Ultrastructure of human neural stem/progenitor cells and neurospheres

BACKGROUND: Biological and morphological characteristics of neural stern/progenitor cells (NSPCs) have been widely investigated.OBJECTIVE: To explore the ultrastructure of human embryo-derived NSPCs and neurospheres cultivated in vitro using electron microscopy.DESIGN, TIME AND SETTING: A cell biology experiment was performed at the Brain Tumor Laboratory of Soochow University, and Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University between August 2007 and April 2008.MATERIALS: Human fetal brain tissue was obtained from an 8-week-old aborted fetus; serum-free Dulbecco's modified Eagle's medium/F12 culture medium was provided by Gibco, USA; scanning electron microscope was provided by Hitachi instruments, Japan; transmission electron microscope was provided by JEOL, Japan.METHODS: NSPCs were isolated from human fetal brain tissue and cultivated in serum-free Dulbecco's modified Eagle's medium/F12 culture medium. Cells were passaged every 5-7 days. After three passages, NSPCs were harvested and used for ultrastructural examination.MAIN OUTCOME MEASURES: Ultrastructural examination of human NSPCs and adjacent cells in neurospheres.RESULTS: Individual NSPCs were visible as spherical morphologies with rough surfaces under scanning electron microscope. Generally, they had large nuclei and little cytoplasm. Nuclei were frequently globular with large amounts of euchromatin and a small quantity of heterochromatin, and most NSPCs had only one nucleolus. The Golgi apparatus and endoplasmic reticulum were underdeveloped; however, autophagosomes were clearly visible. The neurospheres were made up of NSPCs and non-fixiform material inside. Between adjacent cells and at the cytoplasmic surface of apposed plasma membranes, there were vesicle-like structures. Some membrane boundaries with high permeabilities were observed between some contiguous NSPCs in neurospheres, possibly attributable to plasmalemmal fusion between adjacent cells.CONCLUSION: A large number of autophagosomes were observed in NSPCs and gap junctions were visible between adjacent NSPCs.     

纤维连接蛋白促进人神经球内干细胞的迁移

目的研究细胞外基质成份纤维连接蛋白(Fn)对人神经干细胞迁移的作用。方法用Fn包被培养孔,于细胞球转种后的24h、48h、72h、96h、120h、144h、168h和240h各时间点动态观察神经球细胞的迁移变化,测量细胞迁移的最远距离,计算迁移速率,用荧光免疫技术检测迁移细胞的特异性表型标志,计算一定视野内nestin+细胞所占的百分比,同时,平行观察含胎牛血清(FBS)培养诱导的迁移。设立多聚赖氨酸(Ply)包被组以及空白组作为实验对照。结果Ply包被组以及空白组均无细胞迁移发生,FBS组和Fn组的细胞球有细胞从球体向外迁移,二者迁移速率均在前48h达最高峰,但迁移方式不同。Fn组迁移的细胞分化不明显,细胞分裂相多见,nestin+细胞占(30.60±8.06)%,而FBS组迁移出来的细胞成进行性分化梯度,偶见有nestin+细胞,外周边缘区域GFAP+星形胶质细胞是其主要细胞群。结论Fn在体外可促进人胎脑神经干细胞的迁移。     

Regional distribution of neural cell adhesion molecule (N-CAM) and L1 in human and rodent hippocampus

