Human iPSC Derived Ameloblast Organoid Guided by Single-Cell Atlas of Human Tooth Development

Abstract

Tooth enamel secreted by ameloblasts is the hardest material in the human body, acting as a shield protecting the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. In contrast, dentin, the second hardest material in the tooth, continues to be produced by the odontoblasts in the dental pulp. Moreover, undifferentiated dental pulp stem cells (DPSCs), which are present in erupted teeth, are known to participate in the repair of injured dentin, by differentiating into odontoblast-like cells. Here we developed a regenerative strategy to replicate the interface between ameloblast and odontoblasts in vitro utilizing the human induced pluripotent stem cells (hiPSCs) to generate early ameloblasts, and DPSCs to generate odontoblast progenitors. First, we studied the DPSCs from third molars of a diverse patient group. Using high throughput transcriptomic, proteomic analysis, and metabolic flux analysis, we identified the signature for the optimal populations of DPSCs that can be used for expansion and regeneration that do not show rapid cellular senescence phenotype. In particular, we show that the transforming growth factor-beta (TGF-β) pathway and the cytoskeletal proteins are upregulated in rapid aging DPSCs, indicating a loss of stem cell characteristics and spontaneous initiation of terminal differentiation in this particular population. Second, we used sci-RNA-seq to establish a spatiotemporal single-cell atlas for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We revealed key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in a novel human ameloblast in vitro differentiation from hiPSCs. We furthermore developed an enamel organ-like 3D organoid. Finally, we cocultured the optimal DPSCs population we identified with the newly developed ameloblast organoid to form a polarized ameloblast interface with odontoblast progenitors. These studies pave the way for future regenerative dentistry and therapies toward genetic diseases affecting enamel formation.