Immunohistochemical analysis of the developing mouse cortex.

The cerebral cortex is the most complex structure in the mammalian brain, whose development requires coordinated proliferation of neural stem/precursor cells (NPCs) and their differentiation into neurons and glia. Perturbations in NPC homeostasis can lead to abnormal cortical development which is frequently seen in neurodevelopmental disorders. In this chapter, we describe the preparation of cortical tissues from mice and step-by-step protocol for immunohistochemistry to study cortical development. With this technique, we employ commonly used molecular markers and thymidine analog methods to analyze NPC populations. We also discuss assay conditions that can be optimized according to the specific needs to improve experimental outcomes.

SoxE group transcription factor Sox8 promotes astrocytic differentiation of neural stem/precursor cells downstream of Nfia.

The brain consists of three major cell types: neurons and two glial cell types (astrocytes and oligodendrocytes). Although they are generated from common multipotent neural stem/precursor cells (NS/PCs), embryonic NS/PCs cannot generate all of the cell types at the beginning of brain development. NS/PCs first undergo extensive self-renewal to expand their pools, and then acquire the potential to produce neurons, followed by glial cells. Astrocytes are the most frequently found cell type in the central nervous system (CNS), and play important roles in brain development and functions. Although it has been shown that nuclear factor IA (Nfia) is a pivotal transcription factor for conferring gliogenic potential on neurogenic NS/PCs by sequestering DNA methyltransferase 1 (Dnmt1) from astrocyte-specific genes, direct targets of Nfia that participate in astrocytic differentiation have yet to be completely identified. Here we show that SRY-box transcription factor 8 (Sox8) is a direct target gene of Nfia at the initiation of the gliogenic phase. We found that expression of Sox8 augmented leukemia inhibitory factor (LIF)-induced astrocytic differentiation, while Sox8 knockdown inhibited Nfia-enhanced astrocytic differentiation of NS/PCs. In contrast to Nfia, Sox8 did not induce DNA demethylation of an astrocyte-specific marker gene, glial fibrillary acidic protein (Gfap), but instead associated with LIF downstream transcription factor STAT3 through transcriptional coactivator p300, explaining how Sox8 expression further facilitated LIF-induced Gfap expression. Taken together, these results suggest that Sox8 is a crucial Nfia downstream transcription factor for the astrocytic differentiation of NS/PCs in the developing brain.

Evaluation of Fluoro-Jade C Staining: Specificity and Application to Damaged Immature Neuronal Cells in the Normal and Injured Mouse Brain.

Fluoro-Jade C (FJC) staining is widely used for the specific detection of all degenerating mature neurons, including apoptotic, necrotic, and autophagic cells. However, whether FJC staining can detect degenerating immature neurons and neural stem/precursor cells remains unclear. In addition, some conflicting studies have shown that FJC and its ancestral dyes, Fluoro-Jade (FJ) and FJB, can label resting/activated astrocytes and microglia. In the present study, we examined the validity of FJC staining for the detection of neuronal cells in adult and embryonic mouse brains under normal and injured conditions. In the adult rodent subventricular zone (SVZ)-rostral migratory stream (RMS)-olfactory bulb (OB) system, apoptosis associated with neurogenesis occurs under normal conditions. Using this system, we detected FCJ positive (+) cells, some of which were doublecortin (DCX)(+) neuroblasts, in addition to neuronal nuclei (NeuN)(+) mature neurons. FJC negative (-) apoptotic cells expressing activated Caspase 3 were also observed, and a small number of FJC(+)/ionized calcium-binding adaptor molecule 1 (Iba1)(+) microglia and FJC(+)/glial fibrillary acidic protein (GFAP)(+) astrocytes were observed in the normal brain. Next, we analyzed embryonic brains, in which the apoptosis of neural stem/precursor cells was induced by the administration of N-ethyl-N-nitrosourea (ENU) or ethanol at embryonic day 14 or 10, respectively. In those brains, FJC(+) neural stem/precursor cells and neuroepithelial cells expressing SRY-related HMG-box 2 (Sox2) were observed. Surprisingly degenerating mesenchymal cells were also FJC(+). The present study indicates that FJC is a reliable marker for degenerating neuronal cells during all differentiation stages. However, FJC could also label degenerating non-neuronal cells under some conditions.

