Neuroglial Differentiation Research Articles

TMET-02. CLINICAL EFFICACY OF ONC201 IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS IS DRIVEN BY DISRUPTION OF INTEGRATED METABOLIC AND EPIGENETIC PATHWAYS

Abstract H3K27M-mutant diffuse midline glioma (DMG) patients have no proven effective therapies beyond radiation. ONC201, a mitochondrial protease ClpP agonist, has recently demonstrated efficacy in these patients, but the mechanism behind this remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue samples from patients treated in two completed multi-site clinical studies (n=71). Patients treated with ONC201 monotherapy following initial radiation but prior to recurrence (n=35) demonstrated a median overall survival of 21.7 months and a median progression-free survival of 12.2 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. Similar survival benefits were observed using in utero electroporation and orthotopic mouse models of H3K27M-DMG (median 107 versus 77 days [p=0.02] and median 97 versus 141 days [p=0.004], respectively). Integrated transcriptomic and metabolomic analysis of ONC201-treated H3K27M-DMG cells revealed downregulation of TCA cycle, glycolysis, and pyruvate metabolism genes, most notably oxoglutarate dehydrogenase (OGDH), and concomitant changes in levels of associated metabolites including ɑ-ketoglutarate (ɑ-KG). Interestingly, L-2-hydroxyglutarate (L-2HG), a known inhibitor of the Jumonji C domain family of histone lysine demethylases, was also increased. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuro-glial differentiation genes. Genetic knockdown of OGDH recapitulated ONC201-mediated increases in H3K27me3. In contrast, genetic knockdown of ClpP or lactate dehydrogenase A or overexpression of L-2-hydroxyglutarate dehydrogenase (L-2HGDH), but not D-2HGDH, abrogated ONC201-mediated increases in H3K27me3, demonstrating that ONC201 induces the production of L-2HG in H3K27M-DMG cells leading to increased H3K27me3. Overall, our data demonstrates the efficacy of ONC201 in H3K27M-mutant DMG and supports ONC201 as the first monotherapy to improve outcomes in patients with H3K27M-mutant DMG for whom few therapeutic options currently exist. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction.

An Example of Neuro-Glial Commitment and Differentiation of Muse Stem Cells Obtained from Patients with IQSEC2-Related Neural Disorder: A Possible New Cell-Based Disease Model.

Although adult stem cells may be useful for studying tissue-specific diseases, they cannot be used as a general model for investigating human illnesses given their limited differentiation potential. Multilineage-differentiating stress-enduring (Muse) stem cells, a SSEA3(+) cell population isolated from mesenchymal stromal cells, fat, and skin fibroblasts, may be able to overcome that restriction. The Muse cells present in fibroblast cultures obtained from biopsies of patients' skin may be differentiated into cells of interest for analyzing diseases. We isolated Muse stem cells from patients with an intellectual disability (ID) and mutations in the IQSEC2 gene (i.e., BRAG1 gene) and induced in vitro neuroglial differentiation to study cell commitment and the differentiation of neural lineages. The neuroglial differentiation of Muse cells revealed that IQSEC2 mutations may alter the self-renewal and lineage specification of stem cells. We observed a decrease in the percentage of SOX2 (+) neural stem cells and neural progenitors (i.e., SOX2+ and NESTIN+) in cultures obtained from Muse cells with the mutated IQSEC2 gene. The alteration in the number of stem cells and progenitors produced a bias toward the astrocytes' differentiation. Our research demonstrates that Muse stem cells may represent a new cell-based disease model.

