#	Pagina
attuale pagina	/open-h2020/projects/197865/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - STEMMING-FROM-NERVE (Targeted Cell Recruitment During Organogenesis And Regeneration: Glia Makes The Tooth)

Teaser

Summary

â€œDental cell type atlas reveals new stem, intermediate progenitor and differentiated populations in self-renewing mouse incisorâ€

Mouse incisor is the most popular model system to study tooth development, growth, self-renewal, regeneration, hard matrix repair, stem cells and morphogenesis. However, the cellular composition of a tooth is poorly understood. The identity and developmental paths of stem cells are still enigmatic. Using a single cell transcriptomics approach in a combination with lineage tracing and functional studies, we generated an atlas of mature cell types, stem cells and transitory populations from the continuously growing mouse incisor. Our results revealed new subtypes of stem and progenitor cells providing insights into tooth maintenance and self-renewal processes. In the epithelial compartment, we revealed differentiation path of ameloblasts and validated the existence of novel Sox10+ and Fos+/Egr1+ stem-like populations. In the mesenchymal part, we discovered the differentiation trajectory of odontoblasts and pulp cells via a number of previously unknown stages. We provided the detailed knowledge of heterogeneity among regenerative perivascular cells as well as glial, smooth muscle and dental follicle populations. We showed the transition of glial cells into dental mesenchymal populatioins at gene expression level. The profiling of immune cells showed the presence of previously unknown macrophage subtypes and their site-specific spatial distribution conserved also in human teeth. The interactomics mapping suggested that CSF1 secreted by specific pulp cells is a key for hosting dental macrophages. The conditional knockout of Csf1 gene in the neural crest-derived pulp resulted not only in a loss of dental macrophages, but also in a failure of correct incisor development and shaping. Taken together, the unbiased analysis of dental cell types combined with predictions of communications between the cell populations resulted in new discoveries that proved catalyzing power of dental cell type atlas.

Summary of the context

Mammalian teeth are vital for feeding, fending and good quality of life. They are mineralized organs formed by the ectoderm of the first pharyngeal arch and ectomesenchyme of the neural crest. Sophisticated developmental interactions between these tissue types enable the construction of the morphogenetically complex dental structures with pre-defined shape and location. The epithelial part is responsible for the generation of enamel around the crown of the tooth, whereas the mesenchymal part produces the cells forming dentin and root cementum. Neural crest-derived cells also form the soft core, a pulp, which is mainly composed of connective tissue hosting vessels, nerves and immune cells (Zhang et al., 2005, Jussila and Thesleff, 2012; Balic and Thesleff, 2015; (Kapsimali, 2017; Krivanek et al., 2017). An adult mouse incisor is the major model system to study dental stem cell populations because, unlike the other teeth, this type of a tooth grows continuously throughout the entire life of an animal. Thus, the incisor undergoes constant renewal of all cell populations from the apical end to replenish both soft and mineralized tissue that is getting lost due to gnawing and biting at the incisal tip. This allows explorations of stem cell generation, cell differentiation, homeostasis, age-related apoptosis and injury-induced regeneration in the same organ. Despite the major cell types were identified in teeth long time ago, the question about heterogeneity and molecular characterization of rare cell subpopulations, stem cells and their developmental transitions stayed unanswered. Several recent studies expanded our knowledge about the molecular identities of some dental stem cells and their properties in vivo (Kaukua et al, 2014; Balic and Thesleff, 2015; Li et al., 2016; Sharpe, 2016)(Seidel et al., 2017). However, a number of recent publications suggested the existence of multiple types o

