Towards Regenerative Dentistry: Human iOB Differentiation Guided by Single Cell Transcriptomic Atlas of Human Developing Odontoblast.

Abstract

University of WashingtonABSTRACT Towards Regenerative Dentistry: Human iOB Differentiation Guided by Single Cell Transcriptomic Atlas of Human Developing Odontoblast. Sesha Hanson-Drury, DDS Chair of Supervisory Committee:Professor Hannele Ruohola-Baker Departments of Oral Health Science, Biochemistry, Biology, Genome Sciences and Institute for Stem Cell and Regenerative Medicine (ISCRM) Clinicians, scientists, and the general public share the desire to regenerate missing tooth structure. The majority of mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells, odontoblasts. Though odontoblast development has been characterized in mouse models, mice constantly replenish their missing tooth structure though several stem cell niches not present in human teeth, posing translational challenges between the species. Human odontoblast differentiation and maturation remains largely unknown due to the rarity of tissue samples. To bioengineer missing dentin, increased understanding of human tooth development is required. Thus, our lab performed single cell combinatorial indexing RNA sequencing (sci-RNA-seq) followed by RNA Fluorescence in situ Hybridization (RNAScope) of the developing human oral cavity with the hypothesis that: the dental ectomesenchyme is composed of a heterogeneous cell population that each possess a unique transcriptional signature; changes in expression of these specific transcriptional signatures indicate transitions between cell types; and the molecular mechanisms that drive odontoblast developmental transitions occur at specific spatio-temporal intervals. We found that during early tooth development, the dental ectomesenchyme is divided into two transcriptionally unique components of the tooth germ tissue: the dental pulp composed of dental ectomesenchyme and dental papilla surrounded by the dental follicle. Strikingly, multiple computational assays indicate subodontoblasts (SOB) as a novel odontoblast progenitor source during human tooth development. SOB arise from the dental follicle and transition through a preodontoblast state before giving rise to odontoblasts. Further, we revealed the presence of SOB directly beneath the odontoblasts and intermingled with preodontoblasts at the pulpal periphery. SOB are a source of odontoblast renewal in adult mice following injury, but their presence and role in human odontoblast development has eluded researchers. Thus, our finding identifies this regenerative population in developing humans for the first time and holds incredible promise for SOB as a novel odontoblast progenitor not only during injury repair, but during normal human tooth development allowing us to manipulate and better utilize this naturally regenerating population in human dentistry. In order to further study odontoblast development, perform disease modeling, and test therapeutic agents, our lab developed an optimized in vitro human induced pluripotent stem cell to odontoblast differentiation protocol guided by sci-RNA-seq (iOB). Computational analysis identified fibroblast growth factor (FGF), bone morphogenic protein (BMP), and hedgehog (HH) signaling to play critical roles in human odontoblast development, with the majority of signaling ligands secreted by neighboring dental epithelium tissues. We found that activating these signaling pathways in induced neural crest cells by treatment with novel AI-designed FGFR superagonist minibinder, BMP ligand BMP4, and HH pathway agonist SAG signaling ligands produces more mature odontoblasts with increased expression of odontoblast markers and enhanced mineralization capacity. This finding implies great potential for AI-designed minibinders as therapeutic agents to induce odontoblast differentiation in clinical cases of pulp exposure or deep caries, as well as generation of mature iOB to be used for tooth organoid generation. Finally, the congenital disorder Tricho-Dento-Osseous syndrome produces debilitating dental defects associated with mutations in the gene DLX3. While transcription factor DLX3 is known to directly regulate Dspp activity and odontoblast development in mice, its role during human odontoblast differentiation is poorly understood. We generated a DLX3 knockout mutant line from HiPSC and tested their capacity to differentiate to mature odontoblasts. Importantly, we identified that DLX3 mutants are able to successfully differentiate towards a neural crest fate, however, odontoblast differentiation is arrested. This indicates DLX3 plays a critical role in early odontoblast development. The overall significance of this study is threefold: 1) providing unprecedented insight at the single cell level into cell types of the developing tooth dental ectomesenchyme; 2) applying the revealed molecular signaling that controls human odontoblast cell lineage commitment during differentiation to generate a HiPSC-derived odontoblast differentiation method (iOB); 3) utilizing the iOB tool to study the molecular mechanism of human odontoblast differentiation in states of health and disease in order to design appropriate therapies.