). At the cap stage, CD146 was found in the outer enamel epithelium, stellate reticulum, dental follicle, and oral epithelium (E). An intense expression of p75NTR was seen in the dental papilla and dental follicle (F). During the bell stage, the CD146 was positive in the inner enamel epithelium, stratum intermedium, dental follicle, and dental papilla (H), and p75NTR was detected in the inner enamel epithelium, dental follicle and dental papilla (I). In the postnatal group, only the dental pulp cells were positive for CD146 (K), while p75NTR immunostaining was seen in the reduced enamel epithelium and oral mucosa epithelium (L). Magnification: A–I, 400× in the original; H–J, 100×

During the cap stage (E15.5), as the tooth germ grew, the epithelial bud acquired a cap appearance as well as a higher density of ectomesenchymal cells. From this stage on, the enamel organ and the dental papilla were also observed. CD146-positive cells were detected in the oral epithelium, dental lamina, outer enamel epithelium, dental follicle, and dental papilla. The cells of the inner enamel epithelium and stellate reticulum showed weaker staining for CD146. For p75NTR, the dental follicle, dental papilla, and inner enamel epithelium exhibited positive cells. Cells of the stellate reticulum showed a faint immunoreactivity, whereas the staining in oral epithelium, dental lamina, and outer enamel epithelium was absent (Figure 1D–F).

In the bell stage (E17.5), cells undergo differentiation; the tooth germ shows a pronounced concavity and the presence of the stratum intermedium and the cervical loop. CD146 immunostaining was seen in oral epithelium, dental lamina, dental follicle, dental papilla, and in all enamel organ cells. p75NTR-positive cells were detected in the inner enamel epithelium, stratum intermedium, dental follicle, and dental papilla (Figure 1G–I).

In the PN14 group, the alveolar bone resorption was seen around the germs, and the tooth root and crown were formed. Thus, we observed the enamel space because of the decalcification process, besides the dentine deposition and the dental pulp showing blood vessels. CD146 staining was restricted to dental pulp cells, whereas p75NTR-positive cells were detected in the reduced enamel epithelium and in the oral mucosa epithelium, in the basal layer (Figure 1J–L). Table 1 shows the results of the immunohistochemical analysis.

Table 1
Expression of p75NTR and CD146 during tooth development

Discussion

In our study, we showed that all the stages of odontogenesis expressed cells positive for p75NTR and CD146, thus showing the participation of stem cells during tooth initiation, proliferation, morphodifferentiation, and histodifferentiation. Different odontogenic cell populations expressed these markers, which were present since the early stages of tooth development and showed variations in the staining pattern and intensity. Although cell proliferation and mitotic activity are higher in the bud and cap stages,11 - Alappat S, Zhang ZY, Chen YP. Msx homeobox gene family and craniofacial development. Cell Res. 2003;13(6):429-42. doi: 10.1038/sj.cr.7290185 CD146 and p75NTR-positive cells increased as odontogenesis progressed, with an emphasis on the bell stage.

Besides the regulation of cell death and survival, p75NTR seems to be involved in the differentiation of odontoblasts and ameloblasts.2727 - Luukko K, Moshnyakov M, Sainio K, Saarma M, Sariola H, Thesleff I. Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation. Dev Dyn. 1996;206(1):87-99. doi: 10.1002/(sici)1097-0177(199605)206:1<87::aid-aja8>3.0.co;2-x
https://doi.org/10.1002/(sici)1097-0177(...
,2828 - Mitsiadis TA, Magloire H, Pagella P. Nerve growth factor signalling in pathology and regeneration of human teeth. Sci Rep. 2017;7(1):1327. doi: 10.1038/s41598-017-01455-3 In the primary culture of EMSCs, the p75NTR positive cells showed a greater potential to differentiate into odontoblastic-like cells when compared with those that were negative for the marker.2929 - Xing Y, Nie X, Chen G, Wen X, Li G, Zhou X, et al. Comparison of P75 NTR-positive and -negative etcomesenchymal stem cell odontogenic differentiation through epithelial-mesenchymal interaction. Cell Prolif. 2016;49(2):185-94. doi: 10.1111/cpr.12248 Also, p75NTR mediated the differential mineralization in rodent EMSCs77 - Yang K, Wang Y, Ju Y, Li G, Liu C, Liu J, et al. p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells. Cell Prolif. 2017;50(1):e12290. doi: 10.1111/cpr.12290 and has recently been suggested as a possible regulator of dental morphogenesis.3030 - Zhao M, Wen X, Li G, Ju Y, Wang Y, Zhou Z, et al. The spatiotemporal expression and mineralization regulation of p75 neurotrophin receptor in the early tooth development. Cell Prolif. 2019;52(1):e12523. doi: 10.1111/cpr.12523