Cell surface adhesion molecules N-CAM and L 1 are implicated in central nervous system (CNS) cell migration and axon outgrowth in in vitro and in vivo developmental studies. These molecules show a differential distribution during CNS development, thus suggesting that they subserve different roles in process outgrowth and tissue organization. A variety of N-CAM isoforms are known, and individual N-CAMs undergo posttranslational modification. Such changes and the potential for generating numerous molecules may mediate development of specific neural cell contacts and circuitry. We evaluated immunohistochemical staining of polyclonal antibodies to L 1 and N-CAM, as well as monoclonal antibodies directed against embryonic N-CAM and the 140 and 180 kDa species of N-CAM in human, rat, and mouse hippocampus. Staining patterns in the three species were qualitatively similar, but staining in the mouse hippocampus was quantitatively greater for some epitopes. A distinctive pattern of staining was found, corresponding to the known anatomy of the structure. Total N-CAM staining was intense in the hilus and inner molecular layer (ML) of the dentate gyrus with lighter staining in the dentate outer ML. The mossy fiber tract (MFT), comprising axons traveling from the dentate granule cells to CA3 pyramidal cells, was strongly stained by polyclonal antibody to N-CAM. There was abundant staining of the stratum radiatum (SR) and stratum oriens (SO) of CA1, but stratum lacunosum moleculare (LM) showed very little staining. The monoclonal antibody 12F11, which recognizes the 140 and 180 kDa forms of N-CAM, intensely stained the MFT, hilus, and inner ML. With 12F11, SO and SR stained uniformly throughout CA1, with much reduced staining in CA2 and CA3. There was little staining in LM. L1 staining was more evenly distributed throughout the hippocampus and dentate, with light hilar and MFT staining, and even staining through the SR and SO. Stratum LM stained intensely for L1, with a narrow clear zone between SR and LM. The ML of the dentate gyrus stained intensely with anti-L1, with a discrete clear zone separating the inner and outer ML. Embryonic N-CAM had little staining in CA1 but strong hilar and MFT staining; thus, this “embryonic” determinant continues to be expressed in limited regions in the adult. In contrast to the adult rodent hippocampus, human hippocampus exhibited embryonic N-CAM in the outer two-thirds of the ML, but showed very little staining in the hilar region. These distinctive patterns suggest that the distribution of the N-CAM and L1 molecular species have clear-cut structural and functional roles in the laminated circuitry of the hippocampus. (c) 1993 Wiley-Liss, Inc.     

体外培养人类神经干细胞神经球中髓鞘的形成

目的：探索人类神经干细胞的体外培养条件和其形成的神经球的超微结构。方法：从胎脑皮质中机械分离细胞，N2培养基培养和扩增人类神经干细胞，应用免疫细胞化学方法对培养的细胞进行鉴定。对形成的神经球应用透射电镜进行超微结构研究。结果：从胎儿的脑皮质中成功分离培养出神经干细胞，形成典型的神经球，大部分细胞表达神经干细胞的标志物波形蛋白，细胞贴壁后可诱导分化 为神经元和胶质。早期培养的神经球中有典型的髓鞘形成。结论：从胎儿的脑皮质中成功地分离、培养出人类的神经干细胞，细胞呈悬浮状态生长，形成神经球。这种早期培养的神经干细胞有形成髓鞘的能力，可作为治疗人类脱髓鞘病变的潜在细胞来源。     

Regional specification of neurosphere cultures derived from subregions of the embryonic telencephalon

We have studied the molecular specification of precursor cells in expanded neurosphere cultures derived from distinct subregions of the embryonic mouse telencephalon. These regionally derived cultures exhibited differential responses to the mitogens EGF and bFGF, suggesting that the precursors in these cultures were differentially specified as is the case in situ. To examine this further, cultures from each of the telencephalic subregions were expanded in both EGF and bFGF before differentiation. The neurons produced displayed molecular phenotypes similar to those normally derived from each of these regions in vivo. Moreover, analysis of gene expression in the undifferentiated cultures showed that the regionally derived neurospheres express many of the same developmental control genes as their in vivo counterparts. Taken together, the present findings suggest that precursor cells in neurosphere cultures, derived from distinct subregions of the embryonic telencephalon, maintain at least certain aspects of their molecular specification, even after significant expansion in vitro.     

