Organoids from human tooth showing epithelial stemness phenotype and differentiation potential

Abstract

Insight into human tooth epithelial stem cells and their biology is sparse. Tissue-derived organoid models typically replicate the tissue’s epithelial stem cell compartment. Here, we developed a first-in-time epithelial organoid model starting from human tooth. Dental follicle (DF) tissue, isolated from unerupted wisdom teeth, efficiently generated epithelial organoids that were long-term expandable. The organoids displayed a tooth epithelial stemness phenotype similar to the DF’s epithelial cell rests of Malassez (ERM), a compartment containing dental epithelial stem cells. Single-cell transcriptomics reinforced this organoid-ERM congruence, and uncovered novel, mouse-mirroring stem cell features. Exposure of the organoids to epidermal growth factor induced transient proliferation and eventual epithelial-mesenchymal transition, highly mimicking events taking place in the ERM in vivo. Moreover, the ERM stemness organoids were able to unfold an ameloblast differentiation process, further enhanced by transforming growth factor-β (TGFβ) and abrogated by TGFβ receptor inhibition, thereby reproducing TGFβ's known key position in amelogenesis. Interestingly, by creating a mesenchymal-epithelial composite organoid (assembloid) model, we demonstrated that the presence of dental mesenchymal cells (i.e. pulp stem cells) triggered ameloblast differentiation in the epithelial stem cells, thus replicating the known importance of mesenchyme-epithelium interaction in tooth development and amelogenesis. Also here, differentiation was abrogated by TGFβ receptor inhibition. Together, we developed novel organoid models empowering the exploration of human tooth epithelial stem cell biology and function as well as their interplay with dental mesenchyme, all at present only poorly defined in humans. Moreover, the new models may pave the way to future tooth-regenerative perspectives.

Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research

“…Interestingly, whereas perio-ERM and JE clustered together, human DF-ERM grouped with mouse VEE-OEE which gives rise to REE, further strengthening the possibility that REE may also contribute to ERM and/or vice versa ( Figure 4F ). Moreover, the VEE-OEE enriches for stem cells ( Figure 1E ) which is in line with the ERM being proposed, and recently supported, to contain DESC ( Xiong et al, 2012 ; Tsunematsu et al, 2016 ; Hemeryck et al, 2022 ). Lineage tracing studies in mice will be essential to support these hypotheses and to definitively determine the developmental origins of JE and ERM.…”

“…Taken together, the inclusive mouse tooth cell atlas composed here provides a valuable tool to generate new insights, thereby opening the path toward deeper understanding of mouse tooth biology and development. For instance, the novel regulators of AB differentiation surfacing from the bioinformatic analysis of the atlas can now be further examined in vivo (e.g., using genetically modified mice) or in vitro (e.g., using CRISPR/Cas gene editing in tooth organoid models ( Hemeryck et al, 2022 )). Detailed mapping of interactions between cell types (e.g., DE and DM) using the new atlas is expected to shed light on tooth development which is known to encompass key epithelial-mesenchymal interactions underlying the joint differentiation processes of OB and AB ( Hermans et al, 2021 ).…”

“…To complement the mouse tooth sc atlas, we also set out to establish an inclusive sc transcriptomic atlas of healthy human teeth (molars) by combining the at present six publicly available datasets ( Figure 3A ; Table 4 ) ( Krivanek et al, 2020 ; Pagella et al, 2021b ; Shi et al, 2021 ; Yin et al, 2021 ; Hemeryck et al, 2022 ; Opasawatchai et al, 2022 ). Following stringent quality control, 57,906 cells were found suitable for further bioinformatic analysis, with the datasets contributing between 524 and 21,453 cells to the total set, and cells sampled from various dental tissue types (e.g., 24,412 cells from DP and 7,119 from DF) ( Supplementary Figures S3A,B , Table 5 ).…”

“…Regarding mouse, single-cell RNA-sequencing (scRNA-seq) datasets were generated from the constantly (re-)growing incisor and the more static, human-resembling molar at different timepoints, either of whole tooth or specific tissue components ( Sharir et al, 2019 ; Takahashi et al, 2019 ; Chen et al, 2020 ; Chiba et al, 2020 , 2021 ; Krivanek et al, 2020 ; Wen et al, 2020 ; Nagata et al, 2021 ; Zhao et al, 2021 ). Human sc transcriptomic data were predominantly generated from dental pulp and periodontal tissues of molars in both healthy and diseased states ( Krivanek et al, 2020 ; Pagella et al, 2021b ; Shi et al, 2021 ; Yin et al, 2021 ; Hemeryck et al, 2022 ; Lin et al, 2022 ; Opasawatchai et al, 2022 ). All these sc analyses generated deeper insight into the tooth molecular and cellular landscape, furthering the understanding of dental cell type heterogeneity and tooth biology.…”

The Effects of Transforming Growth Factor-β1 on the Differentiation of Cell Organoids Composed of Gingiva-Derived Stem Cells

“…Organoid models have been applied for modeling human diseases, drug screening, and designing drug therapy [ 7 ]. Organoids made from human teeth demonstrated epithelial stemness phenotypes and differentiation potential with long-term expandability [ 8 ]. Organoids possess cellular structures which maintain and provide stability to the key characteristics of the targeted organs [ 9 ].…”