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

PLoS One. 2013 Nov 11;8(11):e80026. doi: 10.1371/journal.pone.0080026. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Retracted Publication

MeSH terms

  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism
  • Animals
  • Biomarkers / metabolism
  • Bone Morphogenetic Protein 4 / chemistry
  • Bone Morphogenetic Protein 4 / metabolism
  • Calcification, Physiologic
  • Cell Adhesion
  • Cell Culture Techniques
  • Cell Differentiation
  • Cell Movement
  • Cell Proliferation
  • Collagen Type I / chemistry
  • Collagen Type I / metabolism
  • Extracellular Matrix Proteins / genetics
  • Extracellular Matrix Proteins / metabolism
  • Fibronectins / chemistry
  • Fibronectins / metabolism
  • Gene Expression Regulation
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Integrin alpha2 / genetics*
  • Integrin alpha2 / metabolism
  • Mice
  • Odontoblasts / cytology*
  • Odontoblasts / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Sialoglycoproteins / genetics
  • Sialoglycoproteins / metabolism
  • Tissue Engineering / methods*
  • Tissue Scaffolds

Substances

  • Biomarkers
  • Bone Morphogenetic Protein 4
  • Collagen Type I
  • Dmp1 protein, mouse
  • Extracellular Matrix Proteins
  • Fibronectins
  • Integrin alpha2
  • Phosphoproteins
  • Sialoglycoproteins
  • dentin sialophosphoprotein
  • Alkaline Phosphatase

Grant support

This work was supported by a grant (No. 22791853 to N.O.) from the program Grants-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.