High Efficiency Of Induction Of Human Keratinocyte Stem Cells Into Enamel-secreting Ameloblasts And RNA-seq Analysis Of Odontogenic Potential

Mammalian tooth development is regulated by reciprocal and sequential tissue interactions that are mediated by diffusible factors between the dental epithelium and the adjacent dental mesenchyme.Both of these tissue components are obligated to participate in tooth development.Based on such developmental principle,bioengineering of a transplantable tooth germ requires both epithelial and mesenchymal cell components with either of them possessing tooth inducing capability.While none of the human postnatal mesenchymal stem cells isolated from dental origin possesses odontogenic potential,these stem cells are regarded promising cell sources with the potential of clinical practice for future tooth replacement and regenerative therapy.Human keratinocyte stem cells and gingival epithelial cells and even iPSC-derived epithelium are able to differentiate into functional ameloblasts and produce enamel.But none of them possesses odontogenic potential,too.To find the important molecular components for odontogenic potential become one of the major challenges of bioengineering of whole tooth.It has been demonstrate that human keratinocyte stem cell is able to be induced into enamel-secreting ameloblasts,recombined with E13.5 mouse molar mesenchyme.But the induction efficiency is only 25 percent.In this study,we built an efficient method for recombinant of human keratinocyte stem cell and mouse E13.5 molar mesenchymal cell.We took different proportion of mouse E13.5 molar mesenchymal cell and human keratinocyte stem cell.Then we find the highest induction efficiency is 92.9%with one proportion of human keratinocyte stem cell combined with nine proportion of mouse E13.5 molar mesenchymal cell.According to this method,we find that E13.5 mouse molar mesenchymal cell lost odontogenic potential after culturing 8 hours.Then we took the RNA from mouse E13.5 molar mesenchymal cells(possessing odontogenic potential)and mouse E13.5 molar mesenchymal cells after culturing 8 hours(losing odontogenic potential)and mouse E13.5 molar mesenchymal cells after culturing 9 days(poor odontogenic competence).After RNA Sequencing and Bioinformatics analysis,we took 12 Transcription factors and 12 growth factors as the important molecular components of odontogenic potential.As the factors were Nfib,Barx1,Sp7,Max,Dlx1,Bcl2,Lef1,Sp3,Pbx1,Pax9,Sp1,Gata6,Mdf2,Msxl,Bcl11b,Bmp2,Fgf12,Gdf1,Cxc12,Hdgfrp2,Fgf8,Bmp6,Fgf3,Fgf9,Ptn,Cspg4,Gdf7.