Neural Differentiation Markers Research Articles

ARMCX3 regulates ROS signaling, affects neural differentiation and inflammatory microenvironment in dental pulp stem cells

BackgroundThe neural differentiation of dental pulp stem cells (DPSCs) exhibits great potential in the treatment of dental pulp repair and neurodegenerative diseases. However, the precise molecular mechanisms underlying this process remain unclear. This study was designed to reveal the roles and regulatory mechanisms of the armadillo repeat-containing X-linked 3 (ARMCX3) in neural differentiation and inflammatory microenvironment in human DPSCs (hDPSCs). MethodsWe treated hDPSCs with porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to simulate the inflammatory microenvironment. Then the lentiviral vectors were introduced to construct stable cell lines with ARMCX3 knockdown or overexpression. The expression of neural-specific markers, ARMCX3 and inflammation factors were estimated by immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) assays. Additionally, we used IF assays and specific kits to investigate the regulatory role of ARMCX3 on reactive oxygen species (ROS) signaling. Moreover, a ROS inhibitor was utilized to verify whether ROS inhibition reversed the effects of ARMCX3 in Pg-LPS-treated hDPSCs. ResultsThis work illustrated that Pg-LPS treatment significantly enhanced ARMCX3 expression and inflammatory response, and inhibited neural differentiation in hDPSCs. ARMCX3 knockdown effectively accelerated neural differentiation and controlled inflammatory cytokines at a lower level in hDPSCs in the presence of Pg-LPS. Additionally, knockdown of ARMCX3 notably reduced ROS production and ROS inhibition effectively eliminated the roles of ARMCX3 overexpression in hDPSCs. Besides, all results were proved to be statistically significant. ConclusionThis investigation proved that ARMCX3 affected neural differentiation and inflammation microenvironment in hDPSCs at least partly by mediating ROS signal. These findings provided a new perspective on the mechanism of neural differentiation of hDPSCs and help to better explore the therapeutic schedule of pulpitis and neurodegenerative diseases.

Undifferentiated versus retinoic acid - differentiated SH-SY5Y cells in investigation of markers of neural function in toxicological research

The SH-SY5Y human neuroblastoma cell line is a standard in vitro experimental model of neuronal-like cells used in neuroscience and toxicological research. These cells can be differentiated into mature neurons, most commonly using retinoic acid (RA). Despite differences in characteristics, both undifferentiated and differentiated SH-SY5Y cells are used in research. However, due to uncertainties regarding the expression of specific markers of neural function in each culture, there is no definite conclusion on which culture is better suited for (neuro)toxicological and/or neuroscience investigations. To address this dilemma, we investigated the basal expression/activity of the key elements of acetylcholine, dopamine, serotonin and GABA neurotransmitter pathways, along with the elements involved in exocytosis of neurotransmitters, and neuron electrophysiological activity in undifferentiated and in RA–differentiated SH-SY5Y cells using a 6-day differentiation protocol. Our findings revealed that both SH-SY5Y cell types are functionally active. While undifferentiated SH-SY5Y cells exhibited greater multipotency in the expression of tested markers, most of those markers expressed in both cell types showed higher expression levels in RA-differentiated SH-SY5Y cells. Our results suggest that the 6-day differentiation protocol with RA induces maturation, but not differentiation of the cells into specific neuron phenotype. The greater multipotency of undifferentiated cells in neural markers expression, together with their higher sensitivity to xenobiotic exposure and more simple cultivation protocols, make them a better candidate for high throughput toxicological screenings. Differentiated neurons are better suited for neuroscience researches that require higher expression of more specific neural markers and the specific types of neural cells.

