Differentiation Of Neural Stem/precursor Cells Research Articles

Tpr Misregulation in Hippocampal Neural Stem Cells in Mouse Models of Alzheimer's Disease.

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-β plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis.

Chronic exposure to aflatoxin B1 increases hippocampal microglial pyroptosis and vulnerability to stress in mice

BackgroundChronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear. MethodsMice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice. ResultsMice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1β) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU+, BrdU-DCX+, and BrdU-NeuN+ cells in the hippocampal dentate gyrus, as well as the percentages of BrdU+ cells that were NeuN+ in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX+ cells correlated negatively with the percentage of ameboid microglia, TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro. ConclusionChronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.

Selective Regulation of Neurons, Glial Cells, and Neural Stem/Precursor Cells by Poly(allylguanidine)-Coated Surfaces.

Poly(allylguanidine) (PAG) was synthesized and characterized as a polycationic coating material for culturing neurons, glial cells, and neural stem/precursor cells (NSPCs) to apply PAG for neural tissue engineering. For comparison, poly-d-lysine (PDL), the golden benchmark of the neuron cell culture system, was also used in this study. When PAG was subjected to a mixed culture of neurons and glial cells, cell adhesion and neurite extension of neuronal cells were clearly observed but only few glial cells could be found alongside the neurons. Compared to PDL, the significantly lower density of the glial fibrillary acidic protein-positive cells implied that PAG suppressed the glial cell development. Likewise, PAG was demonstrated to dominate the differentiation of NSPCs principally into neurons. To investigate whether the different effects of PAG and PDL on neuron and glial cell behaviors resulted from the difference of guanidinium cations and ammonium cations, poly-l-arginine (PLA) was included and compared in this study. Similar to PDL, PLA supported high neuron and glial cell viability simultaneously. Consequently, glial cell growth and viability restrained on PAG was not only affected by the side-chain guanidino groups but also by the backbone structure property. The absence of the peptide structure in the backbone of PAG and the conformation of coated PAG on tissue culture polystyrene possibly determined the polycationic biomaterial to limit the growth of glial cells.

A self-assembled layer-by-layer surface modification to fabricate the neuron-rich model from neural stem/precursor cells

Invitro neural cell-based models have been widely used to mimic the invivo neural tissue environments and quantitatively understand the effects of pharmaceutical molecules on neural diseases. Recently, several biomimetic neural tissue models have been widely developed by using biomaterials or surface modification. However, the complex protocols of material synthesis or surface modification lack an easy execution to fabricate the neuron favorite environment. In this study, we utilized a layer-by-layer technique as a surface modification method for regulating the behaviors of neural stem/precursor cells (NSPCs) on material surfaces. Polyelectrolyte multilayers (PEMs) via alternate deposition of poly (allylamine hydrochloride) (PAH) and poly (sodium-4-styrenesulfonate) (PSS) were used to culture NSPCs. After incubation for 7 days, the neuronal differentiation of NSPCs and synapse function of differentiated neurons were identified by immunocytochemistry for lineage specific markers. Compared with the only PAH film, the PSS-ending film (neuron-rich model) was shown to significantly promote differentiation of NSPCs into neurons (more than 50%), form a neuronal network structure; and differentiated neurons exhibiting functional synaptic activity. This study shows that the PEMs provided an easily alternative approach to modify the surface properties; and might be a method to obtain a neuron-rich model for the biological/pharmaceutical applications.

MiR-21 functions oppositely in proliferation and differentiation of neural stem/precursor cells via regulating AKT and GSK-3β

MicroRNA involves in regulating behavior of neural stem/precursor cells (NSPCs), thus it offers the potential to treat central nervous system disease. However, the effect of miR-21 on NSPCs remains unknown. In this study, we demonstrated that miR-21 reduced proliferation and promoted neural differentiation in NSPCs via regulating the activation of AKT and GSK-3β signaling pathways in vitro. During differentiation of NSPCs, the expression of miR-21 was increased in a time-dependent manner by qRT-PCR. Synthesized pre-miR-21 or anti-miR-21 was transfected into NSPCs, thereby efficiently overexpressing or knocking down miR-21. Overexpression of miR-21 promoted the neural differentiation of NSPCs, as indicated by Tuj1 and PSA-NCAM staining. Interestingly, knocking down miR-21 had the opposite effect of neural differentiation in NSPCs. However, in proliferation area, overexpression of miR-21 decreased the cell viability by 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTT) assay, and inhibited the proliferation of NSPCs, as indicated by 5-Bromo-2-deoxyUridine (BrdU) staining. And likewise, knocking down miR-21 had the opposite effect of cell viability and proliferation. Western blot showed that overexpression of miR-21 enhanced the expression of Cyclin D1, however, knocking down miR-21 prevented its expression. Furthermore, we revealed that protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK-3β) signaling pathways were involved in the proliferation and neural differentiation of NSPCs. Overexpression of miR-21 activated AKT, and the p-GSK-3β was increased. Conversely, knocking down miR-21 blocked the activation of AKT, and decreased the phosphorylation level of GSK-3β. These results demonstrated that miR-21 promotes neural differentiation and reduces proliferation in NSPCs via regulating AKT and GSK-3β pathways. These findings may help to develop strategies for treatment of central nervous system diseases.

