Immunocytochemical characterization of primary teeth pulp stem cells from three stages of resorption in serum-free medium.

Abstract

Dental pulp stem cells from primary teeth cultured in serum-free conditions may have clinical use for the repair and regeneration of teeth as well as other complex tissues and organs. The aim of this study was to test the change in the stem cell markers expression/ stem cell population in human primary pulp cells at the different stages of root resorption. Caries-free human primary canines at defined stages of physiological root resorption were included (n=9). In vitro cultures were established in xeno-free, serum-free Essential 8™ medium with human truncated vitronectin for cell attachment. An embryonic stem cell line (GENEA002) was used as a positive control. The expression of embryonic stem cell markers (Oct4, Nanog and Sox2), neural crest stem cell markers (nestin and Dlx2) and mesenchymal stem cell surface markers (CD90, CD73 and CD105) were investigated by immunocytochemistry. Mesenchymal stem cell markers CD105, CD73 and CD90 and haematopoietic markers: CD45, CD34, CD11b, CD19 and HLA-DR were quantified with flow cytometry. The early neural progenitor markers nestin and Dlx2 were detected in most serum-free cultured dental pulp stem cells, regardless of the tooth resorption stage from which they were harvested. Only isolated cells were found that expressed the embryonic stem cell transcription factors Oct4A, Nanog and Sox2, and in the late stages of resorption, no Oct4A was detected. The majority expressed the mesenchymal stem cell markers CD90, CD73 and CD105. Flow cytometry found positive signals for CD90>97.3%, CD73>99.6% and CD105>82.5%, with no detectable differences between resorption stages. This study identified populations of dental pulp cells in vitro with markers characteristically associated with embryonic stem cells, neural crest-derived cells and mesenchymal stem cells. Flow cytometry found CD105 expressed at lower levels than CD90 and CD73. The consistency of stem cell marker expression in cells cultured from teeth at different resorption stages suggests that pre-exfoliated primary teeth that are free of caries may provide a convenient source of multipotent stem cells for use in regenerative medicine.