STEM-05. CREATING PATIENT-DERIVED TUMOROIDS TO INVESTIGATE RADIORESISTANCE IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Brain cancers are the most frequently diagnosed tumor types in pediatric and young adult populations. Unfortunately, they often carry a poor prognosis and face common challenges such as treatment resistances due to intra- and intertumoral heterogeneity. To fully understand the intrinsic and extrinsic mechanisms which might hinder proper care, there is an emerging need to develop precise models of brain tumors to understand resistances and to predict therapeutic responses. While tumor-derived primary cell lines are relatively easy to produce, fully replicate the complexity of tumors as they grow without interactions with and from the microenvironment. Therefore, the advent of organoid cultures represents a significant advancement in modelling development. Derived from this technology, tumoroids offer a promising solution for modeling 3D tumors and their complex microenvironment. METHODS We develop tumoroids from fresh biopsies and patient-derived tumor xenografts. These models were rigorously validated against to initial tumor using confocal microscopy, methylome analyses, metabolomics and single-cell RNAseq. To assess the intrinsic hypoxic environment and its spatial heterogeneity, characteristic of aggressive cancers, we employed fluorescent oxygen-sensitive dye loaded polymeric nanorods. RESULTS We successfully generated tumoroids using PDX of ependymomas (BT39) and it paired relapse (BT42), two H3.3 mutated high-grade gliomas (BT35 and BT69), one ganglioglioma (BT63) and directly from fresh biopsies of DIPG sample (BT140). Several passages were obtained after primary derivation, and cryopreservation demonstrating the stability of the models over time. Tumoroids displayed similar protein and omics markers as their parental tumors and harbored specific stem cell and neural progenitor markers. The methylation patterns were entirely preserved comparing 3D models and their parental tumors, and other multiomics and metabolic approaches confirmed the accurate reconstitution of tumor complexity. The study of oxygen-sensitive nanorods highlighted the heterogeneous oxygen gradient within tumoroids. CONCLUSIONS All these initial characterized tumoroids helped us to develop radioresistant paired models.

Odontoblasts or odontocytes, expression of stem cells markers and differentiation markers among human adult odontoblasts

ObjectiveDespite that, the odontoblasts of the dental pulp are considered a terminally differentiated type of cell. We were interested in investigating if they express any embryonic, mesenchymal, or neural stem cell markers, along with other differentiation markers they were reported to express previously. Methods: An immunohistochemistry study was performed on wisdom teeth extracted from healthy donors aged between 17 and 19 for dental reasons. Nine markers were tested: c-Myc, SOX2, MCAM, CD73, NCAM1, STRO1, osteocalcin, S100, and Thy1. Results: Odontoblasts expressed the following markers: embryonic stem cell markers SOX2, c-Myc, mesenchymal stem cell marker MCAM, the neural differentiation marker S100, and the osteogenic differentiation marker osteocalcin. Odontoblasts did not express the following markers: mesenchymal stem cell markers CD73, STRO1, Thy1, and neural stem cell marker NCAM1. Conclusion: These findings suggest that odontoblasts’ expression of these stem cell markers may enable them to dedifferentiate under certain conditions. Further investigation is needed into whether dental materials could induce such dedifferentiation for functional dentin regeneration.

Characterizing neuroinflammation and identifying prenatal diagnostic markers for neural tube defects through integrated multi-omics analysis

BackgroundNeural Tube Defects (NTDs) are congenital malformations of the central nervous system resulting from the incomplete closure of the neural tube during early embryonic development. Neuroinflammation refers to the inflammatory response in the nervous system, typically resulting from damage to neural tissue. Immune-related processes have been identified in NTDs, however, the detailed relationship and underlying mechanisms between neuroinflammation and NTDs remain largely unclear. In this study, we utilized integrated multi-omics analysis to explore the role of neuroinflammation in NTDs and identify potential prenatal diagnostic markers using a murine model.MethodsNine public datasets from Gene Expression Omnibus (GEO) and ArrayExpress were mined using integrated multi-omics analysis to characterize the molecular landscape associated with neuroinflammation in NTDs. Special attention was given to the involvement of macrophages in neuroinflammation within amniotic fluid, as well as the dynamics of macrophage polarization and their interactions with neural cells at single-cell resolution. We also used qPCR assay to validate the key TFs and candidate prenatal diagnostic genes identified through the integrated analysis in a retinoic acid-induced NTDs mouse model.ResultsOur analysis indicated that neuroinflammation is a critical pathological feature of NTDs, regulated both transcriptionally and epigenetically within central nervous system tissues. Key alterations in gene expression and pathways highlighted the crucial role of STATs molecules in the JAK-STAT signaling pathway in regulating NTDs-associated neuroinflammation. Furthermore, single-cell resolution analysis revealed significant polarization of macrophages and their interaction with neural cells in amniotic fluid, underscoring their central role in mediating neuroinflammation associated with NTDs. Finally, we identified a set of six potential prenatal diagnostic genes, including FABP7, CRMP1, SCG3, SLC16A10, RNASE6 and RNASE1, which were subsequently validated in a murine NTDs model, indicating their promise as prospective markers for prenatal diagnosis of NTDs.ConclusionsOur study emphasizes the pivotal role of neuroinflammation in the progression of NTDs and underlines the potential of specific inflammatory and neural markers as novel prenatal diagnostic tools. These findings provide important clues for further understanding the underlying mechanisms between neuroinflammation and NTDs, and offer valuable insights for the future development of prenatal diagnostics.