MiR-17-92 facilitates neuronal differentiation of transplanted neural stem/precursor cells under neuroinflammatory conditions.

BackgroundNeural stem/precursor cells (NSCs) are of particular interest because of their potential application in cell therapy for brain damage. However, most brain injury cases are followed with neuroinflammatory stress, which affects the lineage selection of grafted NSCs by promoting astrocytogenesis, thus hampering the potential for neural replacement. The present study investigated the role of miR-17-92 in protecting against detrimental effects of neuroinflammation on NSC differentiation in cell therapy.MethodsNSCs were treated with conditioned medium from lesioned astrocytes with/without neutralizing antibodies of leukemia inhibitory factor (LIF) or/and ciliary neurotrophic factor (CNTF), respectively. Afterward, the levels of p-STAT3 and p-JAK2 were determined by western blotting while expression of glial fibrillary acidic protein (GFAP) and β-tubulin III was assessed by immunostaining. The activation of JAK-STAT pathway and cell differentiation were also evaluated after we overexpressed miR-17-92 in NSCs under different neuroinflammatory conditions. After the transplantation of miR-17-92-overexpressing NSCs into injured mouse cortex, PH3, nestin, GFAP, and NeuN were analyzed by immunostaining. In addition, motor coordination of mice was evaluated by rotarod test.ResultsConditioned medium from lesioned astrocytes activated JAK-STAT pathway and facilitated astrocytic differentiation in NSCs while neutralizing antibodies of LIF and CNTF remarkably attenuated such effects. miR-17-92 cluster repressed the expression of multiple proteins including GP130, CNTFR, JAK2, and STAT3 in JAK-STAT pathway. Overexpression of miR-17-92 in NSCs systematically blocked the activation of JAK-STAT pathway mediated by LIF and CNTF, which facilitated neuronal differentiation in vitro. Furthermore, miR-17-92 increased neuronal generation of grafted NSCs and reduced astrogliosis, which resulted in the improvement of motor coordination of brain-injured mice.ConclusionsOur results suggest that miR-17-92 promotes neuronal differentiation of grafted NSCs under neuroinflammatory condition via inhibition of multiple proteins in JAK-STAT pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0685-5) contains supplementary material, which is available to authorized users.

ID(ealizing) control of adult subventricular zone neural stem/precursor cell differentiation for CNS regeneration.

The adult central nervous system (CNS) was considered a comparatively static tissue with little cell turnover. It is now well established that there is more plasticity than previously thought and that astrocytes act as neural stem/precursor cells (NSPCs) in the subventricular zone (SVZ). The discovery that these NSPCs can give rise to a limited number of new neurons, reactive astrocytes and oligodendrocytes contributing to brain repair in CNS disease, has raised hopes toward harnessing these cells for therapeutic interventions. Here, we will discuss the transcriptional control of adult NSPC differentiation into astrocytes in CNS disease focusing on the helix-loop-helix transcription factor protein family. In our recent study, we reported that elevated BMP-2 levels are translated into an increase in Id3 expression in adult NSPC subpopulations after cortical injury. Id3 then heterodimerizes with the basic helix-loop-helix transcription factor E47 and releases the E47-mediated repression of astrocyte-specific gene expression. Consequently, adult NSPCs preferentially differentiate into astrocytes. We believe that understanding the in vivo differentiation potential and the molecular underpinnings of NSPCs in the adult mammalian brain will help us to evaluate their contributions to brain repair and may lead to new concepts in treating human CNS diseases.

The balance of Id3 and E47 determines neural stem/precursor cell differentiation into astrocytes.

