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Human Hertwig’s Epithelial Root Sheath Cells Play Crucial Roles in Cementum Formation
W. Sonoyama1,2,
B.-M. Seo3,
T. Yamaza1 and
S. Shi1,*
1 Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA;
2 Department of Oral and Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; and
3 Department of Oral and Maxillofacial Surgery, School of Dentistry, Dental Research Institute, Seoul National University, Seoul, Korea
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Figure 1. HERS cells induce PDLSC differentiation. (A) Human HERS cells were isolated and expanded with serum-free medium. (Scale bar = 500 µm for A.) (B) Human HERS cells were cultured under osteoinductive conditions containing L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate for 4 wks. Alizarin red staining showed mineralized nodule formation in the culture. (C) Human PDLSCs were cultured under osteo-inductive conditions and stained with Alizarin red as described in (B). (Scale bar = 100 µm for B and C.) (D–F) Co-culture of HERS cells (D) with PDLSCs (E) in osteo-inductive conditions and stained with Alizarin red, showing an elevated mineralized nodule formation compared with osteo-induced PDLSCs without co-culture with HERS cells (F). (G–I) Co-culture of HERS cells (G) with BMMSCs (H) in the osteo-inductive conditions and stained with Alizarin red, showing the same amounts of mineralized nodule formation with osteo-induced BMMSCs as without co-culture with HERS cells (I). (Scale bar = 10 mm for D–I.) (J) FACS analysis showed that ex vivo expanded BMMSCs and BMMSCs co-cultured with HERS cells (BMMSC/H) expressed similar levels of the STRO-1 molecule (15.7% and 16.1%, respectively). However, PDLSCs co-cultured with HERS cells (PDLSC/H) showed a decreased expression of STRO-1 (6.8%) compared with PDLSCs expanded ex vivo (13.8%).
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Figure 2. Immunohistochemical phenotype of HERS cells. (A) Human HERS cells expressed a variety of epithelial, cemento/osteogenic, and mesenchymal markers (open arrows), including amelogenin, pancytokeratin, E-cadherin, BSP, OCN, vimentin, β-catenin, and N-cadherin. In addition, cementoblasts (triangles) expressed amelogenin, BSP, OCN, vimentin, β-catenin, and N-cadherin. Pre-immunoserum was used as the negative control. HERS (open arrows) cells expressed TGFβ1, TGFβ receptor 1, and TGFβ receptor II. Cementoblasts (triangles) also expressed TGFβ receptors I and II. (Scale bar = 25 µm for A.) (B) Double-immunostaining showed that cytokeratin co-expressed with N-cadherin in HERS clusters and individual HERS cell (upper 2 panels), and E-cadherin co-expressed with osteocalcin (lower middle panel). Pre-immunoserum was used as the negative control (lower panel). (Scale bar = 10 µm for B.)
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Figure 3. TGFβ1-mediated epithelial-mesenchymal transition of HERS cells. (A–D) Under the regular serum-free culture conditions, HERS cells showed the cuboidal morphology of epithelial cells (A,C). When treated with TGFβ1 for 48 hrs, HERS cells underwent transition to an elongated shape (B,D). (Scale bar in A and B = 100 µm; in C and D = 25 µm.) (E) After TGFβ1 treatment, HERS cells showed, by Western blot analysis, a typical epithelial-mesenchymal transition, with down-regulated expression of E-cadherin and β-catenin, and up-regulated expression of N-cadherin and vimentin. (F) Immunocytochemical staining confirmed the down-regulated expression of E-cadherin and the up-regulated expression of N-cadherin and vimentin following a TGFβ1 induction. (Scale bar in F = 50 µm.)
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Figure 4. The TGFβ1/AKT pathway may contribute to the epithelial-mesenchymal transition of HERS cells. (A) Phospho-AKT was detectable in cultured HERS cells, and its expression level was increased when treated with TGFβ1 for 1 hr. Three-hour pre-treatment by LY294002 (an inhibitor of PI3K) significantly decreased phospho-AKT expression. Total AKT expression seemed unaltered by LY294002 in the TGFβ1 treatment group. (B) After 48-hour TGFβ1 treatment, there was an up-regulated expression of vimentin, and LY294002 pre-treatment inhibited this up-regulation. In contrast, the E-cadherin expression level was not recovered by LY294002 pre-treatment. β-actin was used to assess the amounts of protein loading. (C) HERS cells were treated with TGFβ1 for 48 hrs in vitro and then transplanted subcutaneously into immunocompromised mice, with HA/TCP particles as carrier. Eight wks after transplantation, HERS cells generated cementum-like tissue (C, arrows) on the surface of HA/TCP (HA). (D) Immunohistochemical staining showed that cells responsible for forming cementum-like tissue (C) were positive for anti-human-specific mitochondria antibody staining (arrows). (E) Pre-immunoserum control showed negative staining. (F–H) Immunohistochemical staining showed that cells responsible for forming cementum-like tissue were positive for anti-BSP antibody staining (arrows, F) and anti-ALP antibody (arrows, G) staining. Pre-immunoserum control showed negative staining (H). (Scale bar in C–H = 100 mm).
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Journal of Dental Research, Vol. 86, No. 7,
594-599 (2007)
DOI: 10.1177/154405910708600703
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