Comparative evaluation of divergent concoction of NGF, BDNF, EGF, and FGF growth factor's role in enhancing neuronal differentiation of adipose-derived mesenchymal stem cells

MSCs (Mesenchymal Stem Cells) can differentiate into various lineages, including neurons and glial cells. In the past few decades, MSCs have been well explored in the context of neuronal differentiation and have been reported to have the immense potential to form distinct kinds of neurons. The distinguishing features of MSCs make them among the most desired cell sources for stem cell therapy. This study involved the trans-differentiation of Adipose-derived human Mesenchymal Stem Cells (ADMSCs) into neurons. The protocol employs a cocktail of chemical inducers in different combinations, including Brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), and Nerve growth factor (NGF) Fibroblastic growth factor (FGF), in induction media. Both types have been successfully differentiated into neurons, confirmed by morphological aspects and the presence of neural-specific markers through RT-PCR (Reverse transcription polymerase chain reaction) studies and immunocytochemistry assay. They have shown excellent morphology with long neurites, synaptic connections, and essential neural markers to validate their identity. The results may significantly contribute to cell replacement therapy for neurological disorders.

Differential Expression of MicroRNA (MiR-27, MiR-145) among Dental Pulp Stem Cells (DPSCs) Following Neurogenic Differentiation Stimuli.

This study sought to evaluate the expression of previously identified microRNAs known to regulate neuronal differentiation in mesenchymal stem cells (MSCs), including miR-27, miR-125, miR-128, miR-135, miR-140, miR-145, miR-218 and miR-410, among dental pulp stem cells (DPSCs) under conditions demonstrated to induce neuronal differentiation. Using an approved protocol, n = 12 DPSCs were identified from an existing biorepository and treated with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), which were previously demonstrated to induce neural differentiation markers including Sox1, Pax6 and NFM among these DPSCs. This study revealed that some microRNAs involved in the neuronal differentiation of MSCs were also differentially expressed among the DPSCs, including miR-27 and miR-145. In addition, this study also revealed that administration of bFGF and EGF was sufficient to modulate miR-27 and miR-145 expression in all of the stimulus-responsive DPSCs but not among all of the non-responsive DPSCs-suggesting that further investigation of the downstream targets of these microRNAs may be needed to fully evaluate and understand these observations.

A near-infrared-activated heterostructure endowed nerve scaffold with electrical stimulation and antibacterial performance

The weak tissue inducibility and easy implantation infection are still challenges confronted to tissue scaffold. Bismuth sulfide (Bi2S3) possessed favorable photoelectric, photothermal, and photodynamic properties not only enabling to response near-infrared light to generate electrical signals to promote nerve growth but also simultaneously produces reactive oxygen species (ROS) and heat to kill bacteria, which emerges as a promising alternative. However, the easy recombination of electron-hole weakens its photocurrent and ROS generation. Herein, Bi2S3/Ag3PO4 heterostructures are prepared by in situ growing Ag3PO4 on Bi2S3 and then mixed with poly-L-lactic acid powder to fabricate scaffolds by selective laser sintering. Due to the different Fermi levels of Bi2S3 and Ag3PO4, the photogenerated electrons of Bi2S3 transferred to the conduction band of Ag3PO4. Meanwhile, the heterojunction impeded the backflow of electrons, which efficiently achieved electron-hole pair separation. Results indicate that the photocurrent and ROS generated by the scaffold was enhanced. The improved photocurrent effectively induces stem cells to differentiate into nerve cells through up-regulating Ca2+ concentration and neural-specific markers Nestin expression. The produced ROS, photothermal, and Ag+ can synergistically kill bacteria. Ultimately, the scaffold exhibited excellent antimicrobial efficiency with 90.59 % and 91.45 % against E. coli and S. aureus, respectively. This strategy provides a new perspective to realize the integrated preparation of nerve scaffolds with electrical stimulation and antibacterial performance.

