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Epidermoid cysts are infrequent lesions occupying the intracranial space, comprising approximately 1–2% of all intracranial tumors. Brainstem epidermoids are exceptionally uncommon in children; up until now, only a few scattered case reports have been documented in the literature regarding this unique location. These cysts commonly arise from the inclusion of ectodermal elements during neural tube closure. Complete excision of these cysts is challenging due to their close proximity and adherence to the brainstem, which makes it difficult to achieve. As a result, recurrence of the cysts is not uncommon. We have reported a rare case of a 3-year-old with a 5-month history of progressive headache, imbalance while walking and progressive weakness in his right upper limb and lower limb along with difficulty in swallowing. On MRI Brain imaging study he had a pre-pontine epidermoid with intra-axial extension in the pons. The patient underwent retro-sigmoid/suboccipital craniotomy and microsurgical excision of the tumor, including the tumor capsule. After completing the surgery, the cavity was irrigated using a solution containing hydrocortisone and Ringer lactate to prevent the occurrence of aseptic meningitis. In the postoperative, the patient recovered without any complications, as all symptoms showed immediate improvement, and the lower cranial nerves returned to normal functioning. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells (PSCs) are a unique cell type that can differentiate into all cell types in the body. In PSC culture, subpopulations with different levels of pluripotency may exist, which leads to different results during their differentiation. One of the key factors that determine the state of pluripotency and influence the differentiation potential of PSCs is the epigenetic state of cells, including the level of histone deacetylation. Activation of histone deacetylase (HDAC) in human and mouse PSCs increases the percentage of heterochromatin. In this work, we used a protocol for the differentiation of embryoid bodies from induced human pluripotent hiPSC cells designed for the formation of ectoderm and neuroectoderm with their subsequent development into skin organoids. However, after hiPSCs were exposed to HDAC inhibitors (sodium butyrate and valproic acid), the direction of their differentiation changed: mesoderm was formed, which subsequently developed into contracting cardiospheres. [ABSTRACT FROM AUTHOR]

Abstract Background Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. Methods Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety. Results We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19–26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids. Conclusions Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms.

Background: Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. Methods: Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety. Results: We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19–26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids. Conclusions: Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms. [ABSTRACT FROM AUTHOR]

Acetylcholine, a vital neurotransmitter, plays a multifarious role in the brain and peripheral nervous system of various organisms. Previous research has demonstrated the proximity of cholinergic neurons to serotonergic neurons in the apical organ of sea urchin embryos. While several transcription factors have been identified as playing a role in the development of serotonergic neurons in this region of a sea urchin, Hemicentrotus pulcherrimus, comparatively little is known about the specific transcription factors and their spatiotemporal expression patterns that regulate the development of cholinergic neurons. In this study, we establish the requirement of the transcription factor Rx for the development of cholinergic neurons in the apical organ of the species. Furthermore, we investigate the role of the RNA-binding protein Musashi1, known to be involved in neurogenesis, including cholinergic neurons in other organisms, and demonstrate that it is a downstream factor of Rx, and that choline acetyltransferase expression is suppressed in Musashi1 downregulated embryos. Our research also highlights the intricate network formed by neurons and other cells in and around the apical organ of sea urchin larvae through axons and dendrites, providing possibility for a systematic and complexed neural pattern like those of the brain in other organisms.

Characterizing endogenous protein expression, interaction and function, this study identifies in vivo interactions and competitive balance between N-cadherin and E-cadherin in developing avian (Gallus gallus) neural and neural crest cells. Numerous cadherin proteins, including neural cadherin (Ncad) and epithelial cadherin (Ecad), are expressed in the developing neural plate as well as in neural crest cells as they delaminate from the newly closed neural tube. To clarify independent or coordinate function during development, we examined their expression in the cranial region. The results revealed surprising overlap and distinct localization of Ecad and Ncad in the neural tube. Using a proximity ligation assay and co-immunoprecipitation, we found that Ncad and Ecad formed heterotypic complexes in the developing neural tube, and that modulation of Ncad levels led to reciprocal gain or reduction of Ecad protein, which then alters ectodermal cell fate. Here, we demonstrate that the balance of Ecad and Ncad is dependent upon the availability of β-catenin proteins, and that alteration of either classical cadherin modifies the proportions of the neural crest and neuroectodermal cells that are specified.