Regional and temporal specificity of intrinsic plasticity mechanisms in rodent primary visual cortex

Different neocortical regions are functionally specialized, but whether this specialization is reflected in the forms of plasticity present during developmental critical periods (CPs) is largely unknown. In rodent visual cortex, we recently showed that a form of intrinsic plasticity [LTP of intrinsic excitability (LTP-IE)] in the monocular region of the primary visual cortex (V1M) plays an important role in modulating cortical responsiveness following visual deprivation. Here we ask whether LTP-IE is present and similarly regulated by visual experience in the binocular region of the primary visual cortex (V1B), where inputs from the two eyes compete during the CP. In contrast to V1M, where LTP-IE is present throughout the CP, in V1B LTP-IE was only transiently expressed at the onset of the CP. Also distinct from V1M, brief monocular deprivation (MD) was unable to modulate LTP-IE magnitude in V1B, and even binocular deprivation (the equivalent of MD in V1M) could only influence LTP-IE expression during a narrow time window at the peak of the CP. Finally, we asked whether these differences depend on differences in sensory activation of the two areas during development. MD of ipsilateral inputs from before eye opening (to reduce competitive interactions) did not affect the pattern of LTP-IE expression in V1B. Further, the differences in plasticity in the two cortical areas persisted when animals were reared in the dark to remove all patterned visual input. Thus neocortical LTP-IE expression shows dramatic regional and temporal differentiation, and these differences are not driven by differences in sensory experience.     

Gradual specification of response amplitude in human tracking performance

These experiments examine how human subjects use information from a target to trigger a response and to specify its trajectory. We first determined if response initiation is predicated on the prior specification of response amplitude by examining the latencies and trajectories of impulses of isometric elbow flexion aimed to one of three visual targets. We varied target predictability (simple versus choice), the urgency with which the response was required, and the level of practice. With practice, subjects could respond to unpredictable targets with the same latency as to predictable ones; the range of response amplitudes was, however, always constricted. This central tendency bias disappeared when subjects were allowed long latencies to respond to the target, suggesting that with urgency, subjects can respond before specification is complete. To determine the time course of specification, the subjects were trained to initiate force impulses in synchrony with the last of a series of predictable tones. They also attempted to match the amplitude of their force impulses to one of three unpredictable visual targets presented at randomly varying times (50-400 ms) prior to the synchronizing tone. At the shortest stimulus-response intervals, before target information could be processed, the amplitudes of responses to all targets were clustered around that of the middle-sized target. Then, as the stimulus-response interval increased, response amplitudes gradually converged upon their specific targets. Specification started at stimulus-response intervals of about 100 ms and extended until about 350 ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution voxelation mapping of human and rodent brain gene expression

Voxelation allows high-throughput acquisition of multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. To obtain these images, the method employs analysis of spatially registered voxels (cubes). For creation of high-resolution maps using voxelation, relatively small voxel sizes are necessary and instruments will be required for semiautomated harvesting of such voxels. Here, we describe two devices that allow spatially registered harvesting of voxels from the human and rodent brain, giving linear resolutions of 3.3 and 1 mm, respectively. Gene expression patterns obtained using these devices showed good agreement with known expression patterns. The voxelation instruments and their future iterations represent a valuable approach to the genome scale acquisition of gene expression patterns in the human and rodent brain.     

RT-PCR amplification of mRNA from single brain neurospheres

A method is described that allows cDNA production from individual brain cell clones or 'neurospheres'. These culture-generated spheres of stem, progenitor, and differentiated cells have been the focus of interest because they represent an in vitro model of neurogenesis. However, because neurospheres are somewhat resistant, in part due to their enclosure by a dense extracellular matrix, to methods attempting to disrupt them and isolate nucleic acids, there is a need for new technology that affords the simple and efficient RT-PCR for studies of neural gene expression and discovery. A method is described here that uses sonication and an all-in-one approach for the construction of cDNA from single neurospheres. The generation of cDNA from individual adult brain stem/progenitor cell neurospheres is useful for future studies of neurogenic gene expression.     

