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Academic collection --- 591.413 --- 591.413 Vessels. Arteries. Aorta --- Vessels. Arteries. Aorta --- Theses

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Teeth play essential roles in food mastication, speech and psychological well-being. The human tooth consists of highly mineralized tissues of varying density and hardness, such as the enamel. This tissue is the most outer layer of the tooth crown, and is the hardest and highest mineralized tissue in the body. During tooth development, dental epithelial stem cells (DESCs) differentiate into ameloblasts that drive the enamel formation (amelogenesis). Once formed, human enamel cannot be repaired or renewed due to the apoptotic loss of the ameloblasts at the onset of tooth eruption. Restoration of damaged enamel tissue, as caused by trauma or bacterial disease, is currently accomplished using synthetic materials, however troubled by significant shortcomings. Therefore, the use of biologically derived enamel using human-derived DESCs is a highly searched solution. Hence, robust and reliable culture of human DESCs with the capacity to generate ameloblasts and the potential to produce mineralized tissue would be a major step forward in the dental regenerative field. Knowledge on human DESC phenotype and biological function is scarce. Intriguingly, indications for their existence have been found in the epithelial cell rests of Malassez (ERM), a network of cells present in the dental follicle (DF) which encloses unerupted teeth and remains present in the periodontal ligament (PDL) around the root upon tooth eruption. ERM cells co-cultured with dental pulp have been found to differentiate into ameloblast-like cells and generate enamel-like tissue. However, profound studies of the biology and specific role of ERM cells in enamel (re-)generation have been limited due to the lack of reliable study models. Current ERM in vitro culture systems are hampered by limited life span and quick loss of phenotype in the 2D conditions standardly used. Hence, a tractable in vitro system to faithfully expand, study and differentiate human DESCs is strongly needed. During the last decade, the powerful technique of organoid modeling has been on the rise to grow and explore tissue epithelial stem cells in vitro. This technology enables the epithelial stem cells to self-develop into 3D cell constructions when seeded into an extracellular matrix (ECM)-mimicking scaffold (typically, Matrigel) and cultured in a defined medium replicating the tissue's stem cell niche signaling and/or embryogenesis. Typical growth factors needed for organoid development and culture include epidermal growth factor (EGF) and wingless-type MMTV integration site (WNT) activators. The resultant organoids are characterized by high fidelity in mimicking the tissue epithelial (stem) cells of origin, as well as strong expandability while retaining their phenotype and functional properties, thereby overcoming the often-limited human tissue availability as acquired from the clinic. The powerful and versatile technology has led to the development of manifold organoid models from various human tissues. However, tooth-derived organoids, considered to be highly valuable for deep profiling of human tooth development, regeneration and disease, were not established yet. Here, we succeeded in developing a first-in-time tooth-derived organoid model starting from DF tissue of third molars (wisdom teeth) extracted from adolescent patients. Surprisingly, addition of the standardly added EGF did not activate but compromised organoid growth (in particular, their passageability). In the finally optimized medium (thus, without EGF), organoids could be obtained from DF tissue at 100% efficiency, which robustly expanded for more than 4-5 months. The organoids were found to display a tooth epithelial stemness phenotype similar to the DF's ERM. Single-cell transcriptomics reinforced this organoid-ERM congruence, while uncovering novel, mouse-mirroring stem cell features. Then, we analyzed whether this new organoid model could recapitulate reported (stem cell-related) functional properties of the ERM, more specifically the ability to undergo epithelial-to-mesenchymal transition (EMT) and to differentiate toward ameloblasts. Exposure of organoids to EGF induced transient proliferation followed by EMT and cell migration, thereby closely mimicking events taking place in the ERM in vivo, as for instance unfolding upon tooth insult. Subsequently, we investigated whether the organoids could unfold an ameloblast differentiation process in vitro using a specific differentiation medium. The organoids presented molecular changes constituting pathways that underlie ameloblast differentiation during amelogenesis. Moreover, single-cell RNA-sequencing interrogation advanced molecular transitions not revealed before in human amelogenesis. Also, bioinformatical (STRING) analysis projected protein-protein interactions that can further deepen our knowledge on amelogenesis in human tooth, at present only poorly understood. The organoids' ameloblast differentiation process was further enhanced by exposure to transforming growth factor-b (TGFb) and abrogated by TGFb receptor inhibition, thereby reproducing TGFb's known key position in amelogenesis. Interestingly, TGFβ was also found to promote PDL gene expression in the organoids, also reminiscent of ERM function in vivo. Next, we exposed the differentiated tooth organoids to an in vivo environment by transplanting them subcutaneously in immunodeficient mice, seeded into scaffolds. Interestingly, mineralized depositions were found in the organoid grafts as analyzed 4 weeks after transplantation. Although still preliminary, the presence of mineralization, electron-dense calcium-phosphate accumulations and intracytoplasmic deposits suggest that the organoids contribute to amelogenesis. Finally, we explored whether the presence of mesenchyme could enhance the epithelial stem cell differentiation potential by co-culturing organoid-derived epithelial cells with dental mesenchymal stem cells. Development of this assembloid model required the design of a cell-layering approach and the optimization of culture medium that promotes growth of both cell types. Interestingly, ameloblast differentiation of the organoid-derived epithelial stem cells was triggered by the presence of the dental mesenchymal cells in the assembloids, and TGFβ signaling was found to be involved in this process, thereby corroborating in vivo findings of interactive mesenchyme-epithelium importance during tooth development and amelogenesis. Furthermore, when cultured in differentiation medium, ameloblast development was even more pronounced, again mediated by TGFβ signaling. Following in vivo transplantation, the differentiated assembloids deposited mineralized tissue similar to epithelial organoids. In addition, immunohistochemistry suggested pre-dentin deposits to be present within the assembloid grafts. In conclusion, we developed first-in-time organoid models derived from human tooth empowering the exploration of dental epithelial stem cell biology and function as well as the interplay with tooth mesenchyme, all at present only poorly defined in humans. Moreover, the new tools may pave the way to future tooth-regenerative perspectives to replace whole tooth or tooth parts.