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.

A molecular roadmap of reprogramming somatic cells into iPS cells.

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.

Modeling Rett syndrome with stem cells.

The discovery that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) raised the exciting possibility of modeling diseases with patient-specific cells. Marchetto et al. (2010) now use iPSC technology to generate, characterize, and treat an in vitro model for the autism spectrum disorder Rett syndrome.

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.

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.

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.

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.

Small molecules facilitate rapid and synchronous iPSC generation.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-ß inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

Safely Managing Acute Osteoarthritis in the Emergency Department: An Evidence-Based Review.

BACKGROUND: Joint pain caused by acute osteoarthritis (OA) is a common finding in the emergency department. Patients with OA often have debilitating pain that limits their function and ability to complete their activities of daily living. In addition, OA has been associated with a high percentage of arthritis-related hospital admissions and an increased risk of all-cause mortality. Safely managing OA symptoms in these patients can present many challenges to the emergency provider. OBJECTIVES: We review the risks and benefits of available treatment options for acute OA-related pain in the emergency department. In addition, evidence-based recommendations will be made for safely managing pain and disability associated with OA in patients with comorbidities, including cardiovascular disease, renal insufficiency, and risk factors for gastrointestinal bleeding. DISCUSSION: Commonly used treatments for OA include acetaminophen, oral nonsteroidal anti-inflammatory drugs, and opioids, each with varying degrees of efficacy and risk depending on the patient's underlying comorbidities. Effective alternative therapies, such as topical preparations, intra-articular corticosteroid injections, bracing, and rehabilitation are likely underused in this setting. CONCLUSIONS: Emergency providers should be aware of the risks and benefits of all treatment options available for acute OA pain, including oral medications, topical preparations, corticosteroid injections, bracing, and physical therapy.

Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, here we show that cells with low endogenous p19(Arf) (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) protein levels and immortal fibroblasts deficient in components of the Arf-Trp53 pathway yield iPS cell colonies with up to threefold faster kinetics and at a significantly higher efficiency than wild-type cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of Trp53 (also known as p53) in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. Our results show that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscore the similarities between induced pluripotency and tumorigenesis.

Functional genomics identifies therapeutic targets for MYC-driven cancer.

MYC oncogene family members are broadly implicated in human cancers, yet are considered "undruggable" as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ~3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer). Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously "undruggable" oncogene.

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.

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.

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm.

BACKGROUND: We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin-mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. RESULTS: During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenin δ1 mRNAs with a splice-site morpholino caused lateral widening and anterior-posterior shortening of the presomitic mesoderm and somites and a shortened anterior-posterior axis. These phenotypes indicate a cell-migration effect. Co-injection of low amounts of wild-type Cdc42 or wild-type Rac1 or dominant-negative RhoA mRNAs, but not constitutively-active Cdc42 mRNA, rescued these p120 catenin δ1-depleted embryos. CONCLUSIONS: These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development.

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.

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.

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.

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.

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.