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Bone Marrow Cells Can Give Rise to Ameloblast-like Cells

¹ INSERM, U595, Faculty of Medicine, 11 rue Humann, 67085 Strasbourg, France;
² Faculty of Dentistry, Louis Pasteur University, 67085 Strasbourg Cedex, France;
³ Molecular Laboratory for Gene Therapy, Faculty of Stomatology, Capital University of Medical Sciences, 4 Tian Tan Xi Li, Beijing, 100050, PR China; and
⁴ Department of Cell Biology and Histology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa 48940, Vizcaya, Spain

View larger version (110K): [in a new window]   Figure 1. Cultured re-associations between dental mesenchyme and bone marrow cells mixed with dental epithelial cells. (A) Crude EGFP bone marrow cells were mixed with dental epithelial cells (1:1), re-associated with dental mesenchyme, and cultured up to 20 days. As shown after H&E staining (A), a typical dental epithelial histological organization was achieved in 30/45 samples. (B–D) In the samples illustrated in (A), anti-EGFP and anti-cytokeratin 14 (CK-14) antibodies revealed that in only 3/30 samples, bone marrow cells could be detected in the inner dental epithelium (IDE). (E,F) In the dental mesenchyme (Mes), bone marrow cells were abundant. (G) CM-DiI labeling and anti-CK-14 antibody show the engraftment of c-kit+-enriched bone marrow cells in the inner dental epithelium after 6 days. Bone marrow cells also existed in the dental mesenchyme. (H–J) Many c-kit+-enriched bone marrow cells engrafted in the inner dental epithelium and also in the dental mesenchyme after 20 days. (K-M) c-kit–-enriched bone marrow cells could be seen only rarely in the dental mesenchyme. Bar = 40 µm (A–D, H–M) and 10 µm (E–G).

View larger version (107K): [in a new window]   Figure 2. c-kit+-enriched bone marrow cells acquired characteristics of ameloblasts. (A,B) In situ hybridization for amelogenin (Amg) and ameloblastin (Amb) shows strong expression in the ameloblast (Am) layer from post-natal day 7 (P7), in the mouse first lower molar. (C,E) c-kit+-enriched bone marrow cells are present in the newly formed tooth structure from re-associations cultured for 20 days. (D,F) Superimposition of in situ hybridization for amelogenin and ameloblastin with EGFP labeling shows that c-kit+-enriched bone marrow cells engrafted in the inner dental epithelium (IDE) layer expressed the two genes. (G,K) Ameloblasts and dental matrix in the P7 mouse first lower molar were characterized by indirect immunofluorescence with antibodies to amelogenin (AMG) (G) and MMP-20 (K). (H–J) The c-kit+-enriched bone marrow cells in the ameloblast layer secreted amelogenin. (L–N) They also secreted MMP-20. (O–Q) Cultured re-association in the presence of BrdU from day 6 to day 8 shows that the engrafted c-kit+-enriched bone marrow cells in the inner dental epithelium (see Fig. 1g) still divided at this early stage. (R–T) BrdU incorporation for 48 hrs before harvesting showed that engrafted c-kit+-enriched bone marrow cells in the inner dental epithelium were no longer dividing when polarized. BrdU-labeled epithelial cells were observed only in the cervical loop (CL) region. DM, Dental Matrix; E, Enamel; Mes, Dental Mesenchyme. Bar = 40 µm (A–F, O–T) and 10 µm (G–N).

View larger version (90K): [in a new window]   Figure 3. c-kit+-enriched bone marrow cells acquired characteristics of odontoblasts. (A) Odontoblasts (Od) in the post-natal day 7 mouse first lower molar expressed Dsp/Dpp as shown by in situ hybridization. (B,C) Indirect immunofluorescence shows that they also secreted DSP (B) and DMP-1 (C) proteins. (D) In re-associations cultured for 20 days, EGFP-labeled cells derived from c-kit+-enriched bone marrow cells are detected in both epithelial and mesenchymal compartments. (E) Superimposition of Dsp/Dpp in situ hybridization with EGFP+ cells visualized in (D) shows that c-kit+-enriched bone marrow cells engrafted in the odontoblast layer expressed Dsp/Dpp. (F–I) In re-associations cultured for 20 days, the EGFP+ bone marrow cells engrafted in the odontoblast layer (G) developed cell processes (F and G, white arrows) and secreted DSP (H), which was deposited in the dental matrix (DM) between odontoblast processes (white arrows), as better visualized in the merged image (I). (J–M) These cells also secreted DMP-1 proteins. Am, Ameloblasts; CK-14, cytokeratin-14; DM, Dental Matrix; Mes, Dental Mesenchyme. Bar = 40 µm (A, D, E) and 10m (B, C, F–M).

View larger version (58K): [in a new window]   Figure 4. c-kit+-enriched bone marrow cells gave rise to ameloblast- and odontoblast-like cells without cell fusion. (A,B) FISH for the Y chromosome (Y-chm) and staining for EGFP reveal that ameloblast-like c-kit+-enriched bone marrow cells did not have a Y chromosome, (C,D) nor did they have more than 2 X chromosomes (X-chm). (E,F) Odontoblast-like c-kit+-enriched bone marrow cells did not have a Y chromosome, (G,H) nor did they have more than 2 X chromosomes. Am, Ameloblasts; Od, Odontoblasts; Mes, Dental Mesenchyme. Bar = 10 µm.