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Characterization of Dental Pulp Stem Cells of Human Tooth Germs
T. Takeda1,
Y. Tezuka2,
M. Horiuchi3,
K. Hosono2,
K. Iida1,
D. Hatakeyama1,
S. Miyaki3,
T. Kunisada2,
T. Shibata1 and
K. Tezuka2,4,*
1 Department of Oral and Maxillofacial Science and
2 Department of Tissue and Organ Development, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu City, Gifu 501-1194, Japan;
3 Department of Regenerative Biology and Medicine, National Research Institute for Child Health and Development, 2-10-1 Ookura, Setagaya, Tokyo 157-8535, Japan; and
4 PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 322-0012, Japan
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Figure 1. Differentiation of hDPSCs. (A–C) In vitro differentiation of hDPSCs. hDPSCs collected at the crown- (DP1) and root-completed (DP75) stages were cultured and then tested for their ability to differentiate into cells of multiple lineages. Cell morphology of DP1 cells after 5 days in culture is shown in (A). Osteogenesis was assessed by ALP-activity staining (B) and with von Kossa stain (C). Scale bar = 200 µm (A–C). (D–I) Characterization of hDPSCs (crown-completed stage [DP1] and root-formative stage [DP27]) transplanted into NOD-SCID mice for assessment of differentiation in vivo. DP1 (D–F) and DP27 (G–I) cells were seeded onto calcium-phosphate scaffolds and transplanted subcutaneously into NOD-SCID mice. Tissues were isolated at 15 wks post-transplantation and stained with hematoxylin and eosin (D,E,G,H) or with von Kossa (F,I) stain. Polarized light demonstrates alignment of the collagen fibers on the forming surfaces (E,H). In the hDPSC transplants, the scaffolds (Sc) are lined with a dentin-like matrix (d) surrounding connective tissue (CT). Arrows show the calcified matrix (F,I). Scale bar = 500 µm (D–I).
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Figure 2. Growth curves and change in differentiation potential of hDPSCs during long-term culture. (A) Curves for long-term growth obtained from 11 cell lines isolated from individuals of various stages (crown-completed stage, DP1, 2, 11, 28, and 31 [red]; root-formative stage, DP27 [black]; root-completed stage, DP29, 33, 34, 54, and 75 [black]). Cells isolated from the crown-completed stage maintained a high growth rate for more than 2 mos, whereas those from later stages showed a lower growth rate. (B) ALP staining of crown-completed stage (DP1 [top]) and root-formative stage (DP27 [bottom]) cell cultures at P3, 10, 20, and 30, respectively. DP1 and DP27 cells lost their differentiation capability induced by confluence after long-term culture. (C) In vivo differentiation of crown-completed stage (DP1) cells at P4 and P10. Transplants were removed at 8 wks post-transplantation, sectioned, and stained with hematoxylin and eosin. High-magnification view of the boxed area in the left photo is shown in the middle one. In P4 transplants, the scaffolds (Sc) were lined with a layer of dentin-like matrix (d, middle) surrounding connective tissue (CT) and an interface layer of odontoblast-like cells (od, middle); whereas no dentin-like matrix was observed in P10 transplants. Scale bar = 200 µm (left and right), 100 µm (middle).
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Figure 3. Change in expression levels of WNT16, TPD52, ITPR1, and TLR4 mRNA in long-term cultures. Real-time PCR analysis was performed with crown-completed stage (DP2, DP28, and DP31) and root-completed stage (DP33, DP54, and DP75) cells at P4, P10, and P20. The relative amounts of WNT16 (A), TPD52 (B), ITPR1 (C), and TLR4 (D) mRNA were divided by that of GAPDH and used to calculate expression coefficients. Error bars indicate the standard deviation obtained from duplicate analyses.
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Journal of Dental Research, Vol. 87, No. 7,
676-681 (2008)
DOI: 10.1177/154405910808700716
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