第1步<ol><li>板丝裂霉素C ggrowth抑制（10微克/毫升为2.5小时）MS5细胞明胶包30分钟（0.01％）6孔板中的α- MEM媒体的密度在70,000 /平方厘米。 </li><li>当细胞附着并已经形成了单层（过夜增长），切换至SRM。 </li><li>手动隔离ES细胞克隆胚胎干细胞的使用注射器文化与27 ½ G针。 </li><li> 1毫升的蓝色提示和低密度板MS5细胞（通常为2-3％菌落6孔板）仔细Tritrurate殖民地。 </li></ol> 注：胚胎干细胞也可以从使用胶原酶或胰蛋白酶的酶传播 。 第2步<ol><li> MS5馈线的ES细胞成长为14-21天，直到花环含菌落形成。 </li></ol> 注：更改媒体经常。通常是在殖民地和300-500纳克/毫升Noggin被添加到SRM（3）如果他们的形成，可大大提高外缘的花环 。 在一些分化协议，SRM可以用N2媒体交换，并辅以增长或其他因素，以促进细胞的规范（2-5 ）。 第3步<ol><li>隔离和花环半挑选了27 G针，在显微镜下。 </li></ol> 注意：避免削减和采摘MS5基质细胞。如果殖民地玫瑰花包装，也可以隔离整个殖民地。 第4步<ol><li>吸集群的花环，tritrurate 1毫升的蓝色提示，并在聚L -鸟氨酸（0.001％）和纤维连接蛋白（1微克/毫升）或层粘连蛋白（1微克/毫升）涂井板他们仔细N2 - 媒体通常辅以碱性成纤维细胞生长因子（20毫微克/毫升）。花环将改革和扩大。 </li></ol> 注意：要提高细胞的存活，可在水滴的小群，以增加细胞密度。这一步也可以修改，补充额外的增长或其他因素，以促进细胞的规范（2-5）的氮气介质。

<ol>
	<li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 101, 12543-12548 (2004).</li><li>Pruszak, J., Isacson, O., Sullivan, S., Cowan, C., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons.">Directed differentiation of human embryonic stem cells into dopaminergic neurons.</a> Human Embryonic Stem Cells: The Practical Handbook. , (2007).</li><li>Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+and+methods+for+cell+sorting+of+human+embryonic+stem+cell-derived+neural+cell+populations.">Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.</a> Stem Cells. 25, 2257-2268 (2007).</li><li>Sonntag, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yield+of+neuroepithelial+precursors+and+midbrain-like+dopaminergic+neurons+from+human+embryonic+stem+cells+using+the+bone+morphogenic+protein+antagonist+noggin.">Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin.</a> Stem Cells. 25, 411-418 (2007).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+and+pigmented+epithelia+from+primate+ES+cells+by+stromal+cell-derived+inducing+activity.">Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.</a> Proc Natl Acad Sci U S A. 99, 1580-1585 (2002).</li><li>Ko, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neural+precursors+as+a+continuous,+stable,+and+on-demand+source+for+human+dopamine+neurons.">Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.</a> J Neurochem. 103, 1417-1429 (2007).</li><li>Hong, S., Kang, U. J., Isacson, O., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+derived+from+human+embryonic+stem+cells+maintain+long-term+proliferation+without+losing+the+potential+to+differentiate+into+all+three+neural+lineages,+including+dopaminergic+neurons.">Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.</a> J Neurochem. , (2007).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+from+embryonic+stem+cells.">Neural subtype specification from embryonic stem cells.</a> Brain Pathol. 16, 132-142 (2006).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Lazzari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+neural+rosettes+from+cloned+bovine+blastocysts:+a+model+of+early+neurulation+events+and+neural+crest+specification+in+vitro.">Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.</a> Stem Cells. 24, 2514-2521 (2006).</li><li>Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., Goldstein, R. 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The authors have nothing to disclose.

此协议表明，在生成和隔离上皮细胞从人类胚胎干细胞使用SDIA的不同步骤。这种方法的应用是多方面的，在许多协议，并已用于生产特定的神经元（如1，2，5-9）。花环被认为是像神经管细胞与前表型（2，5，10），还含有神经嵴祖细胞（11，12）。此外，他们还保留一定程度的可塑性，因为它们可以在规定的培养条件下的具体因素的图案。因此，SDIA源性神经前体细胞可以引起多种细胞类型，从中央和外周神经系?...

