ARTICLE https://doi.org/10.1038/s41467-020-18512-7 OPEN Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth Jan Krivanek 1,2,18, Ruslan A. Soldatov3,18, Maria Eleni Kastriti1,4, Tatiana Chontorotzea1, Anna Nele Herdina4, Julian Petersen 1,4, Bara Szarowska1, Marie Landova5, Veronika Kovar Matejova6, Lydie Izakovicova Holla 6, Ulrike Kuchler7,8, Ivana Vidovic Zdrilic9, Anushree Vijaykumar 9, Anamaria Balic 10, Pauline Marangoni11, Ophir D. Klein 11,12, Vitor C. M. Neves13, Val Yianni 13, Paul T. Sharpe 13, Tibor Harkany1,14, ✉ Brian D. Metscher 15, Marc Bajénoff16, Mina Mina9, Kaj Fried14, Peter V. Kharchenko 3 & ✉ Igor Adameyko 1,4,17 1234567890():,; Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non- growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, pro- genitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non- growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation. 1 Department of Molecular Neuroscience, Center for Brain Research, Medical University of Vienna, Vienna, Austria. 2 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. 3 Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA. 4 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. 5 Institute of Animal Physiology and Genetics, CAS, Brno, Czech Republic. 6 Clinic of Stomatology, Institution Shared with St. Anne’s Faculty Hospital, Faculty of Medicine, Masaryk University, Brno, Czech Republic. 7 Department of Oral Biology, Medical University of Vienna, Vienna, Austria. 8 Department of Oral Surgery, Medical University of Vienna, Vienna, Austria. 9 Department of Craniofacial Sciences, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT, USA. 10 Research Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland. 11 Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA. 12 Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA. 13 Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral & Craniofacial Sciences. King’s College London, London, UK. 14 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. 15 Department of Evolutionary Biology, University of Vienna, Vienna, Austria. 16 Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, INSERM, CNRS UMR, Marseille, France. 17 Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria. 18These authors contributed equally: Jan Krivanek, Ruslan A. ✉ Soldatov. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2020) 11:4816 | https://doi.org/10.1038/s41467-020-18512-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18512-7 ammalian teeth are formed by the ectoderm of the first growing and self-renewing tooth. We next focused on the most pharyngeal arch and neural crest-derived ectome- striking aspects of population complexity of epithelial (Figs. 2–4) M – senchyme. Developmental interactions between these and mesenchymal (Figs. 5 7) compartments, their human ana- tissue types enable the construction of solid dental struc- logues (Figs. 8, 9), and finally immune (Fig. 10) populations. tures composed of epithelium-derived crown enamel and ectomesenchyme-derived dentin1–4. In humans, teeth primordia are formed in utero and complete their growth before adulthood, Heterogeneity of the epithelial compartment in mouse incisor. at which point the progenitor populations disappear. In contrast The epithelial compartment of the tooth is essential to generate to this, in mice and many other species, teeth can continue to the enamel, as well as for the morphogenetic guidance of tooth grow throughout life, providing the major model system to study development and self-renewal. Focused reanalysis of the epithelial progression of various tooth cell lineages from the dental stem- (Krt14 and Cdh1 co-expressing) subpopulation showed a complex cell populations located in the apical end of the tooth. In mice, the mixture of at least 13 distinct epithelial clusters (Fig. 2a). These incisor stem-cell population continuously self-renews and include mature subpopulations, such as enamel-generating ame- replenishes tissues that are lost due to gnawing, making this loblasts, and a heterogeneous pool of stem/progenitor cells. model attractive for studies of stem-cell generation, cell differ- During sequencing, we enriched for epithelial progenitors by entiation, homeostasis, and injury-induced regeneration. In using Sox2-driven GFP in Sox2GFP animals and subsequent FACS addition, the mouse incisor represents a model of continuously of fluorescent cells. Ameloblasts and enamel development in the self-renewing organ with cell dynamics conceptually similar to incisor is not restricted to early developmental stages as it is in gut epithelium, hair follicles, and nails. Even though major tooth molars. Continuous replenishing of enamel is essential for the cell types have long been identified, the spectrum of rare and incisor growth. Thus, we can find all the ameloblasts’ stages: transient cell populations and interactions that enable tooth closer to the labial cervical loop we can find early stages and growth remain poorly understood. The identity of epithelial and closer to the tip more differentiated stages. Differentiation of mesenchymal stem populations and their possible spatial and ameloblasts starts at the preameloblasts stage (early fate decision functional diversity remains unresolved, especially when it comes and first differentiation), then continues through secretory stage to such populations in growing and nongrowing human teeth. during which the enamel backbone is formed. Subsequently, Besides, whether rodent teeth represent a bioequivalent model the secretory ameloblasts are further differentiated in a matura- system for studying specific aspects of human tooth development tion phase during which the first enamel backbone is fully cal- and physiology is not yet clear. The long held-view is that the cified2. During the last phase, known as a postmaturation phase, human teeth contain mesenchymal stem cells analogous to mouse the enamel epithelium diminishes and enamel production is incisor mesenchymal stem cells5–8. However, at this point, no completed. Consistent with these stages, we observed spatially clear consensus has been reached about the molecular identity of separated stages of ameloblast differentiation, including pre- such cells in vivo6,9. In addition, the role and population structure ameloblasts (Shh+ cluster 11), secretory (Enam+ cluster 5), of other cell types, such as resident cells of the immune system, maturation (Klk4+ cluster 10) and postmaturation (Gm17660+ is unclear in relation to the maintenance of local tissue home- cluster 6) stages (Fig. 2a, e, Fig. 4a, c, h, i)14–16. Our results show ostasis and beyond their major protective function in teeth. There that transitions in gene expression profiles between the canonical is growing evidence that macrophages are important constituents stages are rather abrupt, consistent with the fact that the stages influencing the stem-cell compartments, for instance, in control were previously characterized based on significant morphological of the intestinal stem-cell niche or in promoting wound-induced and functional changes during ameloblast differentiation. The hair follicle regeneration10,11. data show progressive modulation of transcription factor Towards answering these questions, we applied single-cell expression during different stages of ameloblast development transcriptomics and lineage tracing techniques with a specific aim (Fig. 4d–f), connecting known spatial and morphological transi- to examine the organizational complexity and self-renewal of tions associated with the ameloblast differentiation with pre- growing mouse incisor, contrasting it with nongrowing mouse viously uncharacterized intermediate transcriptional states. In molars, and evaluating the extent to which the mouse model addition to these spatially separated populations, we observed a reflects the growth of human teeth. Our data revealed stem and subset of RYR2+ cells scattered in the ameloblasts layer (cluster 3) differentiated cell subtypes in epithelial and mesenchymal com- (Fig. 2a, b). The function of these cells is unknown, however this partments and heterogeneity of tissue-residential immune cells in population expresses different mechanotransduction-related