共查询到20条相似文献,搜索用时 31 毫秒
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Characterization of neurosphere cell phenotypes by flow cytometry 总被引:14,自引:0,他引:14
BACKGROUND: Neural stem cell research regularly utilizes neurosphere cultures as a continuous source of primitive neural cells. Results from current progenitor cell assays show that these cultures contain a low number of neural progenitors. Our goal is to characterize neurosphere cultures and define subpopulations in order to purify neural progenitor cells. METHODS: Cells from embryonic mouse neurosphere cultures were stained with Hoechst 33342 and analyzed by flow cytometry. Subpopulations were sorted based on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 days in culture. RESULTS: Neurosphere cultures contain a relatively high number of cells that stain weakly with Hoechst 33342. This subpopulation is present when cultured as an entire batch in the presence of epidermal growth factor (EGF). When cultured separately, this subpopulation gives rise to a neurosphere population with essentially the same characteristics as freshly isolated embryonic mouse brain cells but contains substantially fewer weakly Hoechst-stained cells. CONCLUSIONS: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells. 相似文献
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Hassan Azari Sharareh Sharififar Jeff M. Fortin Brent A. Reynolds 《Journal of visualized experiments : JoVE》2012,(62)
Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells. 相似文献
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Hassan Azari Sharareh Sharififar Maryam Rahman Saeed Ansari Brent A. Reynolds 《Journal of visualized experiments : JoVE》2011,(47)
In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum‐free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.Download video file.(69M, mov) 相似文献
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There are several known neurogenic areas including subventricular zone and subgranular layer in the dentate gyrus of the hippocampus. Both germinal centers exhibit an age-dependent decline in cell proliferation and neurogenesis, which may be associated with age-related decline in brain function. We recently identified the subcallosal zone (SCZ) as a novel neural stem cell niche with a potential to spontaneously produce new neuroblasts. We examined whether SCZ neurogenesis is also regulated by the age of mice. The number of newly generated neuroblasts was reduced in the SCZ with age, and only marginal number of DCX-labeled neuroblasts was found in 6-month-old SCZ, which is most likely due to reduced proliferation of progenitor cells and loss of neural stem cells (NSCs). This age-dependent changes in the SCZ occurred earlier than that of other neurogenic brain regions. The neurosphere assay in vitro confirmed the depletion of NSCs within the SCZ of young adults. However, marked induction of neuroblast production in the SCZ was seen in 6-month-old mice after traumatic brain injury. Taken together, these results indicate that a rapid decline in SCZ neurogenesis in mice is due to depletion of NSCs and reduced capacity to produce neuroblasts. 相似文献
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PAR-1 kinase phosphorylates Dlg and regulates its postsynaptic targeting at the Drosophila neuromuscular junction 总被引:4,自引:0,他引:4
Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits. 相似文献
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Vanderluit JL Ferguson KL Nikoletopoulou V Parker M Ruzhynsky V Alexson T McNamara SM Park DS Rudnicki M Slack RS 《The Journal of cell biology》2004,166(6):853-863
Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107(-/-) brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107(-/-) neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain. 相似文献
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Brandon C. Shelley Geneviève Gowing Clive N. Svendsen 《Journal of visualized experiments : JoVE》2014,(88)
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products. 相似文献
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Deleyrolle LP Ericksson G Morrison BJ Lopez JA Burrage K Burrage P Vescovi A Rietze RL Reynolds BA 《PloS one》2011,6(1):e15844
Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cell's resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stem-like cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence. 相似文献
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Soon-Tae Lee Kon Chu Keun-Hwa Jung Young-Mi Song Daejong Jeon Seung U. Kim Manho Kim Sang Kun Lee Jae-Kyu Roh 《PloS one》2011,6(7)
Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted. 相似文献
