IGF2 interacts with the imprinted gene Cdkn1c to promote terminal differentiation of neural stem cells.

Adult neurogenesis is supported by multipotent neural stem cells (NSCs) with unique properties and growth requirements. Adult NSCs constitute a reversibly quiescent cell population that can be activated by extracellular signals from the microenvironment in which they reside in vivo. Although genomic imprinting plays a role in adult neurogenesis through dose regulation of some relevant signals, the roles of many imprinted genes in the process remain elusive. Insulin-like growth factor 2 (IGF2) is encoded by an imprinted gene that contributes to NSC maintenance in the adult subventricular zone through a biallelic expression in only the vascular compartment. We show here that IGF2 additionally promotes terminal differentiation of NSCs into astrocytes, neurons and oligodendrocytes by inducing the expression of the maternally expressed gene cyclin-dependent kinase inhibitor 1c (Cdkn1c), encoding the cell cycle inhibitor p57. Using intraventricular infusion of recombinant IGF2 in a conditional mutant strain with Cdkn1c-deficient NSCs, we confirm that p57 partially mediates the differentiation effects of IGF2 in NSCs and that this occurs independently of its role in cell-cycle progression, balancing the relationship between astrogliogenesis, neurogenesis and oligodendrogenesis.

Proneurogenic actions of follicle-stimulating hormone on neurospheres derived from ovarian cortical cells in vitro.

BACKGROUND: Neural stem and progenitor cells (NSPCs) from extra-neural origin represent a valuable tool for autologous cell therapy and research in neurogenesis. Identification of proneurogenic biomolecules on NSPCs would improve the success of cell therapies for neurodegenerative diseases. Preliminary data suggested that follicle-stimulating hormone (FSH) might act in this fashion. This study was aimed to elucidate whether FSH promotes development, self-renewal, and is proneurogenic on neurospheres (NS) derived from sheep ovarian cortical cells (OCCs). Two culture strategies were carried out: (a) long-term, 21-days NS culture (control vs. FSH group) with NS morphometric evaluation, gene expression analyses of stemness and lineage markers, and immunolocalization of NSPCs antigens; (b) NS assay to demonstrate FSH actions on self-renewal and differentiation capacity of NS cultured with one of three defined media: M1: positive control with EGF/FGF2; M2: control; and M3: M2 supplemented with FSH. RESULTS: In long-term cultures, FSH increased NS diameters with respect to control group (302.90 ± 25.20 µm vs. 183.20 ± 7.63 on day 9, respectively), upregulated nestin (days 15/21), Sox2 (day 21) and Pax6 (days 15/21) and increased the percentages of cells immunolocalizing these proteins. During NS assays, FSH stimulated NSCPs proliferation, and self-renewal, increasing NS diameters during the two expansion periods and the expression of the neuron precursor transcript DCX during the second one. In the FSH-group there were more frequent cell-bridges among neighbouring NS. CONCLUSIONS: FSH is a proneurogenic hormone that promotes OCC-NSPCs self-renewal and NS development. Future studies will be necessary to support the proneurogenic actions of FSH and its potential use in basic and applied research related to cell therapy.

Tuberin levels during cellular differentiation in brain development.

Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex (TSC), and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, permits the formation of tumors that can lead to developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that total Tuberin levels are tightly regulated through developmental stages in the embryonic brain. At a cell biology level, we show that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. This functional data supports the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies.

Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro.

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.

CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.

BACKGROUND: Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed. METHODS AND RESULTS: Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1α or BDNF. CONCLUSION: Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1α and BDNF. Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.

The Neurosphere Simulator: An educational online tool for modeling neural stem cell behavior and tissue growth.

Until very recently, distance education, including digital science labs, served a rather small portion of postsecondary students in the United States and many other countries. This situation has, however, dramatically changed in 2020 in the wake of the COVID-19 pandemic, which forced colleges to rapidly transit from face-to-face instructions to online classes. Here, we report the development of an interactive simulator that is freely available on the web (http://neurosphere.cos.northeastern.edu/) for teaching lab classes in developmental biology. This simulator is based on cellular automata models of neural-stem-cell-driven tissue growth in the neurosphere assay. By modifying model parameters, users can explore the role in tissue growth of several developmental mechanisms, such as regulation of mitosis or apoptotic cell death by contact inhibition. Besides providing an instantaneous animation of the simulated development of neurospheres, the Neurosphere Simulator tool offers also the possibility to download data for detailed analysis. The simulator function is complemented by a tutorial that introduces students to computational modeling of developmental processes.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Copper Modulates Adult Neurogenesis in Brain Subventricular Zone.