Ovarian immature teratoma is a rare subtype of germ cell tumour that can be pure or associated with non-teratomatous germ cell tumour elements and is graded based on extent of the immature neuroectodermal component. Immature teratoma (IT) can also be associated with somatic differentiation in the form of sarcoma, carcinoma, or extensive immature neuroectodermal elements and may produce low levels of serum alpha-fetoprotein. Variable interpretation of these issues underlies diagnostic and management dilemmas, resulting in substantial practice differences between paediatric and adult women with IT. The Malignant Germ Cell International Consortium (MaGIC) convened oncologists, surgeons, and pathologists to address the following crucial clinicopathologic issues related to IT: (1) grading of IT, (2) definition and significance of 'microscopic' yolk sac tumour, (3) transformation to a somatic malignancy, and (4) interpretation of serum tumour biomarkers. This review highlights the discussion, conclusions, and suggested next steps from this clinicopathologic conference. • Diagnosis and grading (three- vs. two-tier) of immature teratoma (IT) remains challenging. • IT grade significantly affects treatment options. • Treatment options for IT differ between children and adults. • IT was discussed at a MaGIC-sponsored multidisciplinary conference. • This review presents conference discussion, conclusions, and proposed next steps. [ABSTRACT FROM AUTHOR]

Melanotic neuroectodermal tumor of infancy (MNTI) is a rare, rapidly growing, and pigmented neoplasm of neural crest origin. It predominantly affects the maxilla of infants during the first year of life. A seven-month-old boy presented with a mass approximately 5 cm in diameter in the right oral cavity. On computerized tomography, a lytic expansile lesion was detected in the right maxilla. Microscopically, the tumor consisted of two different neoplastic cell proliferation, located peripherally and centrally, arranged in alveolar clusters within the fibrous connective tissue. Immunohistochemically, peripheral tumor cells showed diffuse staining for Pancytokeratin and HMB-45; the central cells were positive for CD56 and Synaptophysin. MNTI is a rare tumor that can be easily confused with malign small round cell tumors, especially in small biopsies. It has characteristic histomorphological and immunohistochemical findings. Its biological behavior is not fully understood. These tumors can present locally aggressive behavior and a high recurrence rate. [ABSTRACT FROM AUTHOR]

Differentiated cells can be reprogrammed to embryonic stem cell-like cells called induced pluripotent stem cells (iPSCs), in which the natural developmental differentiation process is reversed. It is unclear whether the multi-lineage cells can be isolated and identified during reprogramming. In the current study, we detected the expression of lineage markers, isolated neural lineages, and identified the related microRNAs during iPSC formation. Our results demonstrated that a neuroectoderm appeared earlier than mesoderm and definitive endoderm before forming colonies when mouse embryonic fibroblasts were subjected to iPSC formation using transcription factors (TFs). On day 3, the cells expressed Sox1 and Nestin and had ultrastructure consistent with the transition to identity neural germ layer lineage. Fluorescence-activated cell sorting analysis revealed a peak (40%) in neural progenitor marker–positive cells. When subsequently cultured in a neural precursor cell medium, these cells proliferated slowly, became round and aggregated, generating into neurons and glia. Genome-wide microRNA (miRNA) analysis identified 45 differentially regulated miRNAs. Molecular network analysis demonstrated that these miRNAs validated 6,047 experimental mRNA targets. The GO functional annotation analysis of mRNA targets revealed that most genes were related to neurogenesis, such as growth cone, neuronal cell body, neuron projection, and cell junction synapse. The network of protein–protein interactions was observed, which demonstrated that key nodes of neural lineage reprogramming-associated targets were Sall1, Foxa2, Nf2, Ctnnb1, Shh, and Bmpr1a. Therefore, these data suggested that TFs can drive the reprogramming of somatic cells towards a pluripotent state via neuroectoderm. Moreover, the neural lineage reprogramming system can address how miRNAs influence their target sites. [ABSTRACT FROM AUTHOR]

Planar cell polarity (PCP) is the aligned cell polarity within a tissue plane. Mechanical signals are known to act as a global cue for PCP, yet their exact role is still unclear. In this study, we focused on PCP in the posterior neuroectoderm of Xenopus laevis and investigated how mechanical signals regulate polarity. We reveal that the neuroectoderm is under a greater tension in the anterior-posterior direction and that perturbation of this tension causes PCP disappearance. We show that application of uniaxial stretch to explant tissues can control the orientation of PCP and that cells sense the tissue stretch indirectly through a change in their shape, rather than directly through detection of anisotropic tension. Furthermore, we reveal that PCP is most strongly established when the orientation of tissue stretch coincides with that of diffusion of locally expressed Wnt ligands, suggesting a cooperative relationship between these two PCP regulators. [ABSTRACT FROM AUTHOR]

Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines. [ABSTRACT FROM AUTHOR]

Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.

Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates. [Display omitted] • ESCs naturally differ in their mitochondrial ROS level • ESC fate decisions at the onset of gastrulation are governed by mitochondrial ROS • A high mitochondrial ROS level biases ESCs toward mesendodermal (ME) fates • ME specification is controlled by redox-sensitive ROS/p53-Wnt3/Nrf2-H3K4me3 axis How pluripotent embryonic stem cells (ESCs) choose distinct cell fates during embryonic development is still a mystery. Ulfig and Jakob now demonstrate that ESCs naturally differ in their mitochondrial reactive oxygen species levels that individualize their nuclear redox state, H3K4me3 landscape, and ultimately, cell fate. [ABSTRACT FROM AUTHOR]

Olfactory neuroblastoma (ONB) or esthesioneuroblastoma is a rare malignancy of the nose and paranasal sinuses that originates from the upper nasal vault. We report a rare case of ONB in a 27-year-old female patient with a 2-year history of nasal obstruction, sneezing, nasal discharge, and left eye protrusion. The patient was successfully treated with endoscopic sinus surgery and adjuvant radiotherapy (RT) with 5-year follow-up duration. Studying ONB cases is of great importance due to its rare occurrence, and the lack of literature regarding its management often creates a dilemma leading to nonstandardized treatment methods. Prolonged surveillance is essential due to its late recurrence and endoscopic resection followed by adjuvant RT, which may be a feasible and effective treatment. There is no single case of ONB published yet in Saudi Arabia, and to our knowledge, this is the first case report.

The orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here, we use proximity biotinylation and crosslinking approaches to show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3, also known as Fzd3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. This inhibition required Fz3-dependent Vangl2 phosphorylaton. Consistent with our observations, the complex of Pk3 with nonphosphorylatable Vangl2 did not polarize in the neural plate. These findings provide evidence for in vivo regulation of Vangl2-Pk3 complex formation and localization by a Frizzled receptor. [ABSTRACT FROM AUTHOR]

Olfactory neuroblastoma (ONB) or esthesioneuroblastoma is a rare malignancy of the nose and paranasal sinuses that originates from the upper nasal vault. We report a rare case of ONB in a 27-year-old female patient with a 2-year history of nasal obstruction, sneezing, nasal discharge, and left eye protrusion. The patient was successfully treated with endoscopic sinus surgery and adjuvant radiotherapy (RT) with 5-year follow-up duration. Studying ONB cases is of great importance due to its rare occurrence, and the lack of literature regarding its management often creates a dilemma leading to nonstandardized treatment methods. Prolonged surveillance is essential due to its late recurrence and endoscopic resection followed by adjuvant RT, which may be a feasible and effective treatment. There is no single case of ONB published yet in Saudi Arabia, and to our knowledge, this is the first case report. [ABSTRACT FROM AUTHOR]

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore, an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the "neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42-60 days). With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID) mouse brains compared with the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.

Coordinated polarization of cells in the tissue plane, known as planar cell polarity (PCP), is associated with a signaling pathway critical for the control of morphogenetic processes. Although the segregation of PCP components to opposite cell borders is believed to play a critical role in this pathway, whether PCP derives from egg polarity or preexistent long-range gradient, or forms in response to a localized cue, remains a challenging question. Here we investigate the Xenopus neural plate, a tissue that has been previously shown to exhibit PCP. By imaging Vangl2 and Prickle3, we show that PCP is progressively acquired in the neural plate and requires a signal from the posterior region of the embryo. Tissue transplantations indicated that PCP is triggered in the neural plate by a planar cue from the dorsal blastopore lip. The PCP cue did not depend on the orientation of the graft and was distinct from neural inducers. These observations suggest that neuroectodermal PCP is not instructed by a preexisting molecular gradient but induced by a signal from the dorsal blastopore lip.