Transplantation of Induced Pluripotent Stem Cells (iPSCs) into Cuprizone Model of Demyelination in Rat

Introduction: Multiple Sclerosis (MS) is a neurodegenerative inflammatory disease with a wide range of axonal demyelination and sensory-motor disorders. Today, stem cell therapy is an appropriate choice for MS management. Induced Pluripotent Stem Cells (iPSC) are pluripotent stem cells that have neuroprotective effects with neuroglial differentiation potential. In this study, we used iPSCs to improve remyelination and behavioral function in model of cuprizone multiple sclerosis disease in rats. Method: Human iPSCs were cultured and extended on a mouse embryonic fibroblast feeder cell layer inactivated with mitomycin-C in DMEM/F12 supplemented with 20% knockout serum replacement, 0.1 mmol/L nonessential amino acids, and 10 ng/mL of recombinant human basic fibroblast growth factor in a 5% CO2 and 95% humidity. The demyelination model was induced using a 0.2 oral Cuprizone regime. Thereafter, iPSCs were transplanted after six weeks. Remyelination was investigated via histological assessments and immunocytochemistry following six-week post-transplantation. The functionality was evaluated using behavioral tests, BBB, and Footprint following six-week post-transplantation. Results: The results of in vivo studies showed that after specific myelin tissue staining, the cuprizone diet led to the induction of demyelinated areas in the brain tissue six weeks after the cuprizone diet. Differentiation of transplanted cells was confirmed via the immunocytochemistry technique of PLP. The results of the Footprint test showed that the motor function of the paws of the animals improved compared to the control group. Moreover, BBB behavioral tests showed improved symptoms in the experimental groups that received the cells. Conclusion: In total, iPSCs can improve remyelination and functional recovery following transplantation into the Cuprizone demyelination model in rats. Therefore, iPSC therapy could improve behavioral function in multiple sclerosis disease

Isolation, Expansion, and Endoscopic Delivery of Autologous Enteric Neuronal Stem Cells in Swine

The enteric nervous system (ENS) is an extensive network of neurons and glia within the wall of the gastrointestinal (GI) tract that regulates many essential GI functions. Consequently, disorders of the ENS due to developmental defects, inflammation, infection, or age-associated neurodegeneration lead to serious neurointestinal diseases. Despite the prevalence and severity of these diseases, effective treatments are lacking as they fail to directly address the underlying pathology. Neuronal stem cell therapy represents a promising approach to treating diseases of the ENS by replacing the absent or injured neurons, and an autologous source of stem cells would be optimal by obviating the need for immunosuppression. We utilized the swine model to address key questions concerning cell isolation, delivery, engraftment, and fate in a large animal relevant to human therapy. We successfully isolated neural stem cells from a segment of small intestine resected from 1-month-old swine. Enteric neuronal stem cells (ENSCs) were expanded as neurospheres that grew optimally in low-oxygen (5%) culture conditions. Enteric neuronal stem cells were labeled by lentiviral green fluorescent protein (GFP) transduction, then transplanted into the same swine from which they had been harvested. Endoscopic ultrasound was then utilized to deliver the ENSCs (10,000–30,000 neurospheres per animal) into the rectal wall. At 10 and 28 days following injection, autologously derived ENSCs were found to have engrafted within rectal wall, with neuroglial differentiation and no evidence of ectopic spreading. These findings strongly support the feasibility of autologous cell isolation and delivery using a clinically useful and minimally invasive technique, bringing us closer to first-in-human ENSC therapy for neurointestinal diseases.

Transient cerebral ischemia induces the neuroglial proliferative activity and the potential to redirect neuroglial differentiation

Brain injury triggers a complex response involving morphological changes, cellular proliferation, and differentiation of newly formed neuroglial subpopulations. These processes have been extensively studied in animal stroke models with permanent large vessel occlusion. However, less is known about neuroglial response after transient cerebral ischemia. Herein, we aimed to determine an astrocytic and NG2 glial proliferative response, potential changes in expression of developmental neuroglial markers: vimentin, nestin, oligodendrocyte transcription marker (Olig2), and a role of neuroglial subpopulations as a source of cells replenishing structural deficiencies in the ischemic brain. Results showed an induction of a proliferative neuroglial response in the peri-infarct area reflected in an increased percentage of GFAP/Ki67 + and NG2/Ki67 + cells within 4 weeks after transient MCAO. The peak of GFAP+ astrocytes proliferation of 30.3 ± 10.3% was observed in the first week, and a peak of NG2 + cells proliferation of 23.1 ± 11.8% in the second week after stroke. The presence of GFAP/Vimentin+ and GFAP/Nestin+ cells, as well as GFAP/Olig2 + and NG2/Olig2 + cells indicated an induction of developmental phenotypes with a differentiation potential. Finally, observed between day 1 and week 3 transient GFAP/NG2 + colocalization suggests the heterogeneous source of the reactive neuroglia after transient MCAO. Altogether, one-hour MCAO is a sufficient pathological stimulus to trigger a strong proliferative response of GFAP+ and NG2 + neuroglial cells and induce their early developmental phenotype. Our results suggest that transient ischemia may initiate a change in the direction of differentiation within the neuroglia cell population.

