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Targeted Cell Recruitment During Organogenesis And Regeneration: Glia Makes The Tooth

Periodic Reporting for period 4 - STEMMING-FROM-NERVE (Targeted Cell Recruitment During Organogenesis And Regeneration: Glia Makes The Tooth)

Reporting period: 2020-02-01 to 2020-09-30

In this project, we addressed the problem of a context for glial recruitment into mesenchymal populations giving rise to odontoblasts – cells, producing dentin, and different previously unrecognized subtypes of pulp cells. Thus, we unbiasedly resolved dental tissue cell type heterogeneity and identified new types of stem cells and progenitors including those related to nerve-associated glial cells according to the focus of our ERC proposal and DoA. Finally, we characterized the transition from dental glial cells into mesenchymal progeny at the single cell level within mouse incisor during its self-renewal. This is important for the society because the knowledge of tissue heterogeneity, interactions and inter-conversions between cell types is the key for the bright future of regenerative medicine. In this particular case, we created an extensive knowledge of all cell types inhabiting teeth and participating in self-renewal of this important organ. This knowledge is very important for developments in regenerative dentistry and enhanced dental hard matrix repair technologies especially given that we discovered new previously unknown cell types not seen in the tooth before. Also, we think this is a very interesting topics, and many curious people want to know what our teeth are made of. 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 of dental stem cells waiting to be resolved including glia-derived mesenchymal stem cells.
Our results responded to the overall objectives and addressed how, when and to what extent peripheral glial cells residing in the dental nerves are recruited to produce cells of pulp and odontoblast lineages in the tooth, which might also manifest in additional tooth-inducing properties. For this, we had to describe the existing cell heterogeneity and describe the transitions between cell types (this part is now published in Krivanek et al., Nature Communications 2020). Most recently, we discovered and analyzed the transition of glial cells into dental mesenchymal populatioins at gene expression level (still unpublished data). In parallel, we also demonstrated how the neural crest cells give rise to cranial mesenchymal populations, including those giving rise to teeth, and how such mesenchymal population might be connected lineage-wise with developing glial cells (we published this part in Soldatov et al., Science 2019). Finally, 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.

The conclusion of the action
Taken together, in this study, for the sake of understanding the plasticity of glial and other populations, we generated the unbiased atlas of cell types and subtypes building the human and mouse growing and non-growing teeth, with the special focus on progenitors and glial cells and including continuously self-renewing mouse incisor as the major model system for addressing the mechanisms of growth. We generated polygenic signatures delineating the known and new subtypes, which resulted in new markers and molecular tools for manipulating these populations in the future. Using this dataset, as a proof of principle, we identified and validated previously unknown cell subtypes in epithelial, mesenchymal and immune system compartments. We predicted and validated new stem cell and progenitor populations (including glial) and described differentiation trajectories for ameloblasts, odontoblasts and other terminally differentiated cell types in the tooth, which will become an essential tool for the future regenerative medicine application and in vitro derivation of these cell types. We demonstrated how the denervation influences the conversion of nerve-associated Schwann cells into mesenchymal populations in the tooth by changing differentiation dynamics. To provide functional predictions of cell type integration within dental tissues, we generated the interactomics maps that visualize potential ligand-receptor mediated interactions between all identified populations. Functional validation of one of such predictions showed that Csf1-Csfr1 interaction is essential for previously unanticipated morphogenetic function of macrophages both in epithelial and mesenchymal compartments of the tooth. Finally, the interactomics maps and the cluster structure of all identified populations are available for further exploration and deep data mining online via the intuitive interface Atlas project webpage: http://pklab.med.harvard.edu/ruslan/dental.atlas.html
Overview of the results:

1. We generated the complete and unbiased atlas of dental cell types for mouse and human, and growing and non-growing teeth (see Figure graphical abstract)

2. Thanks to the atlas work, we discovered the new stem and progenitor cell types important for tooth growth and maintenance

3. We profiled the transition from glial cells into mesenchymal populations within the tooth (see Figures 1-2), and described the transitory moments, trauma condition and potential signals influencing plasticity of glia

4. We discovered the importance of dental macrophages for the maintenance of stem cell niches (see Figures 3-4) and overall dental integrity

5. We revealed the potential origins of glial plasticity and its similarity to embryonic multipotent neural crest cells

6. We investigated the tooth-inducing potential of glial cells and did not manage to obtain the proof of this phenomena in vivo.

The results were published in multiple top journals (Science, Nature, Nature Communications) and disseminated via professional academic and non-academic communities (see Publications and Dissemination sections)
According to DoA and main objectives of this project, we achieved the progress beyond the state of the art by creating the first of its kind atlas of all cell types inhabiting the growing and non-growing human and mouse teeth with the power of single cell transcriptomics also to resolve the context and the process of growth including occasional glial-to-mesenchymal transition. Using this atlas, we discovered novel cell subtypes and states, including stem and progenitor cells, and demonstrated the transitions from genetically defined dental mesenchymal cells into odontoblasts, which is the key knowledge for the further development of this scientific directlion.
Address (URL) of the project's public website

http://pklab.med.harvard.edu/ruslan/dental.atlas.html
Figure 1
Dental atlas into figure for incisor
Figure 3
Figure legends for Figures 1-4
Figure 2
Figure 4