Article

Modulation of satellite cell adhesion and motility following BMP2-induced differentiation to osteoblast lineage

Aichi Gakuin University, Nagoya, Aichi, Japan
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 03/2007; 353(1):54-9. DOI: 10.1016/j.bbrc.2006.11.110
Source: PubMed

ABSTRACT

Quiescent satellite cells represent pluripotent stem cells capable of differentiating into other lineages. To define the potential changes in adhesion and motility in these differentiating cells, we utilized an established model system of murine-derived satellite cells induced with BMP2 to undergo osteoblastic differentiation. When mouse myogenic satellite cells were treated with BMP2, myogenesis was inhibited, and interaction with extracellular matrix ligands was altered. alpha7 integrin expression was rapidly downregulated with attenuation of adhesion and migration on laminin substrates. BMP2 also induced alpha2 integrin expression with increased adhesion and motility on collagen substrates as the pluripotent myoblasts develop into the osteogenic lineage. We examined the effect of BMP2 on alpha7 promoter activity in myoblasts using a CAT reporter gene. BMP2 was found to suppress integrin expression through a transcriptional mechanism. The results identify a novel role for BMP2 in modulating satellite cell integrin expression and altering their interactions with the microenvironment during osteoblastic differentiation.

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Available from: Nobuaki Ozeki
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    • "Immunofluorescence staining was performed as previously described [28]. Cells were seeded at 1×104 cells per well on chamber slides (Nalge Nunc Int., Rochester, NY, USA) coated with poly-L-lysine (Sigma-Aldrich) and cultured overnight. "
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    ABSTRACT: 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.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "Immunofluorescence staining was performed as previously described (Ozeki et al, 2007). Cells were seeded at 1 9 10 4 cells per well on chamber slides (Nalge Nunc Int., Rochester , NY, USA) coated with poly-L-lysine (Sigma-Aldrich) and cultured overnight. "
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    ABSTRACT: Objectives: We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Materials and methods: Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Results: Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Conclusions: Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression.
    Full-text · Article · May 2013 · Oral Diseases
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