Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Combined small-molecule treatment accelerates maturation of human pluripotent stem cell-derived neurons.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months to years for reaching adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. Therefore, we designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons which identified multiple compounds that drive neuronal maturation including inhibitors of lysine-specific demethylase 1 and disruptor of telomerase-like 1 and activators of calcium-dependent transcription. A cocktail of four factors, GSK2879552, EPZ-5676, N-methyl-D-aspartate and Bay K 8644, collectively termed GENtoniK, triggered maturation across all parameters tested, including synaptic density, electrophysiology and transcriptomics. Maturation effects were further validated in cortical organoids, spinal motoneurons and non-neural lineages including melanocytes and pancreatic ß-cells. The effects on maturation observed across a broad range of hPSC-derived cell types indicate that some of the mechanisms controlling the timing of human maturation might be shared across lineages.

The prevalence, characteristics, and risk factors of external cervical resorption: a retrospective practice-based study.

OBJECTIVES: External cervical root resorption (ECR) is a poorly understood and aggressive form of resorption. The purpose of this study was to examine the prevalence, characteristics, and risk factors associated with the occurrence of ECR in patients seeking endodontic care from private practice settings. MATERIALS AND METHODS: Records of 343 patients with 390 teeth diagnosed with ECR were identified from 3 private endodontic practices from 2008 to 2022. The patients' demographic information, systemic conditions, and dental history were recorded. The characteristics of the cases including Heithersay classification, pulpal and periapical status, and their management were documented. The association between case severity and potential predisposing factors was examined using chi-square analysis. RESULTS: The overall prevalence of ECR among patients seeking endodontic care was low (< 1%). However, there was a greater than twofold increase in the pooled prevalence from 2016 to 2021 (0.99%) compared to the data from 2010 to 2015 (0.46%). The most commonly affected teeth were anterior teeth (48.7%). Class II (30.0%) and class III (45.4%) defects were the most often identified. Patients with a history of trauma or orthodontic treatment were significantly more likely to be diagnosed with severe cervical resorption (class III or IV) (p < 0.05). CONCLUSIONS: There has been an increase in the prevalence of ECR in patients seeking endodontic care. A history of orthodontic treatment and traumatic dental mechanical injuries may predict the severity of resorption. CLINICAL RELEVANCE: The upward trend in the occurrence of ECR warrants close monitoring of the patients at high risk of developing the condition to facilitate early detection and management.

A retrospective study of initial root canal treatment failure in maxillary premolars via using cone-beam computed tomography.

BACKGROUND: This case-series study examined canal morphology and common factors for endodontic failure in maxillary first and second premolars that were referred for retreatment owing to clinical symptoms or radiographic signs. METHODS: Records were retrospectively searched using Current Dental Terminology codes to identify maxillary first and second premolars with endodontic failure. Periapical and cone-beam computed tomographic images were examined to determine Vertucci classifications and suspected factors related to treatment failure. RESULTS: A total of 235 teeth from 213 patients were included for evaluation. The following Vertucci classification of canal configurations were observed for maxillary first and second premolars: type I (1-1) (4.6% and 32.0%, respectively), type II (2-1) (15.9% and 27.9%, respectively), type III (2-2) (76.1% and 36.1%, respectively), type IV (1-2) (0% and 2%, respectively), and type V (3) (3.4% and 2%, respectively). More treatment failures were noticed in maxillary second premolars than first premolars and in females than in males. The 4 most common factors related to failure were inadequate filling, restorative failure, vertical root fracture, and missed canals. Missed canals were more frequently identified in maxillary second premolars (21.8%) than first premolars (11.4%) (P = .044). CONCLUSIONS: Multiple factors are associated with primary root canal treatment failures in maxillary premolars. Variations in canal morphology appear to be underappreciated in maxillary second premolars. PRACTICAL IMPLICATIONS: Maxillary second premolars have more complicated canal configurations than first premolars. Besides adequate filling, clinicians should give extra attention to anatomic variability in second premolars owing to higher failure incidence.

