Differential Regulation Of Human Keratinocytes Into Enamel-secreting Ameloblasts By PITX2 Isoforms

Tooth morphogenesis and differentiation relies on the interactions between cranial neural crest-derived mesenchymal cells and the stomadial epithelium. Tooth developmental process can be divided into five stages, respectively is the dental placode stage, bud stage, cap stage, bell stage and secretory stage. Tooth develop-mental process between two tissue layers is required for normal differentiation or survival, and the communication is mediated by diffusible signaling molecules. When tooth is non-renewable, the research of tooth regeneration have important scientific value. The enamel of missing teeth cannot repair itself, therefore we need to find an alternative source of the stomadial epithelium. While adult stem cells compared with embryonic stem cells have a considerable advantage in the materials. The studies have shown that human keratinocytes cells can replace dental epithelial cells. Exogenous protein FGF8 added into the recombinants human keratinocytes and the E13.5 dental mesenchyme. Then the human-mouse chimera were transplanted into subrenal capsule of nude mice cultured for 20 days. It is impossible that human keratinocytes into Enamel-secreting Ameloblasts. The efficiency of tooth formation and ameloblasts is low. The reason may be due to the lack of some key factors of ameloblast differentiation when human keratinocytes differentiate into Enamel-secreting Ameloblasts. PITX2 is strongly expressed in the dental germ epithelial. It is one of the symbol of genes involved in the regulation of tooth germ epithelial differentiation. PITX2 is the downstream of FGF8.Using lentiviral expression system can overexpress PITX2 in human keratinocytes stem cells. So we expect to improve the efficiency of human keratinocytes stem cells into ameloblast differentiation.Firstly,the research showed that PITX2 is strongly expressed in the epithelial of human dental germ and possess three subtypes, respectively is PITX2a,PITX2b and PITX2c.Secondly, Human keratinocytes stem cells were separated from pediatric foreskin and identified by immunohistochemical staining. Then the lentiviral vector was used to mediate the genes PITX2 three subtypes into human keratinocytes stem cells.In order to ensure the efficiency of infection, it expand culture that the monoclonal cells from the expression of green fluorescent protein in human keratinocytes stem cells. PITX2 three subtypes cotransfected or each transfection human keratinocytes stem cells and then recombined with E13.5 dental mesenchyme. Then the human-mouse chimera were transplanted into subrenal capsule of nude mice cultured for 20 days.The chimera block for dehydrating embedding and identification. Tissue paraffin sections were prepared for hematoxylin eosin staining and immunohistochemistry. The immunohistochemical detected DSPP, Ameloblastin and Amelogenin protein expression of the chimeric teeth. The results showed that the chimera of PITX2a transfected human keratinocytes stem cells and then recombined with E13.5 dental mesenchyme was the highest the dental formation rate. The efficiency of dental and ameloblast formation is 64.3% and 88.9%. PITX2a is overexpressed in human keratinocytes stem cells. It improve the efficiency of ameloblast differentiation. While PITX2b and PITX2c are overexpressed human keratinocytes, have a little effect improve dental epithelium cells differentiation. It also shows that PITX2a plays the major role in the regulation of differentiation of tooth germ epithelial cells.In summary,PIX2a is one of the key factors genes in the tooth development and regulates human keratinocytes stem cells into odontogenic differentiation. We expe-rimental results provide the most significant experimental data and theoretical reference in the tooth regeneration.