Odontogenic differentiation of hDPSCs induced by preameloblast-derived factors

Objective: The differentiation of odontoblasts from dental papilla is initiated by the organization of differentiating ameloblasts of the enamel organ during tooth formation. However, the exact roles of ameloblast-derived factors in odontoblast differentiation have not yet been characterized. The aim of this experiments are investigation of the effects of preameloblasts conditioned medium (PA-CM) on the odontogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. Methods: We isolated hDPSCs from human third molar and apical bud cells (ABCs) from mouse incisors. We co-cultured hDPSCs/MDPC-23 cells and ABCs simultaneously to measure the effect of ameloblast on odontoblast differentiation by real time PCR and western analysis in vitro. To investigate the effect of PA-CM on mineralized tissue formation in vivo, PA-CM treated hDPSCs were transplanted to the subcutaneous tissue of immunocompromised mice and the formed tissues were analyzed by histology and immunohistochemistry after 6 and 12 weeks. Furthermore, we analyzed the PA-CM by liquid chromatography-mass spectrometry to identify novel factors that facilitate odontoblast differentiation. Results: In the co-culture, ABCs promoted odontoblast differentiation of hDPSCs and MDPC-23 cells. PA-CM was most effective in increasing the dentin sialophosphoprotein promoter activity of odontoblastic MDPC-23 cells. When PA-CM treated hDPSCs were transplanted into immunocompromised mice, they generated pulp-like structure lined with human odontoblast-like cells showing typical odontoblast processes. After proteomics analyses, we identified 113 types of proteins in PA-CM, of which we characterized 23. Particularly, Cpne7, detected molecule in PA-CM proteomics analysis, increased the expression levels of odontogenic genes in MDPC-23 cells. Conclusion: These results suggest that hDPSCs in combination with PA-CM could be valuable in not only odontoblast differentiation but also repair and regeneration of the dentin-pulp complex. In addition, Cpne7 could be a new candidate gene that has an important role in odontoblast differentiation.