Florian Hermans

Abnormal enamel mineralization has been reported in genetic conditions associated with hypocalcemia but never with hypercalcemia. In the present study, we report 10 patients from 2 unrelated families in whom hypomineralized enamel co-segregated with hypercalcemia due to a complete deletion or frameshifting premature truncation (p.(Ser18Argfs*2)) of the guanine nucleotide–binding protein (G-protein) subunit α ₁₁ ( GNA11 ) gene. Both GNA11 variants were heterozygous and predicted to cause a loss of function of Gα ₁₁ . Multiproxy imaging analyses of naturally exfoliated primary teeth and clinically extracted permanent teeth from 2 affected patients revealed abnormal enamel mineralization, with constitutive patches of hypomineralization following no set pattern. In parallel, the study of wild-type mouse tooth germs using combined molecular and protein analyses showed Gna11 and Gα ₁₁ protein expression in ameloblasts at the secretion and maturation stages, as well as in odontoblasts, suggesting a role for Gα ₁₁ in tooth formation. Furthermore, the analysis of a mouse model of Familial Hypocalciuric Hypercalcemia 2 (FHH2) revealed delayed onset of enamel mineralization in the continuously growing incisors and hypomineralized enamel in both incisor and molars of heterozygous ( Gna11 Tm1b ^+/− ) mice, with defects that are very similar to those affecting human FHH2 teeth. In conclusion, our study reveals that abnormal enamel mineralization may occur in association with hypercalcemia due to loss-of-function GNA11 mutations, opening a new field of investigation and highlighting the need to include a dentist in the multidisciplinary team in charge of patients with monogenic calcium disorders.

The aims of this study were 1) to determine how ligature-induced periodontitis (LIP) disrupts barrier functions of the junctional epithelium (JE); 2) to determine, using a genetic approach, the necessity of Wnt signaling for JE barrier functions; and 3) to test, using a biochemical strategy, whether a WNT therapeutic is sufficient to improve barrier functions of a pocket epithelium. In a murine model of LIP, quantitative analyses performed at multiple time points assessed epithelial apoptosis; expression of attachment proteins laminin 5 and β4 integrin, inflammation, and bone resorption. Axin2 ^CreERT2/+ ; R26R ^mTmG/+ mice were used to evaluate how LIP impacted Wnt-responsive cells and their progeny, and K14 ^CreERT2/+ ;Wls ^fl/fl mice were used to determine whether Wnt signaling was required for JE barrier functions. In some cases, LIP was followed by a recovery period to assess molecular changes in pocket epithelium, and in a subset of these mice, a liposomal formulation of human WNT3A protein (L-WNT3A) was tested for its effects on early repair dynamics in pocket epithelium. LIP triggered apoptosis, significantly reduced expression of laminin 5 and β4 integrin in the JE, and disrupted the Wnt-responsive compartment; these epithelial changes were accompanied by inflammation and alveolar bone resorption. Reepithelialization occurred even with a ligature present, but this pocket epithelium had compromised barrier functions. Wntless (Wls) deletion was sufficient to convert a JE into pocket epithelium, while topical L-WNT3A treatment was sufficient to increase hemidesmosomal protein expression in pocket epithelium and reduce inflammation at early time points. LIP destroys barrier functions and thus converts a JE into pocket epithelium. Deletion of epithelial Wls demonstrates that this conversion is a Wnt-dependent event. L-WNT3A restores some early barrier features to pocket epithelium; future studies will focus on the durability of these effects.

Epithelial rests of Malassez (ERMs) constitute a 3-dimensional epithelial meshwork extending from the tooth apex to the junctional epithelium (JE). This position, coupled with observations from a variety of injury models, suggests that ERMs may be involved in sensing disruptions to the mechanical integrity of the periodontium. To test this hypothesis, finite element modeling was employed to predict strain states in tissues surrounding ERMs (e.g., the periodontal ligament [PDL], supracrestal connective tissues, and the JE), which, by virtue of their attachment to the tooth surface, experience distortional strains during mastication. An in vivo hyper-loading mouse model was employed to map these distortional strains to location-specific and time-dependent morphological changes in ERMs. Quantitative immunohistochemistry was used to interrogate ERMs for changes in the expression of Piezo1, β4 integrin, plectin, YAP, TRPV4, and phosphorylated FAK. Collagen and oxytalan fiber organization around ERMs was compared between normal- and hyper-loading conditions. Axin2Cre ^ERT2 ; R26R ^mTmG/+ mice were used to lineage-trace Wnt-responsive ERMs under normal and hyper-loading conditions, and Wnt signaling from ERMs and the JE was disrupted using K14Cre ^ERT2 ; Wls ^fl/fl mice. Collectively, these analyses predicted that hyper-loading produced high distortional strains in the JE and supracrestal connective tissues, which correlated with sites of ERM enlargement. ERMs expressed mechanosensory proteins, whose levels were significantly elevated by hyper-loading. Load-activated ERMs were Wnt-responsive, and if Wnt secretion was blocked, then ERMs no longer expressed mechanosensory proteins and no longer enlarged in response to hyper-loading. A deterioration in the JE attachment and the PDL followed. Thus, the ERM network is positioned to sense distortional strains produced by mastication; they possess the molecular machinery to sense and respond to masticatory forces and, via a Wnt-dependent mechanism, coordinate tissue-level responses to mechanical loading in the PDL, supracrestal connective tissues, and the JE.