Metabolic properties of astrocytes differentiated from rat neurospheres

The metabolic properties of astroglia differentiated from neurospheres have not been fully assessed. In this study, the glycolytic and oxidative metabolism of glucose in astroglia differentiated from rat tertiary neurospheres (astroglia(NS)) was compared with that in astroglia prepared from the striata of embryonic day 16 rats (astroglia(ST)). In addition to the basal condition, we also investigated energy metabolism under Na+,K+-ATPase activation. Furthermore, the effects of glucose concentration in the culture medium were assessed. No significant differences in 2-deoxy-D-[1-(14)C]glucose phosphorylation were observed between astroglia(NS) and astrogliaST. The rates of L-[U-14C]lactate ([14C]lactate) and D-[U-14C]glucose ([14C]glucose) oxidation were 5.74+/-0.82 and 2.83+/-0.4 pmol/60 min/microg protein, respectively, in astrogliaNS grown in low glucose (2 mM) and 3.01+/-1.03 and 1.77+/-0.23 pmol/60 min/microg protein, respectively, in astrogliaNS grown in high glucose (22 mM). Neither the [14C]lactate nor the [14C]glucose oxidation rates in astrogliaNS were significantly different from those in astrogliaST. D-aspartate (500 microM) significantly increased the [14C]lactate and [14C]glucose oxidation rates by 127% and 62%, respectively, in astrogliaNS grown in low glucose and by 217% and 115%, respectively, in astroglia(NS) grown in high glucose. D-aspartate also increased the oxidation of [14C]lactate and [14C]glucose to 236% and 147% of the control values, respectively, in astrogliaST grown in low glucose and to 174% and 144%, respectively, in astrogliaST grown in high glucose. Rat astroglia differentiated from neurospheres might possess an equivalent capacity for utilizing energy substrates under both basal and activated conditions to that of astroglia prepared from striatum.     

A rodent model of human organophosphate exposure producing status epilepticus and neuropathology

Exposure to organophosphates (OPs) often results in seizures and/or status epilepticus (SE) that produce neural damage within the central nervous system (CNS). Early control of SE is imperative for minimizing seizure-related CNS neuropathology. Although standard therapies exist, more effective agents are needed to reduce OP-induced SE and neuronal loss, particularly therapies with efficacy when administered 10’s of minutes after the onset of SE. To evaluate novel antiseizure compounds, animal models should simulate the CNS effects of OP exposure observed in humans. We characterized in rats the effects of the OP, diisopropyl flourophosphate (DFP) as a function of dose and route of administration of supporting agents (pyridostigmine, 2-PAM, atropine); outcome measures were mortality, electrographic seizure activity during SE, and subsequent CNS neuropathology. Doses of DFP between 3 and 7 mg/kg consistently caused SE, and the latency to behavioral tremors and to subsequent initiation of SE were dose related. In distinction, all doses of DFP that resulted in electrographic SE (3–7 mg/kg) produced seizures of similar intensity and duration, and similar CNS neuropathology (i.e., the effects were all-or-none). Although SE was similar across doses, mortality progressively increased with higher doses of DFP. Mortality was significantly lower when the route of administration of therapeutic agents was intramuscular compared to intraperitoneal. This rodent model of OP poisoning demonstrates pathological characteristics similar to those observed in humans, and thus begins to validate this model for investigating potential new therapeutic approaches.     

Differential generation of oligodendrocytes from human and rodent embryonic spinal cord neural precursors