Bioactive superparamagnetic iron oxide-gold nanoparticles regulated by a dynamic magnetic field induce neuronal Ca2+ influx and differentiation

Treating neurodegenerative diseases, e.g., Alzheimer's Disease, remains a significant challenge due to the limited neuroregeneration rate in the brain. The objective of this study is to evaluate the hypothesis that external magnetic field (MF) stimulation of nerve growth factor functionalized superparamagnetic iron oxide-gold (NGF-SPIO-Au) nanoparticles (NPs) can induce Ca2+ influx, membrane depolarization, and enhance neuron differentiation with dynamic MF (DMF) outperforming static MF (SMF) regulation. We showed the that total intracellular Ca2+ influx of PC-12cells was improved by 300% and 535% by the stimulation of DMF (1 Hz, 0.5 T, 30min) with NGF-SPIO-Au NPs compared to DMF alone and SMF with NGF-SPIO-Au NPs, respectively, which was attributed to successive membrane depolarization. Cellular uptake performed with the application of sodium azide proved that DMF enhanced cellular uptake of NGF-SPIO-Au NPs via endocytosis. In addition, DMF upregulated both the neural differentiation marker (β3-tubulin) and the cell adhesive molecule (integrin-β1) with the existence of NGF-SPIO-Au NPs, while SMF did not show these effects. The results imply that noninvasive DMF-stimulated NPs can regulate intracellular Ca2+ influx and enhance neuron differentiation and neuroregeneration rate.

Rapid neurogenic differentiation of human mesenchymal stem cells through electrochemical stimulation

The neurogenic differentiation of human mesenchymal stem cells (hMSCs) has been substantially handicapped with the choice of chemical or electrical stimulations for long durations. We demonstrate an innovative strategy of stimulation with <1.0 V for <200 s to achieve hMSCs differentiation towards neural progenitor cells within 24 h and their commitment towards differentiation to neurons on day 3 with the use of three-electrode electrostimulation. Stimulated hMSCs (ES hMSCs) showed elevated expression of neural-specific markers and mitochondrial membrane potential. A voltage bias of ±0.5 V and ±1.0 V did not show any adverse effect on cell viability and proliferation, whereas cells stimulated with ±1.5 V showed an upsurge in the dead cell populations. With the progression of time after stimulation, a rise in mitochondrial membrane potential (MMP, ΔΨ M) was observed in the ES hMSCs and thereby generating intracellular reactive oxygen species (ROS), acting as a key messenger to induce neuronal differentiation. The stratagem may provide insightful handles to circumvent neurodifferentiation impediments, a focal issue for regenerative medicine.

Hypergravity enhances RBM4 expression in human bone marrow-derived mesenchymal stem cells and accelerates their differentiation into neurons.

The regulation of stem cell differentiation is important in determining the quality of transplanted cells in regenerative medicine. Physical stimuli are involved in regulating stem cell differentiation, and in particular, research on the regulation of differentiation using gravity is an attractive choice. We have shown that microgravity is useful for maintaining undifferentiated mesenchymal stem cells (MSCs). However, the effects of hypergravity on the differentiation of MSCs, especially on neural differentiation related to neural regeneration, have not been elucidated. We induced neural differentiation of human bone marrow-derived MSCs (hbMSCs) for 10 days under normal gravity (1G) or hypergravity (3G) conditions using a gravity controller, Gravite®. HbMSCs were collected, and cell number and viability were measured 3 and 10 days after induction. RNA was also extracted from the collected hbMSCs, and the expression of neuron-associated genes and regulator markers of neural differentiation was analyzed using real-time polymerase chain reaction (PCR). Additionally, we evaluated the NF-M-positive cell rate 10 days after induction using immunofluorescent staining. Neural gene expression and the NF-M-positive cell rate were increased in hbMSCs under the 3G condition 10 days after induction. mRNA expression of RNA binding motif protein 4 (RBM4) and pyruvate kinase M 1 (PKM1) in the 3G condition was also higher than that in the 1G group. Hypergravity can enhance RBM4 and PKM1, promoting the neural differentiation of hbMSCs.