An Alzheimer's Disease Patient-Derived Olfactory Stem Cell Model Identifies Gene Expression Changes Associated with Cognition.

An early symptom of Alzheimer’s disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore changes of early AD-associated pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

Germ cell tumors with neuroglial differentiation do not show molecular features akin to their central nervous system counterpart: experience from extra-gynecological sites.

Teratomas with secondary somatic malignancy showing neuroglial differentiation (central nervous system (CNS)-type tumors) arising from a glial or neuroepithelial component is a very uncommon event and primarily described in the ovary. We aimed to describe the morphological spectrum and molecular features of CNS type of neuroepithelial tumors arising from the germ cell tumors (GCT) in the extra-gynecological sites. All cases of teratoma and mixed GCT arising from the non-gynecological sites over 7years were screened for CNS type of neuroepithelial tumors. Detailed histological and immunohistochemical analysis was performed. IDH1 and 2 sequencings were performed in the glial tumors. Fluorescent in situ hybridization (FISH) was performed for EWSR1 rearrangement, 19/19q co-deletion, CDKN2A homozygous deletion, EGFR amplification, and C19MC amplification, wherever required. Out of 302 GCTs examined, the neuroglial tumor was detected in 15 cases. It included nine cases of glial tumors (including one pilocytic astrocytoma (grade I), two diffuse astrocytomas (grade II), one oligodendroglioma (grade II), one gemistocytic astrocytoma (grade II), three anaplastic astrocytomas (grade III), and one case of glioblastoma (grade IV)) and six cases of the embryonal tumor with multilayered rosettes (ETMR). None of the gliomas showed IDH mutation by immunohistochemistry or sequencing. The ETMR cases did not show Lin28 expression or C19MC amplification. To conclude, the spectrum of neuroglial tumors arising from teratoma in the extragonadal sites is vast and most commonly includes glial neoplasms and embryonal tumors. Our findings indicate that the genotype and pathogenesis of tumors with neuroglial differentiation in teratoma are distinct from their CNS counterpart.

Neural Crest-Derived Stem Cells (NCSCs) Obtained from Dental-Related Stem Cells (DRSCs): A Literature Review on Current Knowledge and Directions toward Translational Applications.

Evidence from dental-related stem cells (DRSCs) suggests an enhanced potential for ectodermal lineage differentiation due to their neural crest origin. Growing evidence that DRSC cultures can produce cells with a neural crest-derived stem cell (NCSC)-like phenotype supports their potential for future therapeutic approaches for neurodegenerative diseases and nerve injuries. However, most of the evidence is limited to the characterization of DRSCs as NCSCs by detecting the expression of neural crest markers. Only a few studies have provided proof of concept of an improved neuro-glial differentiation or direct applicability in relevant models. In addition, a current problem is that several of the existing protocols do not meet manufacturing standards for transferability to a clinical scenario. This review describes the current protocols to obtain NCSCs from DRSCs and their characterization. Also, it provides important considerations from previous work where DRSCs were established and characterized as mesenchymal stromal cells but studied for their neuro-glial differentiation potential. The therapeutic advancement of DRSCs would depend on establishing protocols that can yield a neural crest-like phenotype efficiently, using appropriate manufacturing standards and testing them in relevant models of disease or injury. Achieving these conditions could then facilitate and validate the therapeutic potential of DRSC-NCSCs in regenerative therapies.