The subventricular zone (SVZ) in lateral ventricles is the largest neurogenic region in adult brain containing high amounts of copper (Cu). This study aims to define the role of Cu in adult neurogenesis by chelating labile Cu ions using a well-established Cu chelator D-Penicillamine (D-Pen). A neurosphere model derived from adult mouse SVZ tissues was established and characterized for its functionality with regards to neural stem/progenitor cells (NSPCs). Applying D-Pen in cultured neurospheres significantly reduced intracellular Cu levels and reversed the Cu-induced suppression of NSPC's differentiation and migration. An in vivo intracerebroventricular (ICV) infusion model was subsequently established to infuse D-Pen directly into the lateral ventricle. Metal analyses revealed a selective reduction of Cu in SVZ by 13.1% (p = 0.19) and 21.4% (p < 0.05) following D-Pen infusions at low (0.075 µg/h) and high (0.75 µg/h) doses for 28 days, respectively, compared to saline-infused controls. Immunohistochemical studies revealed that the 7-day, low-dose D-Pen infusion significantly increased Ki67(+)/Nestin(+) cell counts in SVZ by 28% (p < 0.05). Quantification of BrdU(+)/doublecortin (DCX)(+) newborn neuroblasts in the rostral migration stream (RMS) and olfactory bulb (OB) further revealed that the short-term, low-dose D-Pen infusion, as compared with saline-infused controls, resulted in more newborn neuroblasts in OB, while the high-dose D-Pen infusion showed fewer newborn neuroblasts in OB but with more arrested in the RMS. Long-term (28-day) infusion revealed similar outcomes. The qPCR data from neurosphere experiments revealed altered expressions of mRNAs encoding key proteins known to regulate SVZ adult neurogenesis, including, but not limited to, Shh, Dlx2, and Slit1, in response to the changed Cu level in neurospheres. Further immunohistochemical data indicated that Cu chelation also altered the expression of high-affinity copper uptake protein 1 (CTR1) and metallothionein-3 (MT3) in the SVZ as well as CTR1 in the choroid plexus, a tissue regulating brain Cu homeostasis. Taken together, this study provides first-hand evidence that a high Cu level in SVZ appears likely to maintain the stability of adult neurogenesis in this neurogenic zone.

Endogenous Synthesis of Tetrahydroisoquinoline Derivatives from Dietary Factors: Neurotoxicity Assessment on a 3D Neurosphere Culture.

Tetrahydroisoquinoline (THIQ) alkaloids and their derivatives have a structural similarity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-known neurotoxin. THIQs seem to present a broad range of actions in the brain, critically dependent on their catechol moieties and metabolism. These properties make it reasonable to assume that an acute or chronic exposure to some THIQs might lead to neurodegenerative diseases including essential tremor (ET). We developed a method to search for precursor carbonyl compounds produced during the Maillard reaction in overcooked meats to study their reactivity with endogenous amines and identify the reaction products. Then, we predicted in silico their pharmacokinetic and toxicological properties toward the central nervous system. Finally, their possible neurological effects on a novel in vitro 3D neurosphere model were assessed. The obtained data indicate that meat is an alkaloid precursor, and we identified the alkaloid 1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (1-benz-6,7-diol THIQ) as the condensation product of phenylacetaldehyde with dopamine; in silico study of 1-benz-6,7-diol-THIQ reveals modulation of dopamine receptor D1 and D2; and in vitro study of 1-benz-6,7-diol-THIQ for cytotoxicity and oxidative stress induction does not show any difference after 24 h contact for all tested concentrations. To conclude, our in vitro data do not support an eventual neurotoxic effect for 1-benz-6,7-diol-THIQ.

Heterochromatin protein 1γ deficiency decreases histone H3K27 methylation in mouse neurosphere neuronal genes.