There is increasing evidence that long noncoding RNAs (lncRNAs) are among the main regulatory factors of stem cell maintenance and differentiation. They act through various mechanisms and interactions with proteins, DNA, and RNA. This heterogeneity in function increases the capabilities of the lncRNome toolkit but also makes it difficult to predict the function of novel lncRNAs or even rely on biological information produced in animal models. As lncRNAs are species- and tissue-specific, the recent technical advances in self-renewal and differentiation of human embryonic stem cells (ESCs) make these cells the ideal system to identify key regulatory lncRNAs and study their molecular functions. Here we provide an overview of the functional versatility of lncRNA mechanistic heterogeneity in regulating pluripotency maintenance and human differentiation. Embryonic differentiation at the gastrulation stage is a very tightly regulated process in which many long noncoding RNAs (lncRNAs) have been demonstrated to play a crucial role in gene expression modulation. lncRNAs can regulate cellular functions at the transcriptional, post-transcriptional, translational, and post-translational levels. lncRNAs exert a large repertoire of regulatory functions both in the nucleus and in the cytoplasm. [ABSTRACT FROM AUTHOR]

Based on these findings, an embryonal tumor was ultimately diagnosed, most consistent with medulloblastoma. Central nervous system, cerebellum, embryonal tumor, medulloblastoma, neuroectoderm Keywords: central nervous system; cerebellum; embryonal tumor; medulloblastoma; neuroectoderm EN central nervous system cerebellum embryonal tumor medulloblastoma neuroectoderm 283 285 3 06/06/22 20220601 NES 220601 CASE PRESENTATION A 3-year-old neutered male Labrador retriever presented to the neurology service at the Centre Hospitalier Universitaire Vétérinaire of the Université de Montréal for behavioral changes, spastic paresis, episodes of ataxia, and loss of balance over 1.5 months. [Extracted from the article]

During early neural development, changes in signalling inform the expression of transcription factors that in turn instruct changes in cell identity. At the same time, switches in adhesion molecule expression result in cellular rearrangements that define the morphology of the emerging neural tube. It is becoming increasingly clear that these two processes influence each other; adhesion molecules do not simply operate downstream of or in parallel with changes in cell identity but rather actively feed into cell fate decisions. Why are differentiation and adhesion so tightly linked? It is now over 60 years since Conrad Waddington noted the remarkable "Constancy of the Wild Type" (Waddington in Nature 183: 1654–1655, 1959) yet we still do not fully understand the mechanisms that make development so reproducible. Conversely, we do not understand why directed differentiation of cells in a dish is sometimes unpredictable and difficult to control. It has long been suggested that cells make decisions as 'local cooperatives' rather than as individuals (Gurdon in Nature 336: 772–774, 1988; Lander in Cell 144: 955–969, 2011). Given that the cadherin family of adhesion molecules can simultaneously influence morphogenesis and signalling, it is tempting to speculate that they may help coordinate cell fate decisions between neighbouring cells in the embryo to ensure fidelity of patterning, and that the uncoupling of these processes in a culture dish might underlie some of the problems with controlling cell fate decisions ex-vivo. Here we review the expression and function of cadherins during early neural development and discuss how and why they might modulate signalling and differentiation as neural tissues are formed. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, because cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but their functional consequences in differentiating cells are scarcely described. In this work, we show that the loss of chromosome 18q impairs neuroectoderm commitment and that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for this failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, and transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, suggesting that these cells are in an altered state of pluripotency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

• Human ES cell aggregates exhibited morphogenesis under a new differentiation protocol • Transcripts associated with paraxial mesoderm and neuroectoderm were up-regulated • Transcript levels in aggregates were significantly altered by most teratogenic exposures • Size and shape of aggregates were quantifiably altered by many teratogenic exposures Pluripotent stem cells recapitulate many aspects of embryogenesis in vitro. Here, we established a novel culture system to differentiate human embryonic stem cell aggregates (HESCA), and evaluated its utility for teratogenicity assessment. Culture of HESCA with modulators of developmental signals induced morphogenetic and molecular changes associated with differentiation of the paraxial mesoderm and neuroectoderm. To examine impact of teratogenic exposures on HESCA differentiation, 18 compounds were tested, for which adequate information on in vivo plasma concentrations is available. HESCA treated with each compound were examined for gross morphology and transcript levels of 15 embryogenesis regulator genes. Significant alterations in the transcript levels were observed for 94% (15/16) of the teratogenic exposures within 5-fold margin, whereas no alteration was observed for 92% (11/12) of the non-teratogenic exposures. Our study demonstrates that transcriptional changes in HESCA serve as predictive indicator of teratogenicity in a manner comparable to in vivo exposure levels. [ABSTRACT FROM AUTHOR]

