Functional Assays of Human Tooth-Derived Stem Cells

Tooth-derived stem cells are an important resource for tissue engineering approaches that are directed towards matrix regeneration. Whether lineage-specific determination can be accelerated by increased expression of DMP1, a key bone and dentin extracellular matrix (ECM) gene, is not well understood. Objectives: An initial goal was to assess the level of Runx2, Twist-1 and DMP1 expression in three human tooth-derived stem cells: dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs) and stem cells from human exfoliated deciduous teeth (SHEDs). We next evaluated whether increased DMP1 expression in these cell lines would accelerate terminal differentiation as marked by the expression of ECM genes. Methods: Relative levels of expression of Runx2, Twist-1 and DMP1 were determined by semi-quantitative RT-PCR in the dental stem cells cultured with Alpha-MEM/15%FBS. Real-time PCR was used to monitor the mRNA levels of the osteoblast-related factors. DMP1 was transfected into DPSCs, PDLSCs and SHEDs using a novel CMV-mediated adenoviral gene delivery system. Results: Real-time PCR and/or Western blotting showed that baseline expression of DMP1 was seen in DPSCs and SHEDs cells, but not in PDLSCs; Twist-1 was expressed in PDLSCs and SHEDs cells, but not in DPSCs; and Runx2 expression was present in all cell lines. Overexpression of DMP1 did not significantly alter either Runx2 or Twist-1 expression. In addition, expression levels of type I collagen, BSP, DSPP and osteocalcin were unaffected. Conclusions: These preliminary studies indicate that DPSCs, PDLSCs and SHEDs exhibit overlapping yet distinct gene expression profiles and that increased DMP1 expression does not alter the differentiation program of tooth-derived stem cells. Such data provides the framework for tissue engineering strategies involving the use of tooth-derived stem cell lines. (Supported by NIH grant RO1-DE013368 to RDS).