Administration of Epidermal Growth Factor (EGF) and Basic Fibroblast Growth Factor (bFGF) to Induce Neural Differentiation of Dental Pulp Stem Cells (DPSC) Isolates

The aging populations in many countries have developed many chronic illnesses and diseases, including chronic neurologic conditions such as Parkinson's and Azheimer's diseases. Many new lines of research and treatment are focusing on the potential for neurologic regeneration using mesenchymal stem cells (MSCs) in the rapidly growing field of regenerative medicine. This may include dental pulp stem cells (DPSCs), which have recently been demonstrated to produce neuronal precursors. Based upon this evidence, the primary aim of this study was to determine if the growth factors used in MSC-based studies are sufficient to induce neuronal differentiation among DPSCs. Using an existing biorepository, n = 16 DPSC isolates were thawed and cultured for this study, which revealed several subpopulations of rapid-, intermediate-, and slowly dividing DPSCs. Administration of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were sufficient to induce differential changes in growth and viability mainly among some of the rapidly growing DPSCs (n = 4). These phenotypic changes included expression of neural differentiation markers including Sox1, Pax6 and NF-M, which were observed only among those DPSC isolates not expressing early odontoblast-specific biomarkers such as ALP and DSPP. Future studies will be needed to confirm if these methods are sufficient to induce consistent and reliable induction of DPSCs towards neuronal specific differentiation.

The Generation of Human iPSC Lines from Three Individuals with Dravet Syndrome and Characterization of Neural Differentiation Markers in iPSC-Derived Ventral Forebrain Organoid Model.

Dravet syndrome (DRVT) is a rare form of neurodevelopmental disorder with a high risk of sudden unexpected death in epilepsy (SUDEP), caused mainly (&gt;80% cases) by mutations in the SCN1A gene, coding the Nav1.1 protein (alfa-subunit of voltage-sensitive sodium channel). Mutations in SCN1A are linked to heterogenous epileptic phenotypes of various types, severity, and patient prognosis. Here we generated iPSC lines from fibroblasts obtained from three individuals affected with DRVT carrying distinct mutations in the SCN1A gene (nonsense mutation p.Ser1516*, missense mutation p.Arg1596His, and splicing mutation c.2589+2dupT). The iPSC lines, generated with the non-integrative approach, retained the distinct SCN1A gene mutation of the donor fibroblasts and were characterized by confirming the expression of the pluripotency markers, the three-germ layer differentiation potential, the absence of exogenous vector expression, and a normal karyotype. The generated iPSC lines were used to establish ventral forebrain organoids, the most affected type of neurons in the pathology of DRVT. The DRVT organoid model will provide an additional resource for deciphering the pathology behind Nav1.1 haploinsufficiency and drug screening to remediate the functional deficits associated with the disease.

INNV-19. THE DETECTION OF DISSEMINATED PINEAL PARENCHYMAL TUMOR OF INTERMEDIATE DIFFERENTIATION (PPTID) IN CEREBROSPINAL FLUID (CSF) USING CELL CAPTURE AND IMMUNOCYTOCHEMISTRY