Linking phenotypes to genetic components has been an essential part of novel drug discovery, and screening methods have been widely employed to achieve such a goal. Screens can be conducted in either pooled or arrayed formats. Although arrayed screenings provide a better and cheaper alternative in small scale, the larger-scale screenings are conducted in pooled manner. With its adaptability to various models and conditions, CRISPR/Cas9 technology provides an invaluable alternative to classical and RNAi-based screening methods. Combined with high-throughput sequencing and bioinformatics, CRISPR-/Cas9-based pooled screening methods provide unbiased and robust data. In this protocol, we employed CRISPR-/Cas9-based pooled screening for a non-binary and non-immediate readout., (© 2024. Springer Science+Business Media, LLC.)

Mitochondrial disorders (MDs) arise as a result of a respiratory chain dysfunction. While some MDs can affect a single organ, many involve several organs, the brain being the most affected, followed by heart and/or muscle. Many of these diseases are associated with heteroplasmic mutations in the mitochondrial DNA (mtDNA). The proportion of mutated mtDNA must exceed a critical threshold to produce disease. Therefore, understanding how embryonic development determines the heteroplasmy level in each tissue could explain the organ susceptibility and the clinical heterogeneity observed in these patients. In this report, the dynamics of heteroplasmy and the influence in cardiac commitment of the mutational load of the m.13513G>A mutation has been analyzed. This mutation has been reported as a frequent cause of Leigh syndrome (LS) and is commonly associated with cardiac problems. In this report, induced pluripotent stem cell (iPSc) technology has been used to delve into the molecular mechanisms underlying cardiac disease in LS. When mutation m.13513G>A is above a threshold, iPSc‐derived cardiomyocytes (iPSc‐CMs) could not be obtained due to an inefficient epithelial‐mesenchymal transition. Surprisingly, these cells are redirected toward neuroectodermal lineages that would give rise to the brain. However, when mutation is below that threshold, dysfunctional CM are generated in a mutant‐load dependent way. We suggest that distribution of the m.13513G>A mutation during cardiac differentiation is not at random. We propose a possible explanation of why neuropathology is a frequent feature of MD, but cardiac involvement is not always present. [ABSTRACT FROM AUTHOR]

Summary Strandings of live new-born West Indian manatees (WIMs; Trichechus manatus) are one of the main challenges for the conservation of this species in Brazil, particularly in the northeastern states. Congenital malformations (CMs) are rare in sirenians. We identified CMs in two of 19 stranded WIMs that were rescued, rehabilitated and subjected to complete pathological examinations in Ceará and Rio Grande do Norte States between 1992 and 2017. In case 1, dilation of the cerebral lateral and fourth ventricles with abundant cerebrospinal fluid (internal hydrocephalus), was diagnosed. Furthermore, this animal developed necrotizing enterocolitis associated with pneumatosis intestinalis and aspiration pneumonia late during rehabilitation. Cardiac malformations in case 2 included: right ventricle hypoplasia with marked stenosis of the tricuspid outflow, high ventricular septal defect, segmental pulmonary artery aneurysm, mitral valve haemocyst and left ventricular hypertrophy. Herein, we provide the first description of a neural tube defect, specifically a developmental internal hydrocephalus, and multiple cardiac congenital anomalies, together with their respective clinicopathological features in manatees. Although the aetiology of the CMs remains unknown in these cases, a genetic basis is plausible given the low genetic variability in this population. These cases add to the body of knowledge on health and disease aspects of manatees and may provide scientific basis for future medical and conservation efforts on neonatal WIMs. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Melanotic neuroectodermal tumour of infancy (MNTI) is rare, rapidly growing, pigmented neoplasm of neural crest origin. It is generally accepted as a benign tumour despite of its rapid and locally destructive growth. It primarily affects the maxilla of infants during the first year of life. Surgical excision is considered as the treatment of choice. The recurrence rate varies between 10% and 15%, and malignant behaviour has been reported in 6.5% of cases. We report a case of MNTI, associated with an erupted primary tooth in a 5-month-old male child. We discuss the clinical, radiographic and histologic features of this rare tumour, as well as its surgical management and the follow-up.