Managing a Deep Subgingival Cusp Fracture: Case Reports.

Cuspal fractures are relatively common. Fortunately for esthetics, when a cuspal fracture occurs in a maxillary premolar, it most commonly involves the palatal cusp. Fractures with a favorable prognosis may be approached with a minimally invasive treatment to successfully retain the natural tooth. This report describes 3 cases of "cuspidization" to treat maxillary premolars with cuspal fractures. After identifying a palatal cusp fracture, the fractured segment was removed, resulting in a tooth that closely resembles a cuspid. Depending on the extent and location of the fracture, root canal treatment was indicated. Subsequently, conservative restorations sealed the access and covered exposed dentin. Full coverage restorations were neither required nor indicated. The resulting treatment provided practical and functional treatment with a good esthetic outcome. When indicated, the described cuspidization technique can conservatively manage patients with subgingival cuspal fractures. The procedure is minimally invasive and cost-effective and can be done conveniently in routine practice.

Dissection of gastric homeostasis in vivo facilitates permanent capture of isthmus-like stem cells in vitro.

The glandular stomach is composed of two regenerative compartments termed corpus and antrum, and our understanding of the transcriptional networks that maintain these tissues is incomplete. Here we show that cell types with equivalent functional roles in the corpus and antrum share similar transcriptional states including the poorly characterized stem cells of the isthmus region. To further study the isthmus, we developed a monolayer two-dimensional (2D) culture system that is continually maintained by Wnt-responsive isthmus-like cells capable of differentiating into several gastric cell types. Importantly, 2D cultures can be converted into conventional three-dimensional organoids, modelling the plasticity of gastric epithelial cells in vivo. Finally, we utilized the 2D culture system to show that Sox2 is both necessary and sufficient to generate enterochromaffin cells. Together, our data provide important insights into gastric homeostasis, establish a tractable culture system to capture isthmus cells and uncover a role for Sox2 in enterochromaffin cells.

Clinical outcomes of non-surgical root canal obturations using NeoMTA: A retrospective series of case reports.

NeoMTA is a commercially available tricalcium silicate-based cement intended for contact with pulp and periradicular tissues. The purpose of this study was to retrospectively evaluate the outcomes of non-surgical root canal treatments with NeoMTA obturations. Patients were treated in a private endodontic practice between 2015 and 2018. All cases, including initial treatments and retreatments, were either fully obturated with NeoMTA, or using gutta-percha with NeoMTA as an endodontic sealer. Outcomes were assessed using follow-up examination data with digital periapical radiographs with a minimum of a 1-year recall. Teeth were classified based on the clinical examination as: healed/healing (success), or non-healed (failure). 265 teeth were included with an average follow-up time of 1.3 years. The overall success rate was 91.7%. Only the presence of a pre-operative periapical radiolucency was found to significantly affect success. Comparison of obturation techniques demonstrated no effect on outcomes. NeoMTA is suitable for endodontic obturation.

Scleroderma and multiple teeth with invasive cervical resorption: is there a connection?

Scleroderma is a chronic connective tissue disease generally classified as an autoimmune rheumatic disease. Symptoms may include thickening of the skin, calcifications, Raynaud syndrome, and esophageal problems. Invasive cervical resorption is an aggressive form of external resorption localized in the cervical part of the tooth. Its etiology remains uncertain. This case report describes invasive cervical resorption affecting 4 teeth in a 44-year-old woman with scleroderma and speculates on the possible relationships between these disease entities.

Developmental chromatin programs determine oncogenic competence in melanoma.

Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cell­derived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease.

Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APPSWE+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APPSWE+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.

Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo.

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.

Specification of positional identity in forebrain organoids.

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

Histology of NeoMTA Plus and Quick-Set2 in Contact with Pulp and Periradicular Tissues in a Canine Model.