Abstract BACKGROUND Pineal parenchymal tumors of intermediate differentiation (PPTID) account for 21% to 54% of pineal parenchymal tumors and may be complicated by cerebrospinal dissemination (most commonly at time of reoccurrence; up to 4-10 years post resection). The integral membrane protein synaptophysin is expressed in neuroendocrine cells and virtually all neuraxial neurons that contribute to synaptic transmission. PPTID frequently demonstrates synaptophysin independently of other neural differentiation markers. Cerebrospinal fluid (CSF) cell capture assays have demonstrated utility for assessing disseminated intracranial neoplasms, thus we explore this technology for monitoring a patient with PPTID. METHODS A 30-year-old female patient with PPTID diagnosed two years prior was suspected for intracranial dissemination and underwent five CSF collections over the course of 64 days. Per collection, approximately 7 mL of CSF was submitted for each cytology and cell capture. Cytology was performed at Providence Saint John’s Health Center. The CNSide™ platform (Biocept, Inc.) was used to capture and stain cells for DAPI, synaptophysin, and CD45. Multiple capture antibody cocktails (termed 1986, 1822, and gTP1—originally developed for use in carcinoma, melanoma, and glioma) were tested. Cells of interest (COI) were defined by positive immunoreactivity for both DAPI and synaptophysin and no immunoreactivity for CD45. Results were quantified and compared longitudinally. RESULTS CSF analysis on days 0, 9, 21, 36, and 64 demonstrated negative results by cytology and 51, 96, 170, 130, and 43 COI/mL by the CNSide platform, respectively. Cumulative mean capture rates for cocktails 1986, 1822, and gTP1 were 47, 14, and 36 COI/mL/sample, respectively. DISCUSSION A significant amount of PPTID patients will develop cerebrospinal dissemination. The importance of craniospinal control in this setting has been previously demonstrated. Our work suggests that cell capture assays paired with immunocytochemistry can be used as sensitive means to monitor disseminated PPTID and may impact treatment decisions.

Preservation of the Hypoxic Transcriptome in Glioblastoma Patient-Derived Cell Lines Maintained at Lowered Oxygen Tension.

Simple SummaryThe extent of tumour oxygenation is crucial for glioblastoma progression and the effectiveness of radio- and chemotherapy. Patient-derived in vitro cell cultures are the mainstay of molecular biology research, the discovery of new therapeutic targets, and drug testing. Therefore, mirroring as many aspects of in vivo settings as possible, including oxygen concentration, is desired. Here we analyse the effect of oxygen tension on the transcriptome of numerous patient-derived GBM cells and demonstrate that cells cultured in lowered oxygen tension express more genes indicative of higher levels of hypoxia, metabolic adaptation, stemness and tumour progression than cells growing in standard, atmospheric oxygen concentration. The same transcriptomic pattern was also found in primary GBM samples. Its specificity for GBMs was confirmed using the public TCGA dataset. Our data strongly argue for the benefit of lower oxygen tension during culturing of patient-derived GBM cells to preserve oxygen-sensitive pathways in GBM. The proposed approach better mimics certain aspects of GBM pathophysiology than traditional cultures and may advance GBM research in finding a cure.Despite numerous efforts aiming to characterise glioblastoma pathology (GBM) and discover new therapeutic strategies, GBM remains one of the most challenging tumours to treat. Here we propose the optimisation of in vitro culturing of GBM patient-derived cells, namely the establishment of GBM-derived cultures and their maintenance at oxygen tension mimicking oxygenation conditions occurring within the tumour. To globally analyse cell states, we performed the transcriptome analysis of GBM patient-derived cells kept as spheroids in serum-free conditions at the reduced oxygen tension (5% O2), cells cultured at atmospheric oxygen (20% O2), and parental tumour. Immune cells present in the tumour were depleted, resulting in the decreased expression of the immune system and inflammation-related genes. The expression of genes promoting cell proliferation and DNA repair was higher in GBM cell cultures when compared to the relevant tumour sample. However, lowering oxygen tension to 5% did not affect the proliferation rate and expression of cell cycle and DNA repair genes in GBM cell cultures. Culturing GBM cells at 5% oxygen was sufficient to increase the expression of specific stemness markers, particularly the PROM1 gene, without affecting neural cell differentiation markers. GBM spheroids cultured at 5% oxygen expressed higher levels of hypoxia-inducible genes, including those encoding glycolytic enzymes and pro-angiogenic factors. The genes up-regulated in cells cultured at 5% oxygen had higher expression in parental GBMs compared to that observed in 20% cell cultures, suggesting the preservation of the hypoxic component of GBM transcriptome at 5% oxygen and its loss in standard culture conditions. Evaluation of expression of those genes in The Cancer Genome Atlas dataset comprising samples of normal brain tissue, lower-grade gliomas and GBMs indicated the expression pattern of the indicated genes was specific for GBM. Moreover, GBM cells cultured at 5% oxygen were more resistant to temozolomide, the chemotherapeutic used in GBM therapy. The presented comparison of GBM cultures maintained at high and low oxygen tension together with analysis of tumour transcriptome indicates that lowering oxygen tension during cell culture may more allegedly reproduce tumour cell behaviour within GBM than standard culture conditions (e.g., atmospheric oxygen tension). Low oxygen culture conditions should be considered as a more appropriate model for further studies on glioblastoma pathology and therapy.