Human neural precursors are considered to have widespread therapeutic possibilities on account of their ability to provide large numbers of cells whilst retaining multipotentiality. Application to human demyelinating diseases requires improved understanding of the signalling requirements underlying the generation of human oligodendrocytes from immature cell populations. In this study, we compare and contrast the capacity of neural precursors derived from the developing human and rodent spinal cord to generate oligodendrocytes. We show that the developing human spinal cord (6-12 weeks of gestation) displays a comparable ventrodorsal gradient of oligodendrocyte differentiation potential to the embryonic rodent spinal cord. In contrast, fibroblast growth factor 2 (FGF-2) expanded human neural precursors derived from both isolated ventral or dorsal cultures show a reduced capacity to generate oligodendrocytes, whereas comparable rodent cultures demonstrate a marked increase in oligodendrocyte formation following FGF-2 treatment. In addition, we provide evidence that candidate growth factors suggested from rodent studies, including FGF-2 and platelet-derived growth factor (PDGF) do not stimulate proliferation of human oligodendrocyte lineage cells. Finally, we show that the in vivo environment of the acutely demyelinating adult rat spinal cord is insufficient to stimulate the differentiation of immature human spinal cord cells to oligodendrocytes. These results provide further evidence for inter-species difference in the capacity of neural precursors to generate oligodendrocytes.     

In vitro binding of3H-acetylcholine to nicotinic receptors in rodent and human brain

Summary The binding of³H-acetylcholine (³H-ACh) to nicotinic receptors in rodent and human brain was measured in the presence of atropine to prevent binding to muscarinic binding sites.³H-ACh binds specifically and saturably to rodent brain. From saturation binding K_d was 30 nM in rat cerebral cortex, which is close to that calculated from kinetic experiments. The binding was temperature-dependent, being highest at low temperatures and decreasing at higher temperatures. The regional distribution of binding in mouse brain was not uniform. The binding was highest in the midbrain, intermediate in the cerebral cortex and striatum, and lowest in the cerebellum, hippocampus, hypothalamus and medulla oblongata. No significant correlation was found between the regional³H-ACh binding and the regional binding of³H-alpha-bungarotoxin (³H-BTX),³H-nicotine (³H-NIC),³H-tubocurarine and the endogenous acetylcholine content, although the correlation value for³H-ACh/³H-NIC was at the limit for significance.³H-ACh also bound specifically to human cerebral cortical tissue and this binding was approximately three times lower than in rodent brain, when a low³H-ACh concentration was used. In contrast to rat brain there appears to exist multiple binding sites for³H-ACh in human cerebral cortex as suggested by the curvelinear nature of the Scatchard plot. It was calculated that³H-ACh bound with K_d 4 nM and B_max 8 pmol/g protein and K_d 112 nM and B_max 67 pmol/g protein. The Hill number of 1.5 for the binding of low concentration and 2.5 for high concentration of³H-ACh also suggest that the³H-ACh-binding sites interaction exhibit positive cooperativity.     

Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with human

Progress in the development of rat models of human periventricular white matter injury (WMI) has been hampered by uncertainty about the developmental window in different rodent strains that coincides with cerebral white matter development in human premature infants. To define strain-specific differences in rat cerebral white matter maturation, we analyzed oligodendrocyte (OL) lineage maturation between postnatal days (P)2 and P14 in three widely studied strains of rat: Sprague-Dawley, Long-Evans and Wistar (W). We previously reported that late OL progenitors (preOL) are the major vulnerable cell type in human periventricular WMI. Strain-specific differences in preOL maturation were found at P2, such that the W rat had the highest percentage and density of preOL relative to the other strains. Overall, at P2, the state of OL maturation was similar to preterm human cerebral white matter. However, by P5, all three strains displayed a similar magnitude and extent of OL maturation that persisted with progressive myelination between P7 and P14. PreOL were the predominant OL lineage stage present in the cerebral cortex through P14, and thus OL lineage maturation occurred latter than in white matter. The hippocampus also displayed a later onset of preOL maturation in all three strains, such that OL lineage maturation and early myelination was not observed to occur until about P14. This timing of preOL maturation in rat cortical gray matter coincided with a similar timing in human cerebral cortex, where preOL also predominated until at least 8 months after full-term birth. These studies support that strain-specific differences in OL lineage immaturity were present in the early perinatal period at about P2, and they define a narrow window of preterm equivalence with human that diminishes by P5. Later developmental onset of preOL maturation in both cerebral cortex and hippocampus coincides with an extended window of potential vulnerability of the OL lineage to hypoxia-ischemia in these gray matter regions.     

