30 research outputs found

    Mouse-induced pluripotent stem cells differentiate into odontoblast-like cells with induction of altered adhesive and migratory phenotype of integrin.

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    Methods for differentiating induced pluripotent stem (iPS) cells into odontoblasts generally require epithelial-mesenchymal interactions. Here, we sought to characterize the cells produced by a 'hanging drop' technique for differentiating mouse iPS cells into odontoblast-like cells that requires no such interaction. Cells were cultured by the hanging drop method on a collagen type-I (Col-I) scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4) without an epithelial-mesenchymal interaction. We evaluated the expression of odontoblast-related mRNA and protein, and the proliferation rate of these cells using reverse-transcription polymerase chain reaction, immunofluorescence staining, and BrdU cell proliferation enzyme-linked immunosorbent assay, respectively. The differentiated cells strongly expressed the mRNA for dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (Dmp-1), which are markers of mature odontoblasts. Osteopontin and osteocalcin were not expressed in the differentiated cells, demonstrating that the differentiated iPS cells bore little resemblance to osteoblasts. Instead, they acquired odontoblast-specific properties, including the adoption of an odontoblastic phenotype, typified by high alkaline phosphatase (ALP) activity and calcification capacity. The cell-surface expression of proteins such as integrins α2, α6, αV and αVβ3 was rapidly up-regulated. Interestingly, antibodies and siRNAs against integrin α2 suppressed the expression of DSPP and Dmp-1, reduced the activity of ALP and blocked calcification, suggesting that integrin α2 in iPS cells mediates their differentiation into odontoblast-like cells. The adhesion of these cells to fibronectin and Col-I, and their migration on these substrata, was significantly increased following differentiation into odontoblast-like cells. Thus, we have demonstrated that integrin α2 is involved in the differentiation of mouse iPS cells into odontoblast-like cells using the hanging drop culture method, and that these cells have the appropriate physiological and functional characteristics to act as odontoblasts in tissue engineering and regenerative therapies for the treatment of dentin and/or dental pulp damage

    Matrix metalloproteinase-3 in odontoblastic cells derived from ips cells: unique proliferation response as odontoblastic cells derived from ES cells.

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    We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken together, IL-1β induced MMP-3-regulated cell proliferation and suppressed apoptosis in odontoblast-like cells derived from iPS and ES cells

    Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells.

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    We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells

    Expression of odontoblastic functional phenotypic markers.

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    <p>(A, B) Alkaline phosphatase (ALP) activity and calcification capacity are characteristic of differentiated odontoblasts, so were measured in iPS cells and E14Tg2a cells treated with or without CS/BMP-4. (A) ALP activity was measured by absorbance at 405 nm and normalized against total protein. (B) The mineralization of iPS and E14Tg2a cells was assessed by Alizarin Red-S (ARS) staining. ARS was measured as absorbance at 405 nm by an SH-1200 Lab instrument. Scale bars = 100 μm. (*<i>P</i> < 0.01, <i>vs</i>. control).</p

    Flow cytometry analysis of integrin expression.

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    <p>Cells were cultured in the absence (control: grey bars) or presence (black bars) of CS (10% Col-I) and BMP-4 for 7 days in the presence of various anti-integrin monoclonal antibodies (as shown). Detection of the relevant secondary antibody was performed by flow cytometry to estimate the numbers of cells expressing each protein. Negative control values (secondary antibody alone) were subtracted from test values to give the mean fluorescence intensity. Data are mean ± SD (n=3).</p

    Effect of anti-integrin α2 mAb and siRNA on functional activity in odontoblast-like cells.

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    <p>Effect on ALP activity. CS/BMP-4-differentiated iPS cells and E14Tg2a cells were treated with either integrin α2-specific siRNA (or control siRNA) or anti-integrin α2 mAbs (or control mAbs). ALP activity data are presented as the mean ± SD (n=4) of the absorbance at 405 nm, normalized against total protein. </p

    Optimization of ECM components required to differentiate iPS cells into odontoblast-like cells.

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    <p>To evaluate the effect of extracellular matrix (ECM) components on the proliferation of embryoid bodies derived from iPS (A) and E14Tg2a ES cells (B), we cultured these cells on various ECM proteins for 14 days, examining cell proliferation at various time points during this period using a BrdU-based cell proliferation ELISA. Data are the degree of cell proliferation normalized against the number of proliferating cells at the start of the assay period (fold of control). Statistically significant increases in proliferation are shown by * and ** (<i>P</i> < 0.05 and <i>P</i> < 0.01, respectively, <i>vs</i>. control). The optimal concentration of collagen scaffold was also determined by growing iPS (C) and E14Tg2a (D) cells for 14 days <i>in </i><i>vitro</i> on plates pretreated with various concentrations of CS (3%, 5%, 7%, 10%, or 15% Col-I). Proliferation was measured at various time points using a BrdU-based ELISA, as above. Data are the mean ± SD of three independent repeats. Differences between untreated and the various CS-treated groups were assessed using a Mann-Whitney U-test (*<i>P</i> < 0.05; **<i>P</i> < 0.01, <i>vs</i>. untreated).</p

    Odontoblastic differentiation of iPS cells on CS/BMP-4.

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    <p>(A) Immunofluorescence staining for the odontoblastic cell marker, dentin sialoprotein (DSP). Images show iPS cells (a, b) and E14Tg2a cells (c, d) that are undifferentiated (a, c) or differentiated using CS/BMP-4 (b, d). Scale bars = 25 μm. (B) Odontoblastic-related mRNA expression in these differentiated cells. Cells were cultured for 7 days with 10% CS and BMP-4 and the expression of various markers (as shown) assessed by RT-PCR. Rat KN-3 odontoblast-like cells (c) were used as a positive control. Mouse MC3T3-E1 cells (d) were used as an osteoblastic control. Band densities for each cell type cultured in the absence (control: white bars) or presence (black bars) of CS/BMP-4 for 7 days were evaluated using a Multi Gauge-Ver3.X (Fujifilm, Tokyo, Japan). Data are normalized against the housekeeping gene, GAPDH, and are presented as the mean ± SD of at least three independent experiments. </p

    Adhesion and motility of differentiated cells on collagen type-I and fibronectin.

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    <p>(A, B) The adhesion of differentiated (black bars) and undifferentiated (white bars) iPS cells to a substratum of either Fn (5 μg/ml; A) or Col-I (1 μg/ml; B) was assayed in the presence of the indicated anti-integrin antibodies. Data are the number of adherent cells, expressed as a percentage of the total number of cells. Bars indicate the standard deviation. (C, D) Similar experiments were used to investigate motility. The migration of differentiated (black bars) or undifferentiated cells (grey bars) through Transwell inserts coated with Fn (5 μg/ml; C) or Col-I (1 μg/ml; D) was assayed in the presence of the indicated anti-integrin antibodies. Cells were added to the upper chamber and incubated for 3 h. Motility was estimated by counting the number of cells that had migrated to the undersides of the membranes. Data presented are the mean ± SD of at least 10 random microscopic fields.</p
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