Heterochromatin protein (HP) 1γ, a component of heterochromatin in eukaryotes, is involved in H3K9 methylation. Although HP1γ is expressed strongly in neural tissues and neural stem cells, its functions are unclear. To elucidate the roles of HP1γ, we analyzed HP1γ -deficient (HP1γ KO) mouse embryonic neurospheres and determined that HP1γ KO neurospheres tended to differentiate after quaternary culture. Several genes normally expressed in neuronal cells were upregulated in HP1γ KO undifferentiated neurospheres, but not in the wild type (WT). Compared to that in the control neurospheres, the occupancy of H3K27me3 was lower around the transcription start sites (TSSs) of these genes in HP1γ KO neurospheres, while H3K9me2/3, H3K4me3, and H3K27ac amounts remained unchanged. Moreover, amounts of the H3K27me2/3 demethylases, UTX, and JMJD3, were increased around the TSSs of these genes. Treatment with GSK-J4, an inhibitor of H3K27 demethylases, decreased the expression of genes upregulated in HP1γ KO neurospheres, along with an increase of H3K27me3 amounts. Therefore, in murine neurospheres, HP1γ protected the promoter sites of differentiated cell-specific genes against H3K27 demethylases to repress the expression of these genes. A better understanding of central cellular processes such as histone methylation will help elucidate critical events such as cell-specific gene expression, epigenetics, and differentiation.

Postnatal Changes of Neural Stem Cells in the Mammalian Auditory Cortex.

Our previous study reported neural stem cells (NSCs) in the auditory cortex (AC) of postnatal day 3 (P3) mice in vitro. It is unclear whether AC-NSCs exist in vivo. This study aims to determine the presence and changes of AC-NSCs during postnatal development and maturation both in vitro and in vivo. P3, postnatal day 14 (P14), 2-month-old (2M), and 4-month-old (4M) mouse brain tissues were fixed and cryosectioned for NSC marker immunostaining. In vitro, P3, P14, and 2M AC tissues were dissected and cultured in suspension to study NSCs. NSC proliferation was examined by EdU incorporation and cell doubling time assays in vitro. The results show that Nestin and Sox2 double expressing NSCs were observed in the AC area from P3 to 4M in vivo, in which the number of NSCs remarkably reduced with age. In vitro, the neurosphere forming capability, cell proliferation, and percentage of Nestin and Sox2 double expressing NSCs significantly diminished with age. These results suggest that AC-NSCs exist in the mouse AC area both in vitro and in vivo, and the percentage of AC-NSCs decreases during postnatal development and maturation. The results may provide important cues for the future research of the central auditory system.

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need.

Widespread tumor cell invasion is a fundamental property of diffuse gliomas and is ultimately responsible for their poor prognosis. A greater understanding of basic mechanisms underlying glioma invasion is needed to provide insights into therapies that could potentially counteract them. While none of the currently available in vitro models can fully recapitulate the complex interactions of glioma cells within the brain tumor microenvironment, if chosen and developed appropriately, these models can provide controlled experimental settings to study molecular and cellular phenomena that are challenging or impossible to model in vivo. Therefore, selecting the most appropriate in vitro model, together with its inherent advantages and limitations, for specific hypotheses and experimental questions achieves primary significance. In this review, we describe and discuss commonly used methods for modeling and studying glioma invasion in vitro, including platforms, matrices, cell culture, and visualization techniques, so that choices for experimental approach are informed and optimal.

Epidermal growth factor-immobilized surfaces for the selective expansion of neural progenitor cells derived from induced pluripotent stem cells.

Neural progenitor cells (NPCs) are considered to be a promising source for stem cell-based regenerative therapy for central nervous disorders. However, the widespread clinical application of NPCs requires another technology that permits the efficient production of pure NPCs in large quantities. In this study, culture substrates were designed by immobilizing epidermal growth factor (EGF) onto the substrate and evaluated for their feasibility of expanding NPCs obtained through the neurosphere culture of induced pluripotent stem (iPS) cells. After three passages we obtained neurospheres that contained cells abundantly expressing an EGF receptor. The neurospheres were dissociated into single cells and seeded onto the EGF-immobilized substrates. It was observed that neurosphere-forming cells seeded and cultured on the EGF-immobilized surface formed a two-dimensional cellular network characteristic of NPCs. These cells were found to be capable of being subcultured, while remaining their proliferation potential. Furthermore, a majority of cells (~99% of total cells) on the substrate was shown to express an NPC marker, nestin, whereas a limited number of cells (~1% of total cells) expressed neuronal marker, ß-tubulin III. These results as a whole demonstrate that the EGF-immobilized substrate allows for iPS cell-derived NPCs to efficiently proliferate while maintaining the undifferentiated state.

Stigmasterol promotes neuronal migration via reelin signaling in neurosphere migration assays.

