Efficient Induction Of Human IPSc Into Ameloblasts

Tooth morphogenesis is characterized by reciprocal interactions between ectoderm-derived dental epithelium and mesenchymal cells derived from the cranial neural crest.The interaction is comprised of two parts:one is the inducer that equipped with odontogenic potential;another is the responder that can accept the signal and react to inducer.Based on this principle,generation of bio-engineered teeth also requires two cell components,epithelial origin and mesenchymal origin.What’s more,either of them must possess odontogenic potential to initiate regenerative process.Until now,it has been solved that the source of mesenchymal cells in building bio-engineered teeth.However,there is no epithelial stem cells exist when teeth sprouted because it apoptosis after differentiation into ameloblasts and secrete enamel.Therefore,it is necessary to find seed cells to replace the dental epithelium cells that induced by E13.5 mouse molar mesenchyme to form tooth structure.Some researchers have utilized stem cells in tooth regeneration because of its multi-potential differentiation and self-renewal ability.Studies have shown that human keratinocytes can be induced into ameloblasts under the condition of adding foreign proteins.In addition,it was indicated recently that iPSc can differentiate to epithelial sheet under inducing medium,and then combined with E14.5 mouse molar mesenchyme to form tooth-like structures successfully.But both of these induction efficiency are not ideal.On the basis of analysis above and according to the E13.5 mouse molar mesenchymal scattered into cells still has odontogenic potential after reunion again.In this study,we built an efficient method for recombinant human iPSc with E13.5 mouse molar mesenchymal cells in different proportions.Then we find the highest induction efficiency is 93%with one proportion of human iPSc combined with nineteen proportions of mouse molar mesenchymal cells.Then,we cultured the chimeric tooth in the renal capsule membrane and find that it can simulate the development as normal tooth.Human iPSc also experienced aggregation,form dental epithelium and differentiated into ameloblasts successfully by the induced of molar mesenchyme.During 30 days in vivio,chimeric tooth germ development like normal tooth with enamel.iPSc with multi-directional differentiation potential,is it differentiated to keratinocytes first,then form the dental epithelial or differentiated into dental epithelial directly?We text KRT14 and CD29,as mark genes of keratinocytes,while SP6 and SHH as mark genes of dental epithelium.We found that iPSc express keratinocytes mark genes first and then detected the expression of SP6 and SHH.Raman spectra analysis and compare the composition of enamel in chimeric tooth、mouse tooth and human molar tooth found that,in the corresponding wave number has formed Raman characteristic peak indicate that composition of the enamel in these three tissues were consistent.But in the intensity of Raman spectra,there was some difference between teeth,which may be associated with the mineralization degree of chimeric teeth.To sum,in this study,we have established a highly efficient system of inducing iPSc differentiated into ameloblasts and secret enamel,provided a reliable technical solution in the research of engineering regeneration of tooth.