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The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling

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Abstract

Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2’-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.

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Acknowledgments

We are grateful to Mr. Shin-ichi Kenmotsu for his technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research (B) (no. 22390341 to H.O.), Scientific Research (C) (no. 23593026 to K.N.-O.) and Exploratory Research (no. 20659296 to H.O.) from MEXT and JSPS.

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Authors and Affiliations

  1. Division of Oral Science for Health Promotion, Department of Oral Health and Welfare, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Yuko Ishikawa

  2. Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Hiroko Ida-Yonemochi & Hayato Ohshima

  3. Pediatric Dentistry, Niigata University Medical and Dental Hospital, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Kuniko Nakakura-Ohshima

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  1. Yuko Ishikawa
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  2. Hiroko Ida-Yonemochi
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  3. Kuniko Nakakura-Ohshima
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  4. Hayato Ohshima
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Correspondence to Hayato Ohshima.

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Supplementary Fig. 1

Dsp-IR in the sections of the first molars at E13 (a), E15 (b), E18 (c), P0 (d), P5 (e), P2W (f, g), and P5W (h) (B bone, D dentin, DP dental pulp, E enamel or enamel space, OC oral cavity). The tooth germ basically lacks Dsp-positive cells except for some epithelial and mesenchymal cells (arrows) (a). Dsp-IR is observed in the epithelial and mesenchymal cells including endothelial cells (arrows) in addition to bone cells (b). Dsp-IR is almost the same as that in the previous stage (c). Dsp-IR is very weak in the differentiated odontoblasts compared with the bone cells (d). Dsp is expressed in the differentiated odontoblasts and the bone and dentin matrices (arrows) (e-h). The sections are counter-stained with methylene blue. Bar 100 μm (a-d), 250 μm (e-h). (JPEG 287 kb)

High resolution image (TIFF 13573 kb)

Supplementary Fig. 2

Negative control for nestin (a, b: n = 1), BrdU (c, d, e, g: n = 1), STRO-1 (d, e, f: n = 1), and CD146 (d, e, f: n = 1) in the dark- (e-g) or light-fields (a-d) and phase-contrast images (h) at P5 (a), P2W (b), P3W (d), and P5W (c, e-f) (B bone, D dentin, DP dental pulp, E enamel space, OC oral cavity). The immunostained sections of negative controls contain no specific immunoreaction. (JPEG 157 kb)

High resolution image (TIFF 7.93 MB)

Supplementary Fig. 3

Aldehyde-fuchsin-Masson-Goldner (AF-MG) (a, c-e) and Azan (g) staining and double immunohistochemistry for nestin and osteopontin (Opn) (b, f, i) or CD31 (h, j) in the sections of the animal at E18 (n = 1: B bone, DP dental papilla, IEE inner enamel epithelium, M Meckel’s cartilage, OC oral cavity, T tongue). Nestin-IR appears in the CD31-positive endothelial cells of the dental papilla (e, h, j; arrows) in addition to the muscle and Opn-positive osteoblasts (a, c-g, i; arrows). Higher magnification of the boxed area labeled by c, d, e in a (c, d, e). Higher magnification of the boxed area labeled by f, i in b (f, i). Higher magnification of the boxed area labeled by j in h (j). Bars 250 μm (a, b), 50 μm (c-h), 25 μm (i, j). (JPEG 276 kb)

High resolution image (TIFF 12656 kb)

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Ishikawa, Y., Ida-Yonemochi, H., Nakakura-Ohshima, K. et al. The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling. Cell Tissue Res 348, 95–107 (2012). https://doi.org/10.1007/s00441-012-1347-2

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