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore, an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the “neurosphederm”. The large neural tube-type rosette (NTTR) structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42–60 days). With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID) mouse brains compared with the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.

F-53B, as an alternative to the persistent organic pollutant perfluorooctane sulfonate (PFOS), is amply used in the electric plating industry. F-53B and PFOS have similar physicochemical, biochemical and physiological properties, due to the similarity in their chemical structure. Thus, they may also possess similar toxicities. Although epidemiological studies and in vivo assays have shown that prenatal exposure to PFOS may impair the development of the nervous system, toxicity data for F-53B are still scarce. In this study, we employed an embryonic stem cell (ESC) in vitro differentiation system, to detect the potential developmental neural toxicity of F-53B and PFOS, at human exposure relevant doses. We demonstrated that during early mouse ESC (mESC) neural differentiation, F-53B and PFOS disrupted the expression of neural marker genes and affected the morphology of the differentiated cells. However, the very same treatments did not cause any cytotoxic effects. In conclusion, our ESC in vitro differentiation system was able to prove for the first time that F-53B and PFOS at human exposure relevant concentrations, could alter the expression of differentiation biomarkers, indicating a potential developmental neural toxicity. Based on our findings, it is reasonable to deduce that excessive exposure to F-53B and PFOS may cause severe dysfunctions during early stages of embryo development. [ABSTRACT FROM AUTHOR]

Characterizing endogenous protein expression, interaction and function, this study identifies in vivo interactions and competitive balance between N-cadherin and E-cadherin in developing avian ( Gallus gallus ) neural and neural crest cells. Numerous cadherin proteins, including neural cadherin (Ncad) and epithelial cadherin (Ecad), are expressed in the developing neural plate as well as in neural crest cells as they delaminate from the newly closed neural tube. To clarify independent or coordinate function during development, we examined their expression in the cranial region. The results revealed surprising overlap and distinct localization of Ecad and Ncad in the neural tube. Using a proximity ligation assay and co-immunoprecipitation, we found that Ncad and Ecad formed heterotypic complexes in the developing neural tube, and that modulation of Ncad levels led to reciprocal gain or reduction of Ecad protein, which then alters ectodermal cell fate. Here, we demonstrate that the balance of Ecad and Ncad is dependent upon the availability of β-catenin proteins, and that alteration of either classical cadherin modifies the proportions of the neural crest and neuroectodermal cells that are specified. [ABSTRACT FROM AUTHOR]

Transcription factor PRH/Hhex suppresses cell proliferation and contributes to regulation of prenatal and postnatal ontogeny. Neurons of the peripheral nervous system and chromaffin cells were previously considered as non-expressing PRH/Hhex in postnatal development. In our study, the expression of PRH/Hhex in chromaffin cells of rat adrenal glands and association between the decrease of proliferation and activation of PRH/Hhex expression were demonstrated. [ABSTRACT FROM AUTHOR]

Abstract Directed differentiation of pluripotent stem cells provides an accessible system to model development. However, the distinct cell types that emerge, their dynamics, and their relationship to progenitors in the early embryo has been difficult to decipher because of the cellular heterogeneity inherent to differentiation. Here, we used a combination of bulk RNA-Seq, single cell RNA-Seq, and bioinformatics analyses to dissect the cell types that emerge during directed differentiation of mouse embryonic stem cells as embryoid bodies and we compared them to spatially and temporally resolved transcriptional profiles of early embryos. Our single cell analyses of the day 4 embryoid bodies revealed three populations which had retained related yet distinct pluripotent signatures that resemble the pre- or post-implantation epiblast, one population of presumptive neuroectoderm, one population of mesendoderm, and four populations of neural progenitors. By day 6, the neural progenitors predominated the embryoid bodies, but both a small population of pluripotent-like cells and an anterior mesoderm-like Brachyury-expressing population were present. By comparing the day 4 and day 6 populations, we identified candidate differentiation paths, transcription factors, and signaling pathways that mark the in vitro correlate of the transition from the mid-to-late primitive streak stage. Highlights • Single-cell RNA-Seq of 5062 day 4 and 4,842 day 6 embryoid body-derived cells from primitive streak-promoting conditions. • The eight day 4 populations include pre-and post-implantation epiblast, mesendoderm, neuroectoderm, and neural progenitors. • After two more days of differentiation with bFGF, neural progenitors outnumber epiblast and anterior mesoderm cells. • Bioinformatics analyses identitied novel transcription factors and signaling pathways contributing to EB differentaition [ABSTRACT FROM AUTHOR]

