Development of an early tooth organoid via human induced pluripotent stem cells - Project Summary/Abstract Genetic and environment-mediated diseases that affect tooth formation and function can severely compromise quality of life. While mouse models have significantly advanced knowledge of underlying biological processes and genetic drivers, new tractable in vitro systems coupled with gene editing can provide enticing alternatives for tooth bioengineering and disease modeling. Human induced pluripotent stem cells (iPSCs) are such a system since they can in theory differentiate into any somatic cell type, including major dental cell lineages such as odontoblasts and ameloblasts. To this end, the methodology of directed differentiation is used, which is a multistage process with recapitulation of developmental milestones to derive clinically-relevant cell types from iPSCs. Robust, efficient, and reproducible derivation of the earliest dental multipotent progenitors is key for the successful implementation of iPSCs in dental medicine but there exists an incomplete understanding and suboptimal recapitulation in vitro of early tooth development. To address this critical gap, an integrated approach is presented consisting of a logical sequence of experiments that combine human iPSC-based tooth organoid studies with high resolution in vivo studies of early tooth development. First, human iPSC reporter lines for transcription factors that are highly enriched in and define to a great extent the earliest mesenchymal and epithelial progenitors that originate from cranial neural crest and oral ectoderm, respectively, will be generated and used. As the derivation of dental cell types from human iPSCs through the methodology of directed differentiation depends heavily on prior developmental knowledge, the current application proposes the mapping of the earliest dental progenitor populations (both mesenchymal and epithelial) through single-cell RNA-Seq/ATAC-Seq to define genome-wide gene expression and profile regions of open (nucleosome-free) chromatin, respectively. These studies will be complemented with spatial transcriptomics at the same time points of tooth development that will define at exquisite detail the spatial relationships of the emerging dental epithelium and mesenchyme. This knowledge along with information gleaned from the tooth developmental literature, such as signaling pathways, will be used to derive their in vitro human counterparts with high efficiency and fidelity; the latter will be quantified by using state-of-the-art cell similarity methods. The proposed studies will provide new insights in early tooth development and set the basis for a robust human iPSC- based tooth organogenesis system to be used in precision dentistry, including future dental tissue replacement strategies.