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

BACKGROUND/PURPOSE: In vitro neural cell-based models have been widely used to mimic the in vivo 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

Synergistic induction of astrocytic differentiation by factors secreted from meninges in the mouse developing brain.

Astrocytes, which support diverse neuronal functions, are generated from multipotent neural stem/precursor cells (NS/PCs) during brain development. Although many astrocyte-inducing factors have been identified and studied in vitro, the regions and/or cells that produce these factors in the developing brain remain elusive. Here, we show that meninges-produced factors induce astrocytic differentiation of NS/PCs. Consistent with the timing when astrocytic differentiation of NS/PCs increases, expression of astrocyte-inducing factors is upregulated. Meningeal secretion-mimicking combinatorial treatment of NS/PCs with bone morphogenetic protein 4, retinoic acid and leukemia inhibitory factor synergistically activate the promoter of a typical astrocytic marker, glial fibrillary acidic protein. Taken together, our data suggest that meninges play an important role in astrocytic differentiation of NS/PCs in the developing brain.

Cisplatin-induced mitochondrial dysfunction is associated with impaired cognitive function in rats.

PURPOSE: Chemotherapy-related cognitive impairment (CRCI) is commonly reported following the administration of chemotherapeutic agents and comprises a wide variety of neurological problems. No effective treatments for CRCI are currently available. Here we examined the mechanisms involving cisplatin-induced hippocampal damage following cisplatin administration in a rat model and in cultured rat hippocampal neurons and neural stem/progenitor cells (NSCs). We also assessed the protective effects of the antioxidant, N-acetylcysteine in mitigating these damages. EXPERIMENTAL DESIGN: Adult male rats received 6mg/kg cisplatin in the acute studies. In chronic studies, rats received 5mg/kg cisplatin or saline injections once per week for 4 weeks. N-acetylcysteine (250mg/kg/day) or saline was administered for five consecutive days during cisplatin treatment. Cognitive testing was performed 5 weeks after treatment cessation. Cisplatin-treated cultured hippocampal neurons and NSCs were examined for changes in mitochondrial function, oxidative stress production, caspase-9 activation, and neuronal dendritic spine density. RESULTS: Acute cisplatin treatment reduced dendritic branching and spine density, and induced mitochondrial degradation. Rats receiving the chronic cisplatin regimen showed impaired performance in contextual fear conditioning, context object discrimination, and novel object recognition tasks compared to controls. Cisplatin induced mitochondrial DNA damage, impaired respiratory activity, increased oxidative stress, and activated caspase-9 in cultured hippocampal neurons and NSCs. N-acetylcysteine treatment prevented free radical production, ameliorated apoptotic cellular death and dendritic spine loss, and partially reversed the cisplatin-induced cognitive impairments. CONCLUSIONS: Our results suggest that mitochondrial dysfunction and increased oxidative stress are involved in cisplatin-induced cognitive impairments. Therapeutic agents, such as N-acetylcysteine, may be effective in mitigating the deleterious effects of cisplatin.

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

BACKGROUND: Neural 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. METHODS: NSCs 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 ß-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. RESULTS: Conditioned 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. CONCLUSIONS: Our results suggest that miR-17-92 promotes neuronal differentiation of grafted NSCs under neuroinflammatory condition via inhibition of multiple proteins in JAK-STAT pathway.

The role of stem cell therapy in multiple sclerosis: An overview of the current status of the clinical studies.