Stigmasterol (ST) is a multifunctional phytosterol and is found in diverse food. In our previous transcriptomics study, we found ST upregulated migration-related genes. In the present study, we carried out in vitro neurosphere migration assays to investigate the effects of ST on neuronal migration. For this purpose, neurospheres were produced by culturing rat (Sprague-Dawley) E14 cortical neurons. The addition of ST (75 µM) to culture medium increased not only the numbers of migratory neurons by 15% but the distance of movement up to 120 µm from the centers of neurospheres as compared to vehicle cultures. Immunocytochemistry and immunoblotting showed ST upregulated the expressions of Reelin (Reln) and its downstream signaling molecules like phospho-JNK (c-Jun N-terminal kinase), doublecortin (DCX) and dynein heavy chain (DHC) in migratory neurons. Furthermore, in silico molecular docking simulation indicated that ST interacts with Relin receptor ApoER2 by forming a hydrogen bond with Lys2467 and other van der Waals interactions. Taken together, our study shows that ST upregulates Reln signaling and promotes neuronal migration and suggests that ST supplementation is considered as a potential means of treating migration-related CNS disorders.

In vitro neurosphere formation correlates with poor survival in glioma.

Sphere formation is an indicator of tumor aggressiveness independent of the tumor grade; however, its relation to progression-free survival (PFS) is less known. This study was designed to assess the neurosphere forming ability among low grade glioma (LGG) and high-grade glioma (HGG), its stem cell marker expression, and correlation to PFS. Tumor samples of 140 patients, including (LGG; n = 67) and (HGG; n = 73) were analyzed. We used sphere forming assay, immunofluorescence, and immunohistochemistry to characterize the tumors. Our study shows that, irrespective of the pathological sub type, both LGG and HGG formed neurospheres in vitro under conventional sphere forming conditions. However, the number of neurospheres formed from tumor tissues were significantly higher in HGG compared to LGG (P < 0.0001). Different grades of glioma were further characterized for the expression of stem cell marker proteins and lineage markers. When neurospheres were analyzed, CD133 positive cells were identified in addition to CD15 and nestin positive cells in both LGG and HGG. When these neurospheres were subjected to differentiation, cells positive for GFAP and ß-tubulin III were observed. Expression of stem cell markers and ß-tubulin III were prominent in HGG compared to LGG, whereas GFAP expression was higher in LGG than in HGG. Kaplan-Meier survival analysis demonstrated that neurosphere forming ability was significantly associated with shorter PFS (P < 0.05) in both LGG and HGG. Our results supports earlier studies that neurosphere formation may serve as a definitive indicator of stem cell population within the tumor and thus a better predictor of PFS than the tumor grades alone. © 2018 IUBMB Life, 71(1):244-253, 2019.

Micropillar-based microfluidic device to regulate neurite networks of uniform-sized neurospheres.

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar-based microfluidic device to trap the uniform-sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar-based microfluidic device could be a potential tool for screening of neurotoxins.

Stochastic cellular automata model of tumorous neurosphere growth: Roles of developmental maturity and cell death.

The neurosphere assay is a powerful in vitro system for studying stem/progenitor-cell-driven tissue growth. By employing a stochastic cellular automata model, we simulated the development of tumorous neurospheres in response to transformation of a randomly selected progenitor cell into a brain tumor stem cell. Simulated tumorous neurospheres were distinguished from normal neurospheres by their size, which exceeded that of normal neurospheres typically manifold. A decisive factor that determined whether brain tumor stem cells gave rise to tumorous neurospheres was their ability to escape encapsulation by neighboring cells, which suppressed mitotic activity through contact inhibition. In our simulations, the likelihood of tumorigenesis was strongly negatively correlated with the developmental maturity of the neurospheres in which the transformation of a progenitor cell into a brain tumor stem cell was induced. This likelihood was furthermore modulated by the probability of the progeny of dividing cells to undergo cell death. In developmentally immature neurospheres, the number of normal neurospheres, relative to the number of tumorous neurospheres, increased with increasing cell death probability. Markedly, in developmentally mature neurospheres the opposite effect was observed. This dichotomous effect of cell death on simulated tumor progression provides theoretical support for the seemingly paradoxical finding made by other authors in experimental studies that anti-cancer therapies based on induction of apoptosis may both promote and suppress tumor growth.

Pharmacological Notch pathway inhibition leads to cell cycle arrest and stimulates ascl1 and neurogenin2 genes expression in dental pulp stem cells-derived neurospheres.