Misfolded tau protein and disease modifying pathways in transgenic rodent models of human tauopathies

Human tauopathies represent a heterogeneous group of neurodegenerative disorders such as Alzheimer’s disease (AD) that are characterized by the presence of intracellular accumulations of abnormal filaments of protein tau. Presently, AD poses an increasing public health concern, because it affects nearly 2% of the population in industrialized countries and the number of patients is expected to increase threefold within the next 50 years. Therefore, the identification of disease modifying pathways that will lead to the development of novel therapeutic approaches targeting downstream molecular events of the tauopathy is of paramount importance. In order to identify factors that may exacerbate or inhibit the disease phenotype a number of genetically modified rodent models reproducing key clinical, histopathological and molecular hallmarks of human tauopathies were developed. Current tau transgenic rodent models express as a transgene either an individual or all six human wild-type tau isoforms, mutant tau linked to FTDP-17, or structurally modified tau species derived from AD. In this review we will provide an up-to-date account of various facets of the tau neurodegenerative cascade with a special emphasis on the evolution of neurofibrillary tangles, neuronal death and neuroinflammation.     

Dynamic behavior of cells within neurospheres in expanding populations of neural precursors

Large-scale expansion of neural stem and progenitor cells will be essential for clinically treating the large number of patients suffering from neurodegenerative disorders such as Parkinson's disease. Other applications of neural stem cell technology include further research in areas such as neural development or drug testing. Neural stem cells can be grown in vitro as tissue aggregates known as neurospheres, and in the current study, experiments were performed to determine the spatial arrangement and behavior of the cells within the neurosphere structure. A protocol utilizing sulfonated lipophilic fluorescent dyes was developed to effectively label populations of neural stem and progenitor cells without compromising cell density during culture. Cells retained the labels for at least 7 days. Using the labeling protocol, we discovered that the cells within the neurospheres were mobile and, moreover, the cells on the periphery of the neurospheres could migrate into the center of the neurospheres. Most important, the mixing time of two merging neurospheres was observed to be the same order of magnitude as the neural stem cell doubling time (approximately 20 h). This study is the first to show that the neurosphere system is dynamic, and these results will serve as a stepping stone to more in-depth studies of the neurosphere microenvironment.     

Regional potential for oligodendrocyte generation in the rodent embryonic spinal cord following exposure to EGF and FGF-2

The origin of oligodendrocytes in the developing rodent spinal cord has not been fully established, with some evidence that oligodendrocyte progenitors arise exclusively from the ventral neuroepithelium, other studies suggesting that both halves of the spinal cord have oligodendrogenic potential. One way of exploring this issue is to study more primitive oligodendrocyte precursors. Although specific markers are not available, their presence may be inferred using mitogens such as EGF and FGF-2, which stimulate the proliferation of immature neuroepithelial cells, and subsequently studying their differentiation into lineage restricted cells. We used this approach to assess whether the dorsal embryonic rodent spinal cord has the intrinsic potential for oligodendrocyte formation at E14. We confirm that significant numbers of oligodendrocytes and their immediate (A2B5+) precursors are present only in the ventral spinal cord of the E14 rodent, but following exposure to EGF and FGF-2, significant numbers of oligodendrocytes and A2B5+ precursor cells also develop from isolated E14 dorsal derived cells without interaction from the ventral spinal cord. In addition, bromodeoxyuridine studies demonstrate that isolated dorsal derived cells proliferate and express A2B5 following exposure to EGF and FGF-2. The observation that from E14, the dorsal cord already has latent oligodendrogenic potential provides an alternative mechanism for oligodendrocyte formation to ventro-dorsal migration of oligodendrocyte precursors. GLIA 24:382–389, 1998. © 1998 Wiley-Liss, Inc.