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation. [ABSTRACT FROM AUTHOR]

Objective: To observe simultaneous differentiation and analyse possible interactions between co‐cultured human oral mucosal stem cells (hOMSC) and mouse neural stem cells (mNSC). Materials and Methods: hOMSC and mNSC were co‐cultured in mouse and in human medium, and their immunocytochemical characterization to detect survival rate and differentiation pattern was performed. Co‐cultures in different media were compared to hOMSC in human medium and mNSC in mouse medium as controls. Results: Co‐culture of hOMSC and mNSC in medium for human cells led to normal differentiation pattern of human cells, while mNSC were directed towards astrocytes. When the same cells were cultivated in the mouse medium, both cell types succeeded to form neurons, although mNSC showed a tendency to overgrow hOMSC. hOMSC alone in the human‐specific medium differentiated towards ectodermal (Oct4, Map2) and mesodermal (Osterix) cell populations. mNSC in the mouse‐specific medium differentiated towards Map2‐, β3‐tubulin‐ and NeuN‐positive neurons. Conclusions: hOMSC and mNSC can form co‐cultures. Different media considerably affected the differentiation pattern of co‐cultures, whereas one cell population itself modestly influenced differentiation of the other cell type. The in vitro differentiation pattern of hOMSC in the mouse neural tissue environment suggested that hOMSC could be beneficial in the brain tissue affected by ischaemia. [ABSTRACT FROM AUTHOR]

Summary Neuroectodermal developmental anomalies are reported rarely in cetaceans and central nervous system cysts are not described. We describe the gross, microscopical, histochemical and immunohistochemical features of a neuraxial myelencephalic cyst in a stranded neonatal Burmeister's porpoise ( Phocoena spinipinnis ). Grossly, a subdural, extra-axial, well-demarcated, yellow fluid-filled cystic structure (1.9 × 1.6 × 1 cm) expanded the left foramen of Luschka, the left caudolateral cerebellar recess and the left cranioventral myelencephalon. The cyst displaced the ipsilateral ventral paraflocculus and distended the underlying cranial nerves IX, X, XI and XII. Microscopically, the cystic structure was lined by a monolayer of low cuboidal to flattened epithelium supported by a thin fibrovascular matrix. Immunohistochemistry (IHC) revealed strong and diffuse expression of AE1/AE3 and focal positivity for vimentin. IHC for epithelial membrane antigen, glial fibrillary acid protein, synaptophysin and S100 was negative. Based on these findings, an extra-axial cyst of the choroid plexus of the fourth ventricle (CCPFV) was diagnosed. The pathological relevance of the CCPFV in this case is uncertain. The cause of death involved severe perinatal interspecific (shark) trauma. The present case provides the first evidence of a neuroepithelial cyst in cetacean species. [ABSTRACT FROM AUTHOR]

Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, β-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Acetylcholine, a vital neurotransmitter, plays a multifarious role in the brain and peripheral nervous system of various organisms. Previous research has demonstrated the proximity of cholinergic neurons to serotonergic neurons in the apical organ of sea urchin embryos. While several transcription factors have been identified as playing a role in the development of serotonergic neurons in this region of a sea urchin, Hemicentrotus pulcherrimus , comparatively little is known about the specific transcription factors and their spatiotemporal expression patterns that regulate the development of cholinergic neurons. In this study, we establish the requirement of the transcription factor Rx for the development of cholinergic neurons in the apical organ of the species. Furthermore, we investigate the role of the RNA-binding protein Musashi1, known to be involved in neurogenesis, including cholinergic neurons in other organisms, and demonstrate that it is a downstream factor of Rx, and that choline acetyltransferase expression is suppressed in Musashi1 downregulated embryos. Our research also highlights the intricate network formed by neurons and other cells in and around the apical organ of sea urchin larvae through axons and dendrites, providing possibility for a systematic and complexed neural pattern like those of the brain in other organisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yaguchi and Yaguchi.)