OBJECTIVE: Human dental pulp-derived stem cells (hDPSCs) are becoming an attractive source for cell-based neurorestorative therapies. As such, it is important to understand the molecular mechanisms that regulate the differentiation of hDPSCs toward the neuronal fate. Notch signaling plays key roles in neural stem/progenitor cells (NS/PCs) maintenance and prevention of their differentiation. The aim of this study was to address the effects of Notch signaling inhibition on neurosphere formation of hDPSCs and neuronal differentiation of hDPSCs-neurospheres. RESULTS: hDPSCs were isolated from third molar teeth. The cultivated hDPSCs highly expressed CD90 and CD44 and minimally presented CD34 and CD45 surface markers. The osteo/adipogenic differentiation of hDPSCs was documented. hDPSCs were cultured in neural induction medium and N-[N-(3,5-difluorophenacetyl-L-alanyl)]-Sphenylglycine t-butyl ester (DAPT) was applied to impede Notch signaling during transformation into spheres or on the formed neurospheres. Our results showed that the size and number of neurospheres decreased and the expression profile of nestin, sox1 and pax6 genes reduced provided DAPT. Treatment of the formed neurospheres with DAPT resulted in the cleaved Notch1 reduction, G0/G1 arrest and a decline in L-lactate production. DAPT significantly reduced hes1 and hey1 genes, while ascl1 and neurogenin2 expressions augmented. The number of MAP2 positive cells improved in the DAPT-treated group. CONCLUSIONS: Our findings demonstrated the Notch activity in hDPSCs-neurospheres. DAPT treatment positively regulated proneural genes expression and increased neuronal-like differentiation.

[Isolation of neurospheres and neural progenitor cells from the olfactory epithelium]. / Poluchenie neirosfer i neironal'nykh progenitornykh kletok iz oboniatel'nogo épiteliia.

The olfactory epithelium (OE) is an accessible source of neural stem cells and progenitor cells. The objective of the study was to compare the effectiveness of various biopsy sites to isolate and propagate neural progenitor cells from the olfactory epithelium (OE). The authors assessed OE cell count in OE in different sites of the nasal cavity and showed the possibility of isolation neurospheres from nasal biopsies. In total, 45 inpatinets were included in the study. Biopsy specimens were obtained from 30 patients undergoing septoplasty and/or turbinate surgery. Three areas of OE were biopsied: lower third section of the nasal septum (A), anterior part of the middle turbinate (B), upper third of the nasal septum (C). Immunocytochemistry and fluorescence-activated cell sorting showed that OE cells were NCAM-positive. Mean percentage of NCAM+ cells was 7.8% for A, 42.7% for B and 18.2% for C. The difference was significant between A and B (p=0.0001) and B and C (p=0.01). Therefore, the anterior part of the middle turbinate was an easily accessible and safe site to obtain neural cells. To confirm this, neurospheres were obtained in 15 patients with schizophrenia who underwent in-office endoscopy.

Involvement of aquaporin-4 in laminin-enhanced process formation of mouse astrocytes in 2D culture: Roles of dystroglycan and α-syntrophin in aquaporin-4 expression.

In the central nervous system, astrocytes extend endfoot processes to ensheath synapses and microvessels. However, the mechanisms underlying this astrocytic process extension remain unclear. A limitation of the use of 2D cultured astrocytes for such studies is that they display a flat, epithelioid morphology, with no or very few processes, which is markedly different from the stellate morphology observed in vivo. In this study, we obtained 2D cultured astrocytes with a rich complexity of processes using differentiation of neurospheres in vitro. Using these process-bearing astrocytes, we showed that laminin, an extracellular matrix molecule abundant in perivascular sites, efficiently induced process formation and branching. Specifically, the numbers of the first- and second-order branch processes and the maximal process length of astrocytes were increased when cultured on laminin, compared with when they were cultured on poly-L-ornithine or type IV collagen. Knockdown of dystroglycan or α-syntrophin, constituent proteins of the dystrophin-glycoprotein complex that provides a link between laminin and the cytoskeleton, using small interference RNAs inhibited astrocyte process formation and branching, and down-regulated expression of the water channel aquaporin-4 (AQP4). Direct knockdown and a specific inhibitor of AQP4 also inhibited, whereas over-expression of AQP4 enhanced astrocyte process formation and branching. Knockdown of AQP4 decreased phosphorylation of focal adhesion kinase (FAK) that is critically implicated in actin remodeling. Collectively, these results indicate that the laminin-dystroglycan-α-syntrophin-AQP4 axis is important for process formation and branching of 2D cultured astrocytes. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page 436.