Once considered vestigial structures, epithelial rests of Malassez (ERMs) have recently been implicated in periodontal repair, largely based on their location in the periodontal ligament space, adjacent to damaged tissues. This study decodes what ERM activation entails and then tests in a variety of periodontal injury models the consequences of injury activation on ERM behavior and function. A Wnt lineage tracer strain Axin2Cre ^ERT2/+ ; R26R ^mTmG/+ was employed to map the distribution of Wnt-responsive cells and their descendants during root development and in response to injuries to the periodontium. Injury-activated murine ERMs were compared against developmental ERMs, and both were analyzed by histology and immunohistochemistry (IHC). Additionally, ERMs isolated from human tissues were analyzed by single-cell RNA sequencing and IHC. During root development, ERMs were surrounded by Wnt-responsive cells and their progeny. In response to injury, both the number and size of ERMs significantly increased, and this injury-induced enlargement did not involve cell proliferation. The injury-activated state of ERMs was accompanied by expression of Wnt pathway components. Compared to ERMs in uninjured tissues, injury-activated ERMs exhibited a shift toward expression of molecular markers associated with the epithelial-to-mesenchymal transition (EMT). When ERMs were juxtaposed to an injured junctional epithelium (JE) or damaged cementum, some ERM cells adopted a stellate morphology, exhibited evidence of matrix remodeling, and showed a loss of cell–cell adhesion, contributing to the repair of cementum and the JE. A dynamic state of Wnt responsiveness exists in injury-activated ERMs, and a subset of ERM cells undergo EMT. Together, these findings raise the possibility that cells in and around these activated ERMs may adopt an active role in periodontal repair.

Organoid models are a powerful 3D stem cell technology to explore tissue (patho-)biology and development. Tissue-derived (i.e., from tissue biopsies) organoids are long-term and stably expandable while more closely recapitulating key phenotypical and functional characteristics of the tissue-of-origin than traditional 2D culture systems. Additionally, organoids can differentiate into tissue-specific cell types, for instance, following exposure to defined differentiation cues. Although prevailing in vitro cell models have deepened our understanding of mouse tooth development and biology, in vitro representations of the dental epithelium lack (the combination of) these benefits of tissue-derived organoids and are at most derived from one tooth type. Here, we describe the protocol to establish, propagate, and differentiate mouse tooth organoids from both early postnatal molar and incisor teeth. The established organoids display a dental epithelial stemness phenotype and acquire a maturation-stage ameloblast-like phenotype following differentiation.

Cystic fibrosis (CF) is a life-shortening genetic disorder, caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that regulates ion and fluid transport in epithelial tissue. Female CF patients face considerable fertility challenges, with higher prevalence of deficient fertility compared to healthy women. Not much is known about the underlying causes. In particular, the pathobiology of the endometrium, the uterus' inner lining essential for pregnancy and expressing fluctuating CFTR levels during the menstrual cycle, is unexplored in CF. To address this gap, we developed organoid models from CF patient endometrium. The organoids recapitulated CF characteristics and revealed molecular and pathway differences in cycle-recapitulating hormone responses compared to healthy endometrial organoids. Furthermore, specific functional aberrations were restored by CFTR modulator treatment. To further complement human organoid models for unraveling endometrial pathobiology in CF, we also developed organoids from a genetic CF mouse model that were also found to recapitulate CF characteristics. Moreover, single-cell RNA-sequencing analysis of the CF mouse uterus revealed molecular traits in the endometrium similar to the human CF endometrium (as evidenced by its organoid model). Our study provides new endometrium models to advance our understanding of CF-associated endometrial pathobiology, particularly regarding menstrual cycle aberrations that impact fertility. This research is timely since improved CF therapeutics result in increased life expectancy, allowing more CF patients to consider starting a family.