Signaling pathways in the control of embryonic development of the enteric nervous system

The enteric nervous system (ENS) provides intrinsic innervation of the gastrointestinal tract and is the largest and most complex part of the peripheral nervous system. Its functions are vital for life and include control of motility of the digestive tract, secretion, as well as fluid and electrolyte exchange through the intestinal mucosa. ENS is capable of performing most of these functions completely autonomously. A large number of developmental and genetic studies of the most common congenital disease of the ENS, Hischsprung' s disease, has made a major contribution to the understanding of the embryonic development of the ENS. ENS cells raise from the vagal (mostly) and sacral region of the neural crest. These precursor cells migrate along the primitive gut in opposite directions, in order to colonize the entire gut. Proliferation, migration, neuro-glial differentiation, and other processes through which precursor cells of the ENS undergo, are regulated by various signaling pathways. Some of the most important molecules that participate in the regulation of the proper development of the ENS are GDNF (Glial Derived Neurotrophic Fatcor) and its receptor RET (REarranged during Transfection), endothelin 3 and its receptor EDNRB (endothelin receptor type B), transcription factors SOX10 (SRY-box transcription factor 10), PHOX2B (Paired-like Homeobox 2B), morphogens such as BMP 2 and 4 (Bone Morphogenic Proteins) and others. Although our knowledge about control of the development of the ENS has increased significantly in recent years, complexity of structure and function of the ENS requires further research. This review summarizes our current understanding of the most important regulatory mechanisms and signaling pathways involved in the development of the ENS.

Poreklo ćelija enteričkog nervnog sistema i putevi migracije tokom embrionalnog razvoja

The enteric nervous system (ENS) is represented by a complex network of neurons, glial and other cells within the wall of the digestive tract. ENS is responsible for numerous, vital functions in our body. Thus, ENS regulates motility of the digestive tract, secretion into the intestinal lumen, exchange of fluid and electrolytes through the mucosa, as well as mucosal perfusion. In order to perform these important functions, proper embryonic development of ENS is necessary. ENS cells are derived from precursor cells of the neural crest (NCCs - neural crest cells). Two cell populations that contribute to the largest number of future ENS cells are the vagal and sacral NCCs. Vagal NCCs enter the primitive gut tube in the region of the future esophagus (foregut), and begin their migration, through the midgut towards the hindgut and the future anal region. Sacral NCCs enter the hindgut region following the extrinsic nerve fibers and continue their migration rostrally, towards vagal NCCs. Along with the migration process, these cells undergo other important processes, such as proliferation, neuro-glial differentiation, gangliogenesis, axonal pathway formation and synaptogenesis. All these processes are strictly regulated by numerous signaling pathways, which are still being actively researched. Modern lineage tracing and other technologies, that enabled following of individual precursor cells through their development pathways, will significantly contribute to the better understanding of development of ENS. This may have repercussions in improving the diagnosis and treatment of some developmental (Hirschsprung disease) and other ENS disorders.

Spidroin Silk Fibers with Bioactive Motifs of Extracellular Proteins for Neural Tissue Engineering.

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation.

Generation and Characteristics of Glial Cells from Induced Human Pluripotent Stem Cells

Abstract—The technology for producing human induced pluripotent stem cells (iPSCs) and the possibility of directed differentiation into specialized cells of all body tissues have opened unique opportunities for studying the molecular genetic basis of the pathogenesis of neurodegenerative diseases in vitro and effective screening for compounds with neuroprotective activity. The aim of this work was to obtain glial cell cultures from iPSCs of a healthy donor and a patient with the familial form of Parkinson’s disease (G2019S mutation in the LRRK2) and to characterize them. At the first stage, we compared the three previously described protocols for the differentiation of glial cells from neural precursors of human iPSCs and selected the method most acceptable under our conditions in terms of the quality and time necessary for obtaining the desired cultures. Glial cell cultures obtained by this method were characterized by the levels of expression of a number of neuroglial differentiation genes. Also, in the resulting cultures, we analyzed the expression of genes of some neurotrophic factors (GDNF, BDNF, NGF, NT3). It was shown that the culture medium conditioned by glial cells from a patient with Parkinson’s disease had a negative effect on the growth of neurites of dopaminergic neurons in differentiated cultures of a healthy donor, decreasing their length by a factor of 2.

