Identification and Characterization of Side Population Cells from Adult Human Dental Pulp after lschemic Culture

Introduction: Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the side population (SP). Because the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of dental pulp stem cells, this study sought to determine the existence of SP cells and the expression of ABCG2 in human dental pulp and evaluate whether such SP cells had features associated with stem cells. Methods: First, we defined the localization of the SP in healthy and inflammatory human dental pulp. Then, SP cells were isolated from human dental pulp after ischemic culture with flow cytometry and the Hoechst 33342 dye efflux assay. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype, including the colony-forming capacity, the multilineage differentiation ability in vitro, and the expression of stem cell markers. We also evaluated the effect of long-term culture on the marker ABCG2. Results: SP cells in human dental pulp possess mesenchymal stem cell characteristics such as colony-forming efficiency, self-renewal, and multilineage differentiation capabilities and are able to differentiate into odontoblast/osteoblast-like cells, adipocytes, neural-like cells, and endothelial cells. However, under the present conditions, ABCG2 expression decreased along with cell passage. Conclusions: SP cells in human dental pulp were enriched in stem cells compared with main population cells after ischemic culture, suggesting a potential use for these subfractions of human dental pulp stem/progenitor cells in tissue engineering, but the culture condition in vitro should be improved before tissue regeneration. (J Endod 2012;38:1489-1497)