A shared numerical magnitude representation evidenced by the distance effect in frequency-tagging EEG

Humans can effortlessly abstract numerical information from various codes and contexts. However, whether the access to the underlying magnitude information relies on common or distinct brain representations remains highly debated. Here, we recorded electrophysiological responses to periodic variation of numerosity (every five items) occurring in rapid streams of numbers presented at 6 Hz in randomly varying codes—Arabic digits, number words, canonical dot patterns and finger configurations. Results demonstrated that numerical information was abstracted and generalized over the different representation codes by revealing clear discrimination responses (at 1.2 Hz) of the deviant numerosity from the base numerosity, recorded over parieto-occipital electrodes. Crucially, and supporting the claim that discrimination responses reflected magnitude processing, the presentation of a deviant numerosity distant from the base (e.g., base “2” and deviant “8”) elicited larger right-hemispheric responses than the presentation of a close deviant numerosity (e.g., base “2” and deviant “3”). This finding nicely represents the neural signature of the distance effect, an interpretation further reinforced by the clear correlation with individuals’ behavioral performance in an independent numerical comparison task. Our results therefore provide for the first time unambiguously a reliable and specific neural marker of a magnitude representation that is shared among several numerical codes.

Valproate Targets Mammalian Gastrulation Impairing Neural Tissue Differentiation and Development of the Placental Source In Vitro.

The teratogenic activity of valproate (VPA), an antiepileptic and an inhibitor of histone deacetylase (HDACi), is dose-dependent in humans. Previous results showed that VPA impairs in vitro development and neural differentiation of the gastrulating embryo proper. We aimed to investigate the impact of a lower VPA dose in vitro and whether this effect is retained in transplants in vivo. Rat embryos proper (E9.5) and ectoplacental cones were separately cultivated at the air-liquid interface with or without 1 mM VPA. Embryos were additionally cultivated with HDACi Trichostatin A (TSA), while some cultures were syngeneically transplanted under the kidney capsule for 14 days. Embryos were subjected to routine histology, immunohistochemistry, Western blotting and pyrosequencing. The overall growth of VPA-treated embryos in vitro was significantly impaired. However, no differences in the apoptosis or proliferation index were found. Incidence of the neural tissue was lower in VPA-treated embryos than in controls. TSA also impaired growth and neural differentiation in vitro. VPA-treated embryos and their subsequent transplants expressed a marker of undifferentiated neural cells compared to controls where neural differentiation markers were expressed. VPA increased the acetylation of histones. Our results point to gastrulation as a sensitive period for neurodevelopmental impairment caused by VPA.

Augmented Expression of NOGO-A and Its Receptors in Human Retinal Pigment Epithelial Cells Following Treatment with Human Amniotic Fluid.

Background:Nogo-A, a myelin-associated inhibitor for neurite outgrowth, has important role in visual system development. Trans-differentiation ability of human amniotic fluid (HAF) on human retinal pigment epithelial cells (hRPEs) towards neural progenitor cells has been observed in several studies. We aimed to investigate the expression of NOGO-A gene and its receptors as a marker of neural differentiation in HAF-treated hRPE cells.Methods:hRPE cells were cultivated and immune characterized via RPE65 and cytokeratin 8/18 protein markers. Also, the cytotoxicity effect of 30% HAF on hRPE cells was evaluated using ELISA cell death assay. Finally, expression of NOGO-A and its receptors, RTN4R and LINGO1 was evaluated in the cells treated with HAF in comparison with FBS-treated cells using quantitative real-time PCR.Results:Harvested cells showed immunoreactivity for cytokeratin 8/18 and RPE65, confirming the hRPE cell identity. Besides, HAF had no cytotoxic effect on hRPE cells compared with FBS-treated cells. Results showed that NOGO-A and its receptors were expressed in cultured hRPE cells. Besides, comparative gene expression analysis revealed significant increased expression of the investigated genes in HAF-treated hRPE cells compared to FBS-treated cells.Conclusion:Augmented expression of NOGO-A and its receptors can support neural differentiation of hRPE when the cells are treated with HAF. Our outcomes provide more evidences on the trans-differentiation ability of HAF on hRPE cells into neural progenitors and retinal neural cells, but further studies are needed to elucidate the exact mechanism.