During the early stages of tooth development, at first, only the ectomesenchymal condensation exhibited cells positive for p75NTR. Then, from the cap stage onwards, both epithelial and mesenchymal cells showed a positive immunolabeling. This pattern was previously described in human fetal teeth, in which positive cells were initially restricted to the ectomesenchyme and then distributed into the undifferentiated cells of the inner enamel epithelium, dental follicle, and nerve fibers.3131 - Mitsiadis TA, Pagella P. Expression of Nerve Growth Factor (NGF), TrkA, and p75(NTR) in developing human fetal teeth. Front Physiol. 2016;7:338. doi: 10.3389/fphys.2016.00338 In the postnatal stages of odontogenesis, only the oral mucosa epithelium showed cells positive for p75NTR.

In vitro studies showed that p75NTR is expressed in cultured human dental pulp cells,2020 - Pan W, Kremer KL, Kaidonis X, Ludlow VE, Rogers ML, Xie J, et al. Characterization of p75 neurotrophin receptor expression in human dental pulp stem cells. Int J Dev Neurosci. 2016;53:90-8. doi: 10.1016/j.ijdevneu.2016.07.007,3232 - Wang DR, Wang YH, Pan J, Tian WD. Neurotrophic effects of dental pulp stem cells in repair of peripheral nerve after crush injury. World J Stem Cells. 2020;12(10):1196-213. doi: 10.4252/wjsc.v12.i10.1196 but, in our sample, the dental pulp was negative for this marker. This result was previously seen in healthy adult human teeth, in which the p75NTR expression was absent in the dental pulp fibroblasts, but positive in nerve fibers.2828 - Mitsiadis TA, Magloire H, Pagella P. Nerve growth factor signalling in pathology and regeneration of human teeth. Sci Rep. 2017;7(1):1327. doi: 10.1038/s41598-017-01455-3

Neural crest-derived cells and epithelial cells derived from the oral ectoderm are the two populations essential for tooth development. They form, respectively, dental mesenchymal tissues and dental enamel.33 - Yu T, Volponi AA, Babb R, An Z, Sharpe PT. Stem cells in tooth development, growth, repair, and regeneration. Curr Top Dev Biol. 2015;115:187-212. doi: 10.1016/bs.ctdb.2015.07.010 In addition, a third population is involved in this process, the glial-derived cells. They are capable of originating dental MSCs and are positive for S-100 protein, Sox10, and p75NTR.3333 - Couve E, Lovera M, Suzuki K, Schmachtenberg O. Schwann cell phenotype changes in aging human dental pulp. J Dent Res. 2018;97(3):347-55. doi: 10.1177/0022034517733967,3434 - Kaukua N, Shahidi MK, Konstantinidou C, Dyachuk V, Kaucka M, Furlan A, et al. Glial origin of mesenchymal stem cells in a tooth model system. Nature. 2014;513(7519):551-4. doi: 10.1038/nature13536 Our results showed a restricted expression of p75NTR in epithelial cells and a more widespread expression in the ectomesenchymal derived cells. The immunostaining was particularly intense in the dental papilla and dental follicle as odontogenesis progressed. Thus, we speculate that, in tooth development, p75NTR might be a central marker of cells derived from the neural crest and cells with a glial phenotype, such as Schwann cells, but this requires further investigation.

CD146 is an MSC marker expressed in several tissues, including part of those of dental origin. This marker is also present in the pericytes, which are perivascular cells that surround the endothelium of capillaries and microvessels.1313 - Crisan M, Corselli M, Chen WC, Péault B. Perivascular cells for regenerative medicine. J Cell Mol Med. 2012;16(12):2851-60. doi: 10.1111/j.1582-4934.2012.01617.x,3535 - Yianni V, Sharpe PT. Perivascular-derived mesenchymal stem cells. J Dent Res. 2019;98(10):1066-72. doi: 10.1177/0022034519862258 CD146 expression was related to the epithelial-mesenchymal transition in cancer;3636 - Ma Y, Zhang H, Xiong C, Liu Z, Xu Q, Feng J, et al. CD146 mediates an E-cadherin-to-N-cadherin switch during TGF-β signaling-induced epithelial-mesenchymal transition. Cancer Lett. 2018;430:201-14. doi: 10.1016/j.canlet.2018.05.016,3737 - Liu WF, Ji SR, Sun JJ, Zhang Y, Liu ZY, Liang AB, et al. CD146 expression correlates with epithelial-mesenchymal transition markers and a poor prognosis in gastric cancer. Int J Mol Sci. 2012;13(5):6399-406. doi: 10.3390/ijms13056399 however, to the best our knowledge, this marker has not been reported in epithelial-mesenchymal interaction in tooth development. Although CD146 is a surface molecule used to identify mesenchymal cells, at the embryonic stages of odontogenesis, we observed all layers of the enamel organ immunostaining for CD146. This expression by both epithelial and mesenchymal cell types is interesting because the communication between them is an essential process in odontogenesis.22 - Balic A, Thesleff I. Tissue interactions regulating tooth development and renewal. Curr Top Dev Biol. 2015;115:157-86. doi: 10.1016/bs.ctdb.2015.07.006

