Soluble Notch Agonist Enables Human Ameloblast Maturation and Enamel-like Tissue Formation for Tooth Regeneration

Enamel, the hardest material in the human body, is required to protect the living organ, tooth. However, over 90% of adults have lost or damaged enamel and cannot regenerate the protective structure due to lack of enamel producing cells, ameloblasts. iPSC derived secretory Ameloblasts (isAM) have promise in future regenerative dentistry. Today it is not known why iAM maturation requires intimate contact with the dentin producing cell type, odontoblast. Here we reveal that one of the critical signaling ligands emanating from odontoblasts for ameloblast maturation is Delta, the ligand for Notch receptor. We showed that our designed, soluble Notch agonist can induce iAM organoid maturation in an unprecedented manner, without interactions with odontoblast layer. Notably, soluble Notch agonist induces the iAM maturation to a novel, WDR72 positive mature secretory AM stage (ismAM) in our ameloblast organoid model. When transplanted under the kidney capsule of NOD-SCID mice, these ismAM organoids generated enamel-like calcified material, as confirmed by microCT analysis, marking the first demonstration that Notch-activated iAM organoids can form such tissue in vivo. This novel maturation procedure enabled us to analyze the specific requirements of DLX3 function in ameloblasts, independent of its known function in odontoblasts. We now show that DLX3, the gene associated with Amelogenesis Imperfecta, is required on a cell-autonomous manner in human ameloblasts for the expression of Enamelin, MMP20 and WDR72, a role not previously demonstrated in mouse models.