Work performed

Results

Overview of the dental cell type atlas

We isolated dental pulp along the whole length of the adult mouse incisor together with the entire cervical loop area. Then we dissociated these tissues into single cells, which we individually dispensed using FACS into 384-well plates for the following sequencing of their transcriptomes with Smartseq2 protocol (reference). To enrich for epithelial stem cells and their immediate progeny, we took advantage of Sox2-GFP animals since Sox2 is expressed by epithelial stem cells as previously shown (reference).
Unsupervised clustering of 3054 cells that passed quality control (Supplementary Figure 1, Methods) revealed 17 major clusters that correspond to defined cell types, including ameloblasts, odontoblasts, pulp cells, endothelial cells, pericytes, smooth muscle cells of arterial walls, various immune cells and also elements of extra-dental tissues (Figure 1A-D; Figure S1A,B,D). We generated transcriptional signatures for every identified population, thus, providing extensive lists of new markers for all known and unknown cell subtypes (Supplementary data table X, Supplementary Figure 1). Large fraction of proliferating cells, that were identified using cell cycle signature, was associated with the specific sub-clusters inside of the epithelial and pulp populations pointing to the actively operating developmental processes (Figure 2A, 3B, Methods). We next separately re-analyzed every major population including epithelial, mesenchymal and immune cells (Figure 1E,F,G).

Differentiation dynamics and new cell subtypes in the epithelial compartment

The canonical knowledge states that epithelial compartment in continuously growing mouse incisor is represented by dental epithelial stem cells (DESCs), transiently amplifying cells (TACs) residing in the stellate reticulum; outer enamel epithelium, stratum intermedium and, finally, inner enamel epithelium formed by ameloblasts secreting enamel (reference). Separate analysis of the epithelial compartment identified 13 subpopulations that reflect listed cell types as well as the novel subpopulations (Figure 2A).
One of such novel subpopulations formed cluster 7 and expressed Egr1 and Fos transcription factors. Using immunohistochemistry for EGR1, we found positive cells residing inside of the cervical loop stellate reticulum, where the DESCs and TACs are dwelling (Figure 2B). The lineage tracing with FosCreERT2/R26ZsGreen1 demonstrated the presence of the epithelial progeny after 10 days of tracing predominantly inside of the cervical loop and in the outer enamel epithelium. Occasionally, we observed traced cells in the stratum intermedium and among the ameloblasts (Figure 2B). Thus, Egr1+/Fos+ epithelial cells (also co-expressing Fbn2, Ctgf, Pgf, Vrtn, etc.) are novel fate-restricted stem-like progenitors. The population of Egr1+/Fos+ cells appeared to be Sox2 negative, which opens up for the existence of Sox2 negative epithelial progenitor subtypes. At the same time, Sox2 demonstrated the broad expression in other stem and progenitor subtypes in the epithelial dataset (Figure SX). Most of the Sox2+ cells coincided with proliferative markers (Mki67, Pcna, Mcm2, etc.) and transited into Shh+ cells (clusters 2, 5, 11, 12 and 13 corresponding to stellate reticulum, TACs of stellate reticulum, preameloblasts, Lrig1+ TACs and a part of secretory ameloblasts) (Figure S2).
The analysis of expressed transcription factors showed the presence of few Sox10+ cells that did not form a cluster. However, one of these rare cells demonstrated extensive and unique signature (including Ebf1, Jph2, Sema3g, P2rx1, and other genes). To address whether any of the rare Sox10+ epithelial cells could possess stem cell properties, we took advantage of Sox10CreERT2/R26Confetti mouse line (reference) to perform lineage tracing. The results confirmed the existence of long-lasting Sox10+ stem cells and showed all types of the derived epithelial progeny including enam

Final results

According to DoA and main objectives of this project, within 2.5 years of work we achieved the progress beyond the state of the art by creating the first of its kind atlas of all cell types inhabiting the tooth with the power of single cell transcriptomics to resolve the context and the process of glial-to-mesenchymal transition. Using this atlas, we demosntrated the transitions from glial into dental mesenchymal cells and odontoblasts, which is the key knowledge for the further development of the project. Additionally, we discovered novel, previously unknown subtypes of cells in all dental compartments.We are about to submit this manuscript to Nature.
We hope to expand the atlas by the end of the project and also to use the gained knowledge to enhance to transformation of glial cells into odontoblasts inside mouse incisors and molars.