Use of αSMA-GFP to define progenitor cells in dental pulp

The identity of cells involved in the regenerative process of the dental pulp is still unknown. Recent evidence supports the hypothesis that perivascular cells of the postnatal mesenchyme represent a progenitor population capable of repairing damaged tissues. Objectives: To define a progenitor population within dental pulp using a transgenic mouse carrying the α-smooth muscle actin promoter driven expression of green fluorescent protein (αSMA-GFP). Methods: GFP expression in teeth of transgenic mice was examined in tissue sections and correlated to the presence of blood vessels using CD31 immunostaining. Primary cultures were evaluated for the activation of αSMA-GFP and to monitor changes in GFP expression during in vitro mineralization by fluorescence microscopy and FACS analysis. The expression of αSMA-GFP was compared to mineralization detected by Xylenol Orange and von Kossa. Proliferation was determined using supravital Hoechst 33342 staining. Results: αSMA-GFP expression was observed in vivo in a defined population of cells surrounding the vasculature. GFP⁺ cells were nearly undetectable (< 2%) upon plating of primary pulp cells, based on FACS analysis. The number of αSMA-GFP⁺ cells increased rapidly in vitro during the first 5 days, followed by decreased GFP expression after differentiation. There was an inverse correlation between αSMA-GFP expression and areas of mineralization. Furthermore, the GFP⁺ population showed high mitotic activity, whereas GFP^- cells indicated quiescence. Conclusions: The αSMA-GFP model is useful for analysis of dental pulp perivascular cells and for tracking their in vitro pattern of expression. Although in vivo αSMA-GFP expression is limited to a small fraction of dental pulp cells, our data demonstrates a dramatic increase in the numbers of this cell type early in culture. Our collective findings suggest that cells derived from the perivasculature represent a defined population that may have the potential to activate and regenerate damaged tissues. Supported by Grants R01-DE016689, U24-DE06495 and T32-DE007302.