Adult neural stem/precursor cells (NSPCs) of the subventricular zone (SVZ) are an endogenous source for neuronal replacement in CNS disease. However, adult neurogenesis is compromised after brain injury in favor of a glial cell fate, which is mainly attributed to changes in the NSPC environment. Yet, it is unknown how this unfavorable extracellular environment translates into a transcriptional program altering NSPC differentiation. Here, we show that genetic depletion of the transcriptional regulator Id3 decreased the number of astrocytes generated from SVZ-derived adult NSPCs in the cortical lesion area after traumatic brain injury. Cortical brain injury resulted in rapid BMP-2 and Id3 up-regulation in the SVZ stem cell niche. Id3(-/-) adult NSPCs failed to differentiate into BMP-2-induced astrocytes, while NSPCs deficient for the Id3-controlled transcription factor E47 readily differentiated into astrocytes in the absence of BMP-2. Mechanistically, E47 repressed the expression of several astrocyte-specific genes in adult NSPCs. These results identify Id3 as the BMP-2-induced transcriptional regulator, promoting adult NSPC differentiation into astrocytes upon CNS injury and reveal a molecular link between environmental changes and NSPC differentiation in the CNS after injury.

Valproic Acid Arrests Proliferation but Promotes Neuronal Differentiation of Adult Spinal NSPCs from SCI Rats.

Although the adult spinal cord contains a population of multipotent neural stem/precursor cells (NSPCs) exhibiting the potential to replace neurons, endogenous neurogenesis is very limited after spinal cord injury (SCI) because the activated NSPCs primarily differentiate into astrocytes rather than neurons. Valproic acid (VPA), a histone deacetylase inhibitor, exerts multiple pharmacological effects including fate regulation of stem cells. In this study, we cultured adult spinal NSPCs from chronic compressive SCI rats and treated with VPA. In spite of inhibiting the proliferation and arresting in the G0/G1 phase of NSPCs, VPA markedly promoted neuronal differentiation (β-tubulin III(+) cells) as well as decreased astrocytic differentiation (GFAP(+) cells). Cell cycle regulator p21(Cip/WAF1) and proneural genes Ngn2 and NeuroD1 were increased in the two processes respectively. In vivo, to minimize the possible inhibitory effects of VPA to the proliferation of NSPCs as well as avoid other neuroprotections of VPA in acute phase of SCI, we carried out a delayed intraperitoneal injection of VPA (150mg/kg/12h) to SCI rats from day 15 to day 22 after injury. Both of the newborn neuron marker doublecortin and the mature neuron marker neuron-specific nuclear protein were significantly enhanced after VPA treatment in the epicenter and adjacent segments of the injured spinal cord. Although the impaired corticospinal tracks had not significantly improved, Basso-Beattie-Bresnahan scores in VPA treatment group were better than control. Our study provide the first evidence that administration of VPA enhances the neurogenic potential of NSPCs after SCI and reveal the therapeutic value of delayed treatment of VPA to SCI.

3,6-Bis(1-methyl-4-vinylpyridinium)-carbazole diiodide as a marker for tracking living neural stem/precursor cells.

Until now, there has been no appropriate marker to specifically and effectively label living neural stem/precursor cells (NSPCs) to investigate NSPC migration and differentiation. Therefore, the purpose of this study was to develop a specific method for tracking NSPCs by a cell marker, 3,6-bis(1-methyl-4-vinylpyridinium)-carbazole diiodide (BMVC). It was found that the bright fluorescence spots could be rapidly observed in the nuclei of NSPCs, isolated from the embryonic rat cerebral cortex, after incubation with BMVC for just 5 min and could be maintained for exceeding 7 days without obvious decay. Even NSPCs were induced to undergo differentiation; their daughter cells also expressed bright fluorescence spots. In contrast, foreskin fibroblasts, adipose-derived stem cells (ADSCs), bone marrow mesenchymal stem cells (BMMSCs), neurons, and glial cells did not express the bright fluorescence spot in the presence of BMVC. In addition, BMVC-labeled NSPCs could be tracked in wound healing and decellularized rat brain models with minimal cell toxicity. Therefore, BMVC possessed a specific function to distinguish between NSPCs and other stem and neuron-related cells. These results are very encouraging, since a cell marker favorable for long-term labeling NSPCs should be useful in the development of strategies for tracking the development of NSPCs in neuroscience research.