The complexity of multiple sclerosis (MS) and the incompetence of a large number of promised treatments for MS urge us to plan new and more effective therapeutic approaches that aim to suppress ongoing autoimmune responses and induction of local endogenous regeneration. Emerging data propose that hematopoietic, mesenchymal, and neural stem cells have the potential to restore self-tolerance, provide in situ immunomodulation and neuroprotection, as well as promote regeneration. Thus, in this article, we will first provide an overview of the cell sources for proposed mechanisms that contribute to the beneficial effects of stem cell transplantation, the ideal route and/or timing of stem cell-based therapies for each main stem cell group, and finally, an overview of the current status of stem cell research in clinical trial stages in MS by comparable and healthy therapeutic effects of different stem cell therapies for MS patients.

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

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-3ß 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-3ß) signaling pathways were involved in the proliferation and neural differentiation of NSPCs. Overexpression of miR-21 activated AKT, and the p-GSK-3ß was increased. Conversely, knocking down miR-21 blocked the activation of AKT, and decreased the phosphorylation level of GSK-3ß. These results demonstrated that miR-21 promotes neural differentiation and reduces proliferation in NSPCs via regulating AKT and GSK-3ß pathways. These findings may help to develop strategies for treatment of central nervous system diseases.

Ankfy1 is dispensable for neural stem/precursor cell development.

There are few studies on the membrane protein Ankfy1. We have found Ankfy1 is specifically expressed in neural stem/precursor cells during early development in mice (murine). To further explore Ankfy1 function in neural development, we developed a gene knockout mouse with a mixed Balb/C and C57/BL6 genetic background. Using immunofluorescence and in situ hybridization, neural defects were absent in mixed genetic Ankfy1 null mice during development and in adults up to 2 months old. However, Ankfy1 gene knockout mice with a pure genetic background were found to be lethal in the C57/BL6 inbred mice embryos, even after seven generations of backcrossing. Polymerase chain reaction confirmed homozygotes were unattainable as early as embryonic day 11.5. We conclude that Ankfy1 protein is dispensable in neural stem/precursor cells, but could be critical for early embryonic murine development, depending on the genetic background.

PPARß/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone.

The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARß/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARß/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARß/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARß/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARß/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis.

The interaction between microglia and neural stem/precursor cells.

In the adult mammalian brain, neurogenesis from neural stem/precursor cell occurs within two regions, the subgranular zone (SGZ) in the dentate gyrus (DG) and the subventricular zone (SVZ) lining the lateral ventricles. The function of neural stem cell is enhanced by external stimuli, such as injury and inflammation. Microglia, as the main immune modulating cells, play important roles in the central nervous system (CNS). Recently, select discoveries reported that microglia might influence the proliferation, differentiation and survival of neural precursor cells (NPCs). Other studies revealed that NPCs might reversibly regulate the function of microglia. Accordingly, in this review we focus on the interaction between microglia and NPCs.

Low-doses of cisplatin injure hippocampal synapses: a mechanism for 'chemo' brain?

Chemotherapy-related cognitive deficits are a major neurological problem, but the underlying mechanisms are unclear. The death of neural stem/precursor cell (NSC) by cisplatin has been reported as a potential cause, but this requires high doses of chemotherapeutic agents. Cisplatin is frequently used in modern oncology, and it achieves high concentrations in the patient's brain. Here we report that exposure to low concentrations of cisplatin (0.1µM) causes the loss of dendritic spines and synapses within 30min. Longer exposures injured dendritic branches and reduced dendritic complexity. At this low concentration, cisplatin did not affect NSC viability nor provoke apoptosis. However, higher cisplatin levels (1µM) led to the rapid loss of synapses and dendritic disintegration, and neuronal-but not NSC-apoptosis. In-vivo treatment with cisplatin at clinically relevant doses also caused a reduction of dendritic branches and decreased spine density in CA1 and CA3 hippocampal neurons. An acute increase in cell death was measured in the CA1 and CA3 neurons, as well as in the NSC population located in the subgranular zone of the dentate gyrus in the cisplatin treated animals. The density of dendritic spines is related to the degree of neuronal connectivity and function, and pathological changes in spine number or structure have significant consequences for brain function. Therefore, this synapse and dendritic damage might contribute to the cognitive impairment observed after cisplatin treatment.

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.