<table><tbody><tr><td>L-Glutamine</td><td></td><td>GIBCO, by Life Technologies</td><td>25030</td><td></td></tr><tr><td>alpha-MEM</td><td></td><td>GIBCO, by Life Technologies</td><td>12571</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td></td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>Knockout-DMEM</td><td></td><td>GIBCO, by Life Technologies</td><td>10829</td><td></td></tr><tr><td>Knockout serum replacement</td><td></td><td>GIBCO, by Life Technologies</td><td>10828</td><td></td></tr><tr><td>MEM nonessential amino acid solution</td><td></td><td>GIBCO, by Life Technologies</td><td>12383</td><td></td></tr><tr><td>DMEM/F12</td><td></td><td>GIBCO, by Life Technologies</td><td>11330</td><td></td></tr><tr><td>N2-A supplement</td><td></td><td>Stem Cell Technologies</td><td>07152</td><td></td></tr><tr><td>Mitomycin-C</td><td></td><td>Sigma-Aldrich</td><td>M0503</td><td></td></tr><tr><td>Gelatine type-A</td><td></td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>poly-L-ornithine</td><td></td><td>Sigma-Aldrich</td><td>P4957</td><td>0.01% solution</td></tr><tr><td>Laminin</td><td></td><td>Sigma-Aldrich</td><td>L-2020</td><td></td></tr><tr><td>Fibronectin</td><td></td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td></td><td>Invitrogen</td><td>13256</td><td></td></tr><tr><td>1 mL Syringe with 27 1/2 G needle</td><td></td><td>BD Biosciences</td><td>309623</td><td></td></tr><tr><td>N2-A media</td><td>medium</td><td></td><td></td><td>DMEM/F12 + 1% N2-A supplement </td></tr><tr><td>Serum replacement media (SRM)</td><td>medium</td><td></td><td></td><td>Knockout-DMEM + 20% Knockout serum replacement +1% MEM nonessential amino acid solution + 2 mM L-glutamine</td></tr><tr><td>&amp;alpha;-MEM media</td><td>medium</td><td></td><td></td><td>&amp;alpha;-MEM + 10% FBS + 2 mM L-glutamine + 1% penicillin/streptomycin</td></tr><tr><td>MS5</td><td>cell line</td><td></td><td></td><td>stromal cells </td></tr><tr><td>Microscope</td><td></td><td></td><td></td><td></td></tr><tr><td>6-well Plates</td><td></td><td></td><td></td><td>for tissue culture</td></tr></tbody></table>

es cell-derived neuroepithelial cell cultures

胚胎干细胞有潜力分化成所有胚层，这使得他们的新疗法的发展有吸引力的工具细胞。在一般情况下，ES细胞的分化如下的概念，首先生成不成熟的祖细胞，然后就可以繁殖成成熟的细胞表型分化。这也适用于ES细胞源性神经元的神经细胞的发展，其中以下两个主要步骤：首先，推导和扩大，不成熟的神经上皮前体和第二，其分化为成熟的神经细胞。从ES细胞产生的神经前体细胞胚体（EB）的形成，从而揭示了所有胚层，包括神经外胚层细胞的分化是基于一个共同的方法。另一种更有效的方法诱导上皮细胞的发育，使用基质细胞衍生诱导活性（SDIA），可实现与颅骨骨骨髓来源的基质细胞（1）共培养的ES细胞。两个EB形成和SDIA，揭示发展的莲座状结构，这被认为是类似于神经管和/或神经嵴祖。可以分离出神经前体，扩大和进一步分化成特定的神经元和神经胶质细胞，使用定义的文化条件。在这里，我们描述的一代在与基质细胞MS5（2-5）共培养的实验和隔离等花环。

Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

ES Cell-derived Neuroepithelial Cell Cultures

从胚胎干细胞（ES）细胞基质细胞衍生诱导活性（SDIA）上皮前体的推导。

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

es-cell-derived-neuroepithelial-cell-cultures

Research

JoVE Journal

Biology

13.5K 次观看.  Harvard Medical School. 从胚胎干细胞（ES）细胞基质细胞衍生诱导活性（SDIA）上皮前体的推导。

视频: ES Cell-derived Neuroepithelial Cell Cultures

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

培养小鼠神经干细胞的前体

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

科研

生物学

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

发育生物学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

神经科学

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

<meta charset="utf8"></head><body>对从小鼠脑室下区分离的 NSC 进行高效成核

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>作者聚焦：改进的核转染技术，在小鼠脑室下区衍生的神经干细胞培养物中实现高效基因递送

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...