INTRODUCTION: NeoMTA Plus (Avalon Biomed Inc, Bradenton, FL) is a tricalcium silicate material similar to the first mineral trioxide aggregate product, ProRoot MTA (Dentsply Sirona, York, PA), but with improvements such as decreased setting time, increased ion release, increased water sorption, and nonstaining radiopacifiers. Quick-Set2 (Avalon Biomed Inc) is a newly formulated calcium aluminosilicate material that has a faster setting time and increased acid resistance and is nonstaining. The purpose of this study was to compare the healing of pulpal and periapical tissues in dogs after exposure to NeoMTA Plus and Quick-Set2 after pulpotomy and root-end surgery procedures. METHODS: Seventy-two teeth (36 for each procedure) in 6 beagle dogs received pulpotomy or root-end surgery using either NeoMTA Plus or Quick-Set2. The dogs were sacrificed at 90 days, and the teeth and surrounding tissues were prepared for histologic evaluation. Sixty teeth were evaluated and scored histologically (29 with pulpotomies and 31 with root-end resections). Specimens were scored for inflammation, quality and thickness of dentin bridging, pulp tissue response, cementum and periodontal ligament formation, and apical bone healing. RESULTS: Both materials displayed favorable healing at 90 days. The only significant difference was the quality of dentin bridge formation in pulpotomies using NeoMTA Plus compared with Quick-Set2. CONCLUSIONS: Quick-Set2 and NeoMTA Plus had similar effects on inflammation, pulp response, periodontal ligament and cementum formation, and apical tissue healing in dogs. NeoMTA Plus had superior dentin bridge quality compared with Quick-Set2.

Prospective Isolation of Poised iPSC Intermediates Reveals Principles of Cellular Reprogramming.

Cellular reprogramming converts differentiated cells into induced pluripotent stem cells (iPSCs). However, this process is typically very inefficient, complicating mechanistic studies. We identified and molecularly characterized rare, early intermediates poised to reprogram with up to 95% efficiency, without perturbing additional genes or pathways, during iPSC generation from mouse embryonic fibroblasts. Analysis of these cells uncovered transcription factors (e.g., Tfap2c and Bex2) that are important for reprogramming but dispensable for pluripotency maintenance. Additionally, we observed striking patterns of chromatin hyperaccessibility at pluripotency loci, which preceded gene expression in poised intermediates. Finally, inspection of these hyperaccessible regions revealed an early wave of DNA demethylation that is uncoupled from de novo methylation of somatic regions late in reprogramming. Our study underscores the importance of investigating rare intermediates poised to produce iPSCs, provides insights into reprogramming mechanisms, and offers a valuable resource for the dissection of transcriptional and epigenetic dynamics intrinsic to cell fate change.

Bioactive endodontic materials for everyday use: a review.

Bioceramic materials are at the forefront of modern dentistry. Bioactive bioceramic endodontic materials promote pulpal and periapical tissue healing and are easy to use. Dentists can choose among many endodontic materials, depending on their needs. This article highlights the major differences among commercially available bioactive tricalcium silicate bioceramics, commonly known as mineral trioxide aggregate materials, to enable dentists to make appropriate decisions in the selection of these materials.

Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

Phf8 loss confers resistance to depression-like and anxiety-like behaviors in mice.

PHF8 is a histone demethylase with specificity for repressive modifications. While mutations of PHF8 have been associated with cognitive defects and cleft lip/palate, its role in mammalian development and physiology remains unexplored. Here, we have generated a Phf8 knockout allele in mice to examine the consequences of Phf8 loss for development and behaviour. Phf8 deficient mice neither display obvious developmental defects nor signs of cognitive impairment. However, we report a striking resiliency to stress-induced anxiety- and depression-like behaviour on loss of Phf8. We further observe misregulation of serotonin signalling within the prefrontal cortex of Phf8 deficient mice and identify the serotonin receptors Htr1a and Htr2a as direct targets of PHF8. Our results clarify the functional role of Phf8 in mammalian development and behaviour and establish a direct link between Phf8 expression and serotonin signalling, identifying this histone demethylase as a potential target for the treatment of anxiety and depression.