Functional Cross-Talk Between the Cellular Prion Protein and the Neural Cell Adhesion Molecule is Critical for Neuronal Differentiation of Neural Stem/Precursor Cells

Cellular prion protein (PrP) is prominently expressed in brain, in differentiated neurons but also in neural stem/precursor cells (NPCs). The misfolding of PrP is a central event in prion diseases, yet the physiological function of PrP is insufficiently understood. Although PrP has been reported to associate with the neural cell adhesion molecule (NCAM), the consequences of concerted PrP-NCAM action in NPC physiology are unknown. Here, we generated NPCs from the subventricular zone (SVZ) of postnatal day 5 wild-type and PrP null (-/-) mice and observed that PrP is essential for proper NPC proliferation and neuronal differentiation. Moreover, we found that PrP is required for the NPC response to NCAM-induced neuronal differentiation. In the absence of PrP, NCAM not only fails to promote neuronal differentiation but also induces an accumulation of doublecortin-positive neuronal progenitors at the proliferation stage. In agreement, we noted an increase in cycling neuronal progenitors in the SVZ of PrP-/- mice compared with PrP+/+ mice, as evidenced by double labeling for the proliferation marker Ki67 and doublecortin as well as by 5-bromo-2'-deoxyuridine incorporation experiments. Additionally, fewer newly born neurons were detected in the rostral migratory stream of PrP-/- mice. Analysis of the migration of SVZ cells in microexplant cultures from wild-type and PrP-/- mice revealed no differences between genotypes or a role for NCAM in this process. Our data demonstrate that PrP plays a critical role in neuronal differentiation of NPCs and suggest that this function is, at least in part, NCAM-dependent.

A neural stem/precursor cell monolayer for neural tissue engineering

The purpose of this study was to prepare a monolayer of neural stem/precursor cells (NSPCs) for neural tissue engineering applications. Two components present in serum, fibronectin and epidermal growth factor (EGF) were added into DMEM/F12 medium (termed medium B) to examine the effect of the migration-, proliferation- and differentiation-promoting potential on the cultured NSPCs, isolated from embryonic rat cerebral cortex. Compared with the serum effect, medium B also permitted neurosphere attachment onto the substrate surface and cell migration out of neurospheres extensively, but enhanced more extensive cell division and slowed down NSPC differentiation to generate a confluent NSPC monolayer. It was found the medium B-treated NSPCs possessed the capability to form typical neurospheres or to undergo differentiation into neuron-related cell types on various biomaterial surfaces. Therefore, we proposed a two-stage process for wound healing or nerve conduit preparation. Extensive NSPC division and MAP2-positive neuron differentiation were manipulated in NSPCs cultured in the medium B followed by the neuronal differentiation-favorable medium. These results should be useful for controlling the proliferation and differentiation of NSPCs on various biomaterials and conduits in neuroscience research.

Kv3.1 channels stimulate adult neural precursor cell proliferation and neuronal differentiation

Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs, voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However, the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1, a high voltage-gated KDR channel, was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties, such as resting membrane potential, of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation, not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation.

Covalent bonding of GYIGSR to EVAL membrane surface to improve migration and adhesion of cultured neural stem/precursor cells

In the present study, we modified poly (ethylene-co-vinyl alcohol) (EVAL) membranes with the covalent bonding of the laminin-derived peptides, GYIGSR by using carbodiimidazole (CDI) to activate the hydroxyl groups on the membrane surface. The resulting GYIGSR-immobilized EVAL (EVAL–GYIGSR) membrane was analyzed in terms of the effect of immobilized peptide sequence on the behaviors of neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, in the serum-free medium. Compared to the unmodified EVAL, GYIGSR immobilized on the EVAL membrane was shown to significantly increase NSPCs migrating out of neurospheres (p<0.05). In addition, NSPCs on the EVAL–GYIGSR membrane were able to differentiate into neural lineage cells and differentiated neurons expressed functional synaptic activity. Basically, there was no significant difference between GYIGSR-immobilized and laminin-coated substrates for their ability to enhance migration and differentiation of NSPCs, suggesting that the immobilization of GYIGSR on the EVAL membrane was successful and the laminin-derived peptide YIGSR and laminin had similar effect on NSPC behaviors. However, it is non-permanent modification for coating laminin on the substrates to support cell survival after a long-term culture. In this study, differentiated neurons could still adhere to the EVAL–GYIGSR surface with functional synaptic activity after incubation for 20 days. Therefore, the bioactive EVAL–GYIGSR provided an alternative approach to improve migration and survival of NSPCs for neural tissue engineering applications.

