Enamel Regeneration: Dissecting Notch Signaling, DLX3 Function, and Hydroxyapatite Mineralization in Human iPSC-Derived Ameloblast Organoids

Abstract

Tooth enamel is the hardest mineralized tissue in the human body and the primary protective barrier of the tooth. Over 90% of adults worldwide experience enamel loss due to dental caries, erosion, trauma, or congenital disease, yet enamel cannot be regenerated because the cells responsible for its formation, ameloblasts, undergo programmed apoptosis upon tooth eruption. Congenital disorders of enamel formation, collectively termed Amelogenesis Imperfecta, affect millions of individuals worldwide and cause significant functional and psychosocial burden. Despite decades of research, the molecular signals governing human ameloblast maturation remained unknown, and no human cellular model of ameloblast biology existed prior to the work described in this dissertation.Using single-cell combinatorial indexing RNA sequencing of developing human fetal tooth tissue spanning gestational weeks 9 through 22, our laboratory established the first comprehensive spatiotemporal single-cell atlas of human tooth development and used the signaling pathways identified therein to develop a hiPSC-derived ameloblast organoid platform (iAM). Building on this foundation, the work described in this dissertation addresses three interconnected questions at the frontier of human enamel biology. First, I identify Notch signaling as both necessary and sufficient for human ameloblast maturation. Single-cell transcriptomic analysis of hiPSC-derived ameloblast and odontoblast co-cultures identified DLL1 and DLL4 as the dominant odontoblast-derived Notch ligands received by NOTCH1, NOTCH2, and NOTCH3 receptors on ameloblasts. Pharmacological inhibition of Notch signaling by DAPT reduced terminal maturation marker expression in co-cultures, establishing necessity. A computationally designed soluble Notch agonist, C3-DLL4, was then generated and shown to drive full ameloblast maturation in the complete absence of odontoblasts, inducing a novel WDR72-positive mature secretory ameloblast stage designated ismAM. When transplanted under the kidney capsule of NOD-SCID mice, C3-DLL4 matured iAM organoids generated enamel-like calcified material as confirmed by microCT analysis, marking the first demonstration that Notch-activated hiPSC-derived ameloblast organoids can form enamel-like tissue in vivo. Second, I demonstrate that the transcription factor DLX3, associated with Tricho-Dento-Osseous syndrome and Amelogenesis Imperfecta, is required cell-autonomously in Human Ameloblasts for terminal maturation. Two independent DLX3 knockout hiPSC lines were generated by CRISPR-Cas9 genome editing and differentiated through the iAM protocol. Early ameloblast markers including SP6 and AMBN were unaffected by DLX3 loss, while terminal maturation markers ENAM, MMP20, and WDR72 failed to upregulate upon C3-DLL4 treatment despite equivalent Notch pathway activation. This cell-autonomous requirement for DLX3 in Human Ameloblasts was not previously demonstrable in mouse models, where ameloblast and odontoblast defects are intertwined, and establishes the first human cellular model of DLX3-associated Amelogenesis Imperfecta. Third, I demonstrate that computationally designed hydroxyapatite minibinder proteins expressed by iAM organoids enhance enamel-like mineralization in vivo. A Tet-ON inducible hiPSC line expressing a secreted HAp minibinder protein was generated and differentiated through the iAM protocol with C3-DLL4 maturation. Kidney capsule transplantation experiments showed progressive and enhanced mineralization in HAp minibinder expressing organoids compared to C3-DLL4 treatment alone, establishing proof of concept for a combined cellular maturation and protein engineering approach to enamel biomineralization. Finally, this dissertation describes broader applications of the hiPSC-derived oral epithelial platform to craniofacial biology, including functional validation of orofacial cleft-associated genetic variants in two genome-wide association studies published in Nature Communications, application of a computationally designed DNMT3A epigenome editor to enhance oral epithelial and ameloblast differentiation efficiency, and contribution to a spatial transcriptomic atlas of human craniofacial development. Together these findings establish Notch signaling as a master regulator of human ameloblast maturation, define the cell-autonomous role of DLX3 in human enamel formation, demonstrate proof of concept for engineered protein-guided enamel biomineralization, and position the hiPSC-derived oral epithelial and ameloblast platform as a broadly applicable tool for craniofacial biology and regenerative dentistry.