In the postnatal group, scattered dental pulp cells expressed CD146 while the odontoblasts were negative. A previous study showed that the cells around the pulp blood vessels are CD146-positive in deciduous teeth.3838 - Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100(10):5807-12. doi: 10.1073/pnas.0937635100 Keller, et al.3939 - Keller LV, Kuchler-Bopp S, Lesot H. Restoring physiological cell heterogeneity in the mesenchyme during tooth engineering. Int J Dev Biol. 2012;56(9):737-46. doi: 10.1387/ijdb.120076hl (2012) observed an important heterogeneity in the immunostaining of dental pulp cells in development. The blood vessels and pericytes in the fully formed tooth pulp were positive for CD146, while, in the cap stage of odontogenesis, CD146-positive cells were present in the odontogenic epithelium. Further, Xiao, et al.4040 - Xiao L, Ide R, Saiki C, Kumazawa Y, Okamura H. Human dental pulp cells differentiate toward neuronal cells and promote neuroregeneration in adult organotypic hippocampal slices in vitro. Int J Mol Sci. 2017;18(8):1745. doi: 10.3390/ijms18081745(2017) showed that the cells around odontoblasts, in the cell-rich zone of the pulp of permanent teeth, exhibited a positive staining for CD146.4040 - Xiao L, Ide R, Saiki C, Kumazawa Y, Okamura H. Human dental pulp cells differentiate toward neuronal cells and promote neuroregeneration in adult organotypic hippocampal slices in vitro. Int J Mol Sci. 2017;18(8):1745. doi: 10.3390/ijms18081745 In our sample, these cells were negative for the marker. We speculate that both the odontoblast layer and the subodontoblastic region may be positive for CD146 in later stages, when the dental pulp is fully developed.

Figure 2 summarizes our findings with a schematic representation of the relationship between the markers in the stages of odontogenesis. In the enamel knot signaling centers, which participate in regulating tooth morphogenesis, p75NTR was expressed by both epithelial and ectomesenchymal cells, whereas CD146 was absent. This absence of cells positive for CD146 in these zones of the tooth germ may indicate its association with more undifferentiated states of the odontogenic epithelium. These findings may be useful in selecting cell populations for stem cell-based therapies for tooth repair and regeneration.

Figure 2
Schematic representation of the immunopositivity for CD146 and p75NTR in the stages of odontogenesis. In the box, we represent an enamel knot during the early bell stage. These signaling centers are responsible for the epithelial-mesenchymal interaction necessary for the regulation of tooth morphogenesis. In this region, both epithelial and ectomesenchymal cells were positive for p75NTR

Conclusions

In conclusion, the four stages of odontogenesis in mice exhibited positive cells for the stem cell markers CD146 and p75NTR. Furthermore, the staining patterns of both markers underwent temporal and spatial changes during tooth development. In general, the number of positive cells increased with the progression of odontogenesis. Our study demonstrated, for the first time, the expression of the CD146 marker in this process and confirmed the immunostaining for p75NTR in odontogenic and neural crest-derived cells. In addition, both markers were expressed by cells of epithelial and mesenchymal origin, which is interesting due to the crucial epithelial-mesenchymal interactions during odontogenesis.

Acknowledgements

This study was supported by CNPq – National Council for Scientific and Technological Development (grant #429985/2016-9). Ms. A. Queiroz received a scholarship from FAPESP – São Paulo Research Foundation (#2016/02055-5) during the period in which this study was carried out.

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Publication Dates

  • Publication in this collection
    20 Sept 2021
  • Date of issue
    2021

History

  • Received
    21 Mar 2021
  • Reviewed
    29 May 2021
  • Accepted
    28 June 2021
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