Neural Differentiation of Human Dental Mesenchymal Stem Cells Induced by ATRA and UDP-4: A Comparative Study.

Human mesenchymal stem cells (MSC) are multipotent stem cells, which are isolated from various sources. Currently, there is a worldwide interest for dental MSC to be used against neurodegenerative diseases, since they derive from the neural crest and express embryonic stem cell markers. This fact prompted us to explore their potential for neural trans-differentiation in culture. We employed all-trans-retinoic acid (ATRA) and 2-(3-ethylureido)-6-methylpyridine (UDP-4) to induce neural differentiation of human MSC from the dental apical papilla (SCAP). The SCAP were exposed to either agent separately and assessed for proliferation, viability, morphology, and gene expression of the following neural-specific markers: neuron-specific enolase (ENO2), neurofibromin 1 (NF1), choline acetyltransferase (CHAT), tyrosine hydroxylase (TH), and the vesicular GABA transporter (SLC32A1). They were also assessed for the expression of glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) by immunofluorescence. ATRA or UDP-4 treatment inhibited the cell growth and promoted limited cell death, but to a different extent. The addition of the neuroprotective agent recombinant human erythropoietin-alpha (rhEPO-α) enhanced the UDP-4-inducing capacity for more than three weeks. ATRA or UDP-4 treatment significantly upregulated ENO2 and NF1 expression, indicating neuronal differentiation. Moreover, the ATRA treatment significantly induced the upregulation of the GABAergic-specific SLC32A1, while the UDP-4 treatment led to the significant upregulation of the adrenergic-specific TH. The UDP-4 treatment induced the expression of NeuN and GFAP after four and three weeks, respectively, while the ATRA-treatment did not. Our findings indicate that SCAP can be differentiated into neural-like cells after treatment with ATRA or UDP-4 by exhibiting a disparate pattern of differentiation. Therefore, UDP-4 is suggested here as a new potent neural-differentiation-inducing compound, which, when combined with rhEPO-α, could lay the foundation for robust stem-cell-based therapies of neurodegeneration.

Primary desmoplastic small round cell tumor of the submandibular gland: a case report and literature review

BackgroundDesmoplastic small round cell tumor (DSRCT) is a sporadic, highly malignant tumor with a poor prognosis. The abdomen and pelvis have been reported as the primary localization sites. However, to the best of our knowledge, there are few reports on primary DSRCT in the submandibular gland.Case presentationWe report a case of a 26-year-old Chinese man with a mass in the right submandibular gland. Imaging studies showed a hypoechoic mass in the right submandibular region. Intraoperative pathology revealed that the tumor tissue was composed of small round tumor cells and a dense desmoplastic stroma. On immunostaining, the tumor cells showed markers of epithelial, mesenchymal, myogenic, and neural differentiation. The EWSR1 gene rearrangement was detected by fluorescence in situ hybridization. Based on the overall morphological features and immunohistochemical findings, a final diagnosis of DSRCT was made. The patient was treated with comprehensive anti-tumor therapy mainly based on radiotherapy and chemotherapy.ConclusionsDSRCT is an uncommon malignant neoplasm with rare submandibular gland involvement. In this report, we have described a case of DSRCT in the submandibular gland and reviewed the literature on DSRCT over the past 5 years. Considering the importance of differential diagnosis between DSRCT, especially with rare extra-peritoneal involvement, and small round blue cell tumors, a full recognition of the clinicopathological features will help to better diagnose this neoplasm.