MicroRNA‑29a promotes the neural differentiation of rat neural stem/progenitor cells by targeting KLF4

Neural stem/progenitor cells (NSPCs) remain in the mammalian brain throughout life, where they have the ability to self-renew and generate different types of cell in the central nervous system (CNS). Therefore, NSPCs may be a potential novel therapeutic strategy following damage to the CNS. Previous research has reported that microRNA (miR)-29a served an important role in regulating cell proliferation, differentiation and survival; however, to the best of our knowledge, little is known of the effect of miR-29a in neural differentiation. The present study aimed to investigate the effect of miR-29a on the differentiation of NSPCs, determined via RNA interference, immunostaining, reverse transcription-quantitative PCR and western blotting. The present study discovered that the expression levels of miR-29a were significantly upregulated in a time-dependent manner during neural differentiation. Immunostaining showed that overexpression of miR-29a promoted neural differentiation, which manifested in increased expression levels of neuron-specific class III β-tubulin (Tuj1); however, miR-29a had no effect on neuroglial differentiation. The expression levels of Kruppel-like factor 4 (KLF4) were downregulated following overexpression of miR-29a, whereas the inhibition of miR-29a demonstrated the opposite effect. These results suggested that the overexpression of miR-29a may promote neural differentiation in cultured rat NSPCs by decreasing the expression levels of KLF4. Thus indicating that targeting KLF4, a crucial regulatory factor for the maintenance of stemness, may be a potential underlying mechanism of action for miR-29a. In conclusion, the findings of the present study identified a potential mechanism of action for miR-29a in NSPC differentiation and provided a novel insight into the treatment strategies for CNS damage.

Human skin-derived precursor cells xenografted in aganglionic bowel

PurposeOne in 5000 newborns is diagnosed with Hirschsprung disease each year in the United States. The potential of employing neural crest stem cells to restore the enteric nervous system has been investigated. Skin-derived precursor cells (SKPs) are multipotent progenitor cells that can differentiate into neurons and gliocytes in vitro and generate enteric ganglion-like structures in rodents. Here we examined the behavior of human SKPs (hSKPs) after their transplantation into a large animal model of colonic aganglionosis. MethodsJuvenile minipigs underwent a chemical denervation of the colon to establish an aganglionosis model. The hSKPs were generated from human foreskin and were cultured in neuroglial-selective medium. Cells were labeled with a fluorescent dye and were injected into the porcine aganglionic colon. After one week, transplanted hSKPs were assessed by immunofluorescence for markers of multipotency and neuroglial differentiation. ResultsIn culture, hSKPs expressed nestin and S100b indicative of neuroglial precursors. After xenografting in pigs, hSKPs were identified in the myenteric and submucosal plexuses of the colons. The hSKPs expressed nestin and early neuroglial differentiation markers. ConclusionsHuman SKPs transplanted into aganglionic colon demonstrated immunophenotypes of neuroglial progenitors, suggesting their potential use for Hirschsprung disease.Level of evidence

Low concentrations of morphine enhanced the neuroglia-like differentiation

The present study was undertaken to evaluate the effects of different concentrations of morphine in chronic manner on neuroglial differentiation in PC12 cells. PC12 cells were cultured in RPMI 1640 culture medium including 0.02 % bovine serum albumin together with different concentrations of morphine for 12 days. Cytotoxicity was performed by lactate dehydrogenase assay. Cell death was performed by PI/Hoechst staining assay. Neuroglial differentiation was performed by Nestin, Tuj-1, MAP-2, S-100 and GFAP Immunocytochemistry assay. Data showed that morphine either at low or high concentration activated opioid receptors, which resulted in a decrease of cytotoxicity and cell death and induction of Nestin, Tuj-1, MAP-2, Neurofilament-M (NF-M), GFAP and S-100 protein expression as compared in treated cells with the control (untreated cells) (p<005). It can be concluded that low concentrations of morphine in chronic manner stimulate the neuroglial-like differentiation by activating protein expression and survival-promoting signaling in PC12 cells with opioid receptor-dependent mechanism (Fig. 8, Ref. 36). Text in PDF www.elis.sk.

Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model

BackgroundHirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. MethodsJuvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. ResultsPrior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. ConclusionsAutologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of StudyBasic science.

Neuroglial Differentiation and Neoplasms in Testicular Germ Cell Tumors Lack Immunohistochemical Evidence of Alterations Characteristic of Their CNS Counterparts: A Study of 13 Cases.

Overgrowth of neuroglial tissue is rare in testicular germ cell tumors and mostly reported as isolated cases. We retrospectively reviewed 13 cases of testicular germ cell tumors from 2 institutions from 1995 to 2018. Hematoxylin and eosin slides were collected and reviewed. Immunohistochemistry was performed in all cases with available material. The series included 4 primary tumors and 9 metastases, including 8 retroperitoneal and 1 axillary lymph node (LN). The average age was 34 (range: 19 to 54). Five of the LN dissections were postchemotherapy, with one a recurrence 5 years after the initial diagnosis. The average tumor size for primary tumors was 5.15 cm (range: 1.7 to 7.3) and for metastases was 6.4 cm (range: 0.6 to 15). The largest size of the neuroglial component was 4.5 cm in the primary tumors and 7.5 cm in metastatic sites. The neuroglial component in the primary site was associated with pure teratoma (n=2) and with a mixed germ cell tumor (teratoma, seminoma, and embryonal carcinoma) (n=2). Cases involving LNs were associated with teratoma (n=4), seminoma (n=2), rhabdomyosarcoma (n=2), primitive neuroectodermal tumors (n=1), and high-grade sarcoma (n=1) (some with >1 other component). Two cases were pure glial tumor. Histologically, the neuroglial components included low-grade astrocytoma (n=3) (both with microcysts formation and pilocytic features), gemistocytic astrocytomas (n=3), anaplastic astrocytoma (n=2), ganglioglioma (n=1), glioblastoma (n=2), gliosarcoma (n=1), and developing central nervous system (CNS) (n=2). By immunohistochemistry, 13/13 (100%) cases were GFAP(+), 10/10 (100%) cases showed retained ATRX, 10/10 were IDH1 pR132H (-), 5/10 (50%) were p53 (+). A single case 1/10 (10%) was BRAF p.V600E (+), but a mutation was not identified by polymerase chain reaction. Follow-up was available in 6 patients; 4 were confirmed to have received chemotherapy with BEP; 1 had a local recurrence and the patient with gliosarcoma developed a lung metastasis morphologically similar to the gliosarcoma of the retroperitoneum. In conclusion, neuroglial differentiation and neoplasms are rare in testicular germ cell tumors and are most commonly associated with teratomas; they can be seen in primary and metastatic sites. They exhibit the full range of neuroglial differentiation including developing CNS to gliomas/glioneuronal tumors WHO grades I-IV. None of the cases showed results consistent with ATRX, IDH or BRAF alterations, suggesting they have different oncogenic mechanisms than their CNS counterparts.