Interferon Gamma and Sonic Hedgehog Signaling Are Required to Dysregulate Murine Neural Stem/Precursor Cells

BackgroundThe pro-inflammatory cytokine interferon gamma (IFNγ), a key player in various neurological diseases, was recently shown to induce a dysregulated phenotype in neural stem/precursor cells (NSPCs) that is characterized by the simultaneous expression of glial and neuronal markers and irregular electrophysiological properties. Thus far, the mechanisms of this phenomenon have remained unclear.Methodology/Principal FindingsTo determine if binding of the signal transducers and activators of transcription (Stat 1) to the sonic hedgehog (SHH) promoter is important for this phenomenon to occur, chromatin immunoprecipitation and pharmacological inhibition studies were performed. We report here that the activation of both the Stat 1 and SHH pathways is necessary to elicit the dysregulated phenotype.Conclusions/SignificanceThus, blocking these pathways might preserve functional differentiation of NSPCs under inflammatory conditions leading to more effective regeneration.

Combination of media, biomaterials and extracellular matrix proteins to enhance the differentiation of neural stem/precursor cells into neurons

The purpose of this study was to induce the differentiation of neural stem/precursor cells (NSPC) more towards neurons than glial cells by the combination of media, biomaterials and extracellular matrix (ECM) proteins. Considering the role of serum, 10% fetal bovine serum or its fractions were added to DMEM/F12 medium to examine the effect of the differentiation-promoting potential on cultured NSPC isolated from embryonic rat cerebral cortex. The NSPC were cultured for 7days, after which differentiation was assayed using immunocytochemistry for lineage specific markers. It was demonstrated that molecules promoting neuron differentiation were present in serum with molecular weight <100kDa, which could dominate the differentiation of NSPC principally into neurons in the presence of basic fibroblast growth factor. In contrast, NSPC were induced to differentiate predominantly into glial cell phenotypes in the presence of whole serum components. Based on medium containing serum fraction, semi-quantification showed that the MAP2-positive percentage of the immunoreactive ratio within migrated cells could be promoted over 85% by combining poly(ethylene-co-vinyl alcohol) biomaterial and fibronectin matrix protein. These results are very encouraging, since an environment favorable for neuronal differentiation should be useful in the development of strategies for controlling the behavior of NSPC in neuroscience research.

Effects of the CNTF-collagen gel-controlled delivery system on rat neural stem/progenitor cells behavior

The injury of central nervous system (CNS) usually causes the cavity formation. Although transplantation of neural stem/precursor cells (NSPCs) into the lesioned area of CNS has been shown to be implicated in the functional restoration, the therapeutic result is limited by the poor survival of NSPCs as well as their insufficient proliferation and differentiation abilities. Type-1 collagen is considered as a candidate scaffold or drug delivery system to overcome the aforementioned obstacle. This study observed the effects of the CNTF (ciliary neurotrophic factor)-collagen gel-controlled delivery system and daily addition of soluble-form CNTF on the NSPC survival, migration, proliferation and differentiation. The results showed that, within 12 h of the initial co-culture, CNTF was released in a burst pattern, then the CNTF-collagen gel-controlled delivery system stably released CNTF for up to 12 d. The cell viability test, together with immunohistochemistry, RT-PCR and Western blotting, showed that the CNTF-collagen gel-controlled delivery system supported the NSPCs seeded on the surface of collagen gel survival and facilitated their migration and proliferation. The daily addition of soluble-form CNTF to the medium had similar effects to the CNTF-collagen gel-controlled delivery system, but large quantities of soluble-form CNTF were consumed during the entire process. Taken together, the CNTF-collagen gel-controlled delivery system not only provides a physical scaffold for the transplanted NSPCs to adhere and migrate, but also facilitates the NSPC survival, growth and proliferation, simultaneously reducing the consumption of the expensive growth factors. This system may be used to enhance the microenvironment in the lesioned area of CNS.

Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex

In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly- d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3β (GSK-3β) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.

Region-specific differentiation potential of adult rat spinal cord neural stem/precursors and their plasticity in response to in vitro manipulation.

This study characterized the differentiation of neural stem/precursor cells (NSPCs) isolated from different levels of the spinal cord (cervical vs lumbar cord) and different regions along the neuraxis (brain vs cervical spinal cord) of adult male Wistar enhanced green fluorescent protein rats. The differentiation of cervical spinal cord NSPCs was further examined after variation of time in culture, addition of growth factors, and changes in cell matrix and serum concentration. Brain NSPCs did not differ from cervical cord NSPCs in the percentages of neurons, astrocytes, or oligodendrocytes but produced 26.9% less radial glia. Lumbar cord NSPCs produced 30.8% fewer radial glia and 6.9% more neurons compared with cervical cord NSPCs. Spinal cord NSPC differentiation was amenable to manipulation by growth factors and changes in in vitro conditions. This is the first study to directly compare the effect of growth factors, culturing time, serum concentration, and cell matrix on rat spinal cord NSPCs isolated, propagated, and differentiated under identical conditions.

Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.