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Vanesa Nieto-Estévez Jaime Pignatelli Marcos J. Araúzo-Bravo Anahí Hurtado-Chong Carlos Vicario-Abejón 《PloS one》2013,8(1)
Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation. 相似文献
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Can Chen Yi Wang Sally S. L. Goh Jing Yang Dang Hoang Lam Yukti Choudhury Felix Chang Tay Shouhui Du Wee Kiat Tan Yovita Ida Purwanti Weimin Fan Shu Wang 《Journal of neurochemistry》2013,126(3):318-330
The breakthrough in derivation of human‐induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC‐derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC‐NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non‐migratory hiPSC‐NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down‐regulated in migratory hiPSC‐NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC‐NSCs toward cancer cells, and that inhibition of its activity or down‐regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell‐mediated cancer therapy. 相似文献
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Sun Shim Choi 《Genes & genomics.》2010,32(3):225-231
Neural stem cell (NSC) culture offers a renewable resource for cell replacement treatment of neurodegenerative diseases. In a NSC culture, the frequency of NSCs is estimated to be only a few percent, at most, while the majority of cells are neuroprogenitor cells (NPCs), the progeny of NSCs with a limited capability to proliferate. Here, the effects of several cell culture parameters on the cell composition of NSC cultures were illustrated over time using a simple model. The model shows thatvarious initial cell compositions in NSC cultures converge into a single steady state. The model also implies that the rate of increase in total cell number entirely depends on the self-renewal rate of NSCs, regardless of the proliferative capacity of NPCs. Furthermore, the model predicts that difference sin cell passage methods between neurosphere and monolayer cultures results in a change in the frequency of NSCs in culture. 相似文献
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Neuregulin-1 increases the proliferation of neuronal progenitors from embryonic neural stem cells 总被引:4,自引:0,他引:4
Neuregulins are a family of proteins expressed in the developing brain and in brain regions that continue to undergo neurogenesis in adult animals. We investigated the effects of neuregulins on embryonic neural stem cells (NSCs) isolated from E11 mouse telencephalon. Treatment of basic fibroblast growth factor (bFGF)-expanded neurosphere cultures with the EGF-like domain of neuregulin1-beta1 (NRG-1(177-244)) resulted in a 4-fold increase of bromodeoxyuridine (BrDU)-labeled cells, suggesting that NRG-1 stimulated proliferation. The majority of the BrdU-positive cells co-labeled with an antibody against MAP2, indicating that the proliferating cells were neuronal. No BrDU labeling was seen in GFAP- or O4-positive cells. In NRG-1-treated cultures, many of the MAP2-positive cells co-labeled with an anti-nestin antibody, suggesting that these cells are neuron-restricted progenitors (NRPs). Few MAP2/nestin-positive cells were seen in control cultures. The increase in the number of neuronal cells in NRG-1-treated cultures was due to increased proliferation of MAP2-positive cells rather than the regulation of cell survival or fate determination. These results suggest that neuregulins are mitogenic to NRPs, thus endogenous neuregulins may play important roles during CNS neurogenesis. 相似文献
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Strengths and limitations of the neurosphere culture system 总被引:8,自引:0,他引:8
After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a nearperfect system for large-scale
production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More
than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become
apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications.
Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances
and for the appropriate purposes, neurospheres hold up to their initial promise.
This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay
for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for
transplantation purposes. 相似文献
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Hassan Azari Maryam Rahman Sharareh Sharififar Brent A. Reynolds 《Journal of visualized experiments : JoVE》2010,(45)
Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.Download video file.(159M, mp4) 相似文献
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神经干细胞是一类具有多向分化潜能、自我复制能力,在特定条件诱因下,能够向特定类型神经元或神经胶质细胞分化的未分化或低分化细胞。神经干细胞的研究对于我们阐明神经发生机制,进行神经变性疾病的细胞替代治疗都有重要意义。目前,从人胚组织分离神经干细胞的方法不尽相同,对于大脑皮层神经干细胞的分离,一些学者认为机 相似文献