Effects of norfloxacin exposure on neurodevelopment of zebrafish (Danio rerio) embryos

In view of the wide application of fluoroquinolones (FQs), a group of broad-spectrum synthetic antibacterial agents, and their large ingress into the environment, the toxic effects on non-target organisms caused by FQs have received great attention. In this study, we used zebrafish embryo as a model, measured the general toxic effects of norfloxacin, a commonly used FQs, and investigated the effects of norfloxacin on the neurodevelopment of zebrafish embryos. Our data showed that norfloxacin significantly inhibited the hatching rate of zebrafish embryos, and increased the mortality and malformation rate of the embryos. To discuss the developmental neurotoxicity of norfloxacin, we measured the expression of several stem cell and neuron lineage markers in the zebrafish embryos. We found that norfloxacin exposure inhibited the expression of GFAP (glial cell marker), and enhanced the expression of Sox 2 (stem cell marker) and Eno2 (mature neuron marker). By measuring the level of active Caspase 3 and the expression ratio of Bax to Bcl2, we discovered that norfloxacin induced obvious cell apoptosis in the brain of zebrafish embryos. To explore the mechanism of the developmental neurotoxic effects of norfloxacin, we applied MK-801, a non-competitive NMDA receptors antagonist, to block the actions of NMDA receptors. The results indicated that MK-801 could rescue the upregulated cell apoptosis and disrupted balance of neuro-glial differentiation induced by norfloxacin in the brain of zebrafish embryos. Our results suggest that the activation of NMDA receptors mediates the developmental neurotoxicity of norfloxacin.

Multiple effects of the herbicide glufosinate-ammonium and its main metabolite on neural stem cells from the subventricular zone of newborn mice

The globally used herbicide glufosinate-ammonium (GLA) is structurally analogous to the excitatory neurotransmitter glutamate, and is known to interfere with cellular mechanisms involved in the glutamatergic system. In this report, we used an in vitro model of murine primary neural stem cell culture to investigate the neurotoxicity of GLA and its main metabolite, 4-methylphosphinico-2-oxobutanoic acid (PPO). We demonstrated that GLA and PPO disturb ependymal wall integrity in the ventricular-subventricular zone (V-SVZ) and alter the neuro-glial differentiation of neural stem cells. GLA and PPO impaired the formation of cilia, with reduced Celsr2 expression after PPO exposure. GLA promoted the differentiation of neuronal and oligodendroglial cells while PPO increased B1 cell population and impaired neuronal fate of neural stem cells. These results confirm our previous in vivo report that developmental exposure to GLA alters neurogenesis in the SVZ, and neuroblast migration along the rostral migratory stream. They also highlight the importance of investigating the toxicity of pesticide degradation products. Indeed, not only GLA, but also its metabolite PPO disrupts V-SVZ homeostasis and provides a novel cellular mechanism underlying GLA-induced neurodevelopmental toxicity. Furthermore, we were able to demonstrate a neurotoxic activity of a metabolite of GLA different from that of GLA active substance for the very first time.

Enteric Neural Stem Cells Expressing Insulin-Like Growth Factor 1: A Novel Cellular Therapy for Hirschsprung's Disease in Mouse Model.

Transplanting enteric neural stem cells (ENSCs) is an innovative approach for replacing enteric neurons in Hirschsprung's disease (HSCR). However, posttransplantation cell survival and differentiation limit efficacy. We aimed to investigate whether transplantation of ENSCs engineered with insulin-like growth factor 1 (IGF-1) could improve survival and differentiation of the engrafted cells and promote functional recovery of the aganglionic colon. ENSCs were isolated from the intestine of neonatal mice and genetically modified to express IGF-1. After implantation into the mice aganglionic colon induced by benzalkonium chloride, survival and differentiation of engrafted cells were assessed by immunohistochemistry for GFP, neuronal, and glial cell markers. Colonic motility was quantified by colonic bead expulsion time and response to electrical field stimulation (EFS). Expression of the neural marker nNOS and the glial cell marker (glial fibrillary acidic protein [GFAP]) was increased in IGF-1-ENSCs. IGF-1 had also improved neuroglial differentiation of ENSCs following transplantation in vivo. Colonic motility was significantly improved after IGF-1-ENSC transplantation, as demonstrated by reduction of colonic bead expulsion time and recovery of EFS-induced relaxation. IGF-1 has enhanced neurogenesis and function of ENSCs upon transplantation for enteric nervous system replacement, which may provide a potential novel therapy for the treatment of HSCR.