Ameloblasts are the specialized dental epithelial cell type responsible for enamel formation. Following completion of enamel development in humans, ameloblasts are lost and biological repair or regeneration of enamel is not possible. In the past, in vitro models to study dental epithelium and ameloblast biology were limited to freshly isolated primary cells or immortalized cell lines, both with limited translational potential. In recent years, large strides have been made with the development of induced pluripotent stem cell and organoid models of this essential dental lineage – both enabling modeling of human dental epithelium. Upon induction with several different signaling factors (such as transforming growth factor and bone morphogenetic proteins) these models display elevated expression of ameloblast markers and enamel matrix proteins. The advent of 3D bioprinting, and its potential combination with these advanced cellular tools, is poised to revolutionize the field – and its potential for tissue engineering, regenerative and personalized medicine. As the advancements in these technologies are rapidly evolving, we evaluate the current state-of-the-art regarding in vitro cell culture models of dental epithelium and ameloblast lineage with a particular focus toward their applicability for translational tissue engineering and regenerative/personalized medicine. Graphical Abstract Future perspectives for in vitro modeling of dental epithelium and ameloblasts. Development of iPSC and organoid models that can reliably generate dental epithelium and ameloblast-like cells, together with advances in 3D bioprinting, provide promising tools for enamel research. Advanced models will provide new avenues for development of enamel repair/regeneration approaches, for testing of dental materials or drugs, studying host-pathogen and/or cell-cell interactions, in vitro modeling of enamel diseases (e.g. amelogenesis imperfecta) and developing novel insights in fundamental tooth biology (e.g. regulation of amelogenesis, lineage specification). Abbreviations: iPSC: induced pluripotent stem cells; TO: tooth organoids; DE: dental epithelium; AB: ameloblast.

Single-cell (sc) omics has become a powerful tool to unravel a tissue’s cell landscape across health and disease. In recent years, sc transcriptomic interrogation has been applied to a variety of tooth tissues of both human and mouse, which has considerably advanced our fundamental understanding of tooth biology. Now, an overarching and integrated bird’s-view of the human and mouse tooth sc transcriptomic landscape would be a powerful multi-faceted tool for dental research, enabling further decipherment of tooth biology and development through constantly progressing state-of-the-art bioinformatic methods as well as the exploration of novel hypothesis-driven research. To this aim, we re-assessed and integrated recently published scRNA-sequencing datasets of different dental tissue types (healthy and diseased) from human and mouse to establish inclusive tooth sc atlases, and applied the consolidated data map to explore its power. For mouse tooth, we identified novel candidate transcriptional regulators of the ameloblast lineage. Regarding human tooth, we provide support for a developmental connection, not advanced before, between specific epithelial compartments. Taken together, we established inclusive mouse and human tooth sc atlases as powerful tools to potentiate innovative research into tooth biology, development and disease. The maps are provided online in an accessible format for interactive exploration.

The pituitary represents the endocrine master regulator. In mouse, the gland undergoes active maturation immediately after birth. Here, we in detail portrayed the stem cell compartment of neonatal pituitary. Single-cell RNA-sequencing pictured an active gland, revealing proliferative stem as well as hormonal (progenitor) cell populations. The stem cell pool displayed a hybrid epithelial/mesenchymal phenotype, characteristic of development-involved tissue stem cells. Organoid culturing recapitulated the stem cells’ phenotype, interestingly also reproducing their paracrine activity. The pituitary stem cell-activating interleukin-6 advanced organoid growth, although the neonatal stem cell compartment was not visibly affected in Il6^−/− mice, likely due to cytokine family redundancy. Further transcriptomic analysis exposed a pronounced WNT pathway in the neonatal gland, shown to be involved in stem cell activation and to overlap with the (fetal) human pituitary transcriptome. Following local damage, the neonatal gland efficiently regenerates, despite absence of additional stem cell proliferation, or upregulated IL-6 or WNT expression, all in line with the already high stem cell activation status, thereby exposing striking differences with adult pituitary. Together, our study decodes the stem cell compartment of neonatal pituitary, exposing an activated state in the maturing gland. Understanding stem cell activation is key to potential pituitary regenerative prospects.