马库斯Pajtler和罗伯特Bohrer的支持和意见，对本议定书的可视化，是具有非常重要的价值。

作者状态，对动物的实验是根据欧洲共同体理事会指令（86/609/EEC），美国国立卫生研究院的机构动物所规定的实验程序和法规的动物护理和使用准则保养和使用委员会（IACUC）在杜伊斯堡 - 埃森大学（德国）。 第1部分:设置（视频所示） 准备材料和解决方案（前鸡蛋孵化器） <ul><li>鸡蛋必须在培养箱培养11天，于37℃（100℉）。 </li><li> 70％的乙醇喷雾，洁净板，精确的清洁钳，4 ° C（40 ° F）的冷无菌冰DMEM和60毫升无菌注射器</li><li> 50 ml无菌康宁管必须有4个半满° C冷DMEM培养液以1:1的比例（浸渍质量:DMEM）混合浸渍胚胎。只要鸡蛋是鸡胚的孵化器清扫已经开始。 </li><li>消毒的高压灭菌器或一天清扫的清扫工具，沉浸在70％乙醇20分钟的工具。 </li></ul>第2部分:夹层鸡胚（视频演示） <ol><li>湿至少30秒，用70％乙醇喷雾的孵化鸡蛋，干后用干净的软纸巾仔细而不是晃动鸡蛋过分。 注意:我们建议不超过60鸡胚同时解剖。 </li><li>打开鸡蛋仔细横飞对锋利的边缘，并采取了谨慎不破坏胚胎的黄麻袋或鸡胚本身。 </li><li>脐带迅速开始搜索和明确的包装不破坏蛋黄麻袋或任何一个小的船只开放。然后解剖精确的清洁钳的脐带。 </li><li>以胚胎蛋黄麻袋。 注:胚胎应迅速断头（在视频中未显示） 。 </li><li>收集胚胎在最短的重要介质，在4 ° C。 </li></ol>第3部分:浸渍鸡胚<ol><li>以60毫升无菌注射器的柱塞。 </li><li>约10胚胎转移到一个60毫升注射器。 </li><li>小心把柱塞进入注射器，为了不失去浸渍材料的风险。 </li><li>浸渍倒推柱塞。 </li><li>康宁用DMEM半满的比例1:1（浸渍群众:DMEM培养）管在准备收集浸渍质量。使用约25毫升的体积是10胚胎生产。 </li><li>在4 ° C为45分钟的混合物放置在摇床</li></ol>第4部分:离心鸡胚 - DMEM培养液暂停<ol><li>传输鸡胚 - 到离心管的DMEM悬浮。 </li><li>添加无菌透明质酸酶浓度1毫克每25毫克的胚胎。 </li><li>皮的离心管非常精确。 注意:这一步，应谨慎进行离心运行在非常高的G - Force和材料或离心机可能发生，如果使用不平衡管损坏。一个高准确度称重机按照关于三个所有管的小数点后应该用DMEM调整失踪金额收到的职位。 </li><li>离心机的温度降低到4 ° C。 注:离心转子也应存放在一个冷却室或冰箱过夜。 </li><li>离心机6个小时，至少为每小时180.000 xgx注:对于单相分离的骄人业绩，使用266.000 xgx H.如果可能的话，使用真空调整。 </li></ol>第5部分:鸡胚提取物过滤离心后，三个阶段可以注意到:上灰溜溜的脂肪片段在中间，明确液相和颗粒在离心管底部的液相注意 :摇晃或移动轻快的离心混合物严格避免，因为这将破坏，失去了明确的中央液相的离心步骤的结果。 注:以下所有步骤都必须在无菌条件下使用空气层流替补执行: <ol><li>移液器0.45微米的孔进入过滤系统，明确的液相。然后交换机上连接的真空泵。 注意:请勿触摸底部的沉淀。这将导致提取物的污染和堵塞过滤系统。 </li><li>转移到0.22微米和交换机连接真空泵再次毛孔与过滤系统的预提取。 </li><li>分装的取得鸡胚提取物在无菌15毫升康宁管。 </li><li>快速的分装保存于-80 ° C（-62 ° F）。注:中欧和东欧的最后两年，如果保存于-80 ° C。 </li></ol>

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

该协议是基于已在过去的10,11中所述的程序进行修改。除了前出版物，我们在这里展示个人更加扩大的过程中不同步骤。支持重要的细节，确保正确的程序和可靠的结果，实验者。此外，由于解剖过程是中欧和东欧提取的重要组成部分，我们描述蛋黄麻袋的胚胎中的每一个细节的过程。有几个重要的点要注意下面讨论。 解剖鸡胚孵化结束后应尽快开始。胚胎死到清扫前，不应该用于本议定书作为酶的活化和蛋白降解的任何步骤，可能会造成不利影响。 在开始之前有些手培训，应作为解剖过程的所有步骤进行快速，准确地执行。重要的是整个过程的成功，避免污染是伴随着一个值得注意的负面干扰蛋白质与蛋黄。后胚胎蛋黄麻袋，他们应该迅速断头（在视频中未显示）。 温度以及Celerity的转子是非常重要的的。为了避免酶的活化和减少NCSCM需要因素的质量和数量的离心机转子的温度应减少到4 ° C。明确中央液相后离心步骤是重要的先决提取物含有的NCSCM增长的基本因素。要达到一个相当大的不同组分的分离和富集的因素，需要我们建议Celerity的不低于180.000 xgx H. 由于之间的透明液体相，离心管顶部灰溜溜的液相密度差异并不妨碍混音两部分，要避免任何晃动。灰溜溜液相的组成部分，不能用0.45微米的过滤系统过滤掉，因为它的脂肪部分会很快导致过滤系统堵塞。离心机的底部还沉淀在不应该在所有被感动，因为它的某些部分会导致过滤系统堵塞。 在最后一步中东欧NCSCs种植的潜力已被验证。这种干细胞的多能的流动人口，而无需添加足够的生产中东欧失去​​其在媒体的可行性迅速。因此，它表达了一个基因的活性依赖性早期的非公务员合约标记和用于非公务员合约雇员的生物学研究转基因小鼠线（JoMa1）是种植在新获得的每一个中东欧10负责。培育成功的NCSCs代表中欧和东欧的一个适当的验证。

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>11 days incubated chicken eggs</td>
<td>&nbsp;</td>
<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Alcoholic disinfection spray</td>
<td>&nbsp;</td>
<td>Sch&uuml;lke &amp; Mayr GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEMGlutamax</td>
<td>&nbsp;</td>
<td>GIBCO Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe sterile (60 ml)</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tubes sterile (50; 15 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipet tips sterile</td>
<td>&nbsp;</td>
<td>Starlab GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase typeIV-S</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
<td>&nbsp;</td>
<td>Fisher Scientific AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipettes sterile (25; 10; 5 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge L5-50</td>
<td>&nbsp;</td>
<td>Rotor type Ti-45</td>
<td>&nbsp;</td>
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production of chick embryo extract for the cultivation of murine neural crest stem cells

来自神经外胚层神经嵴的胚胎发育过程中，仍然存在只是暂时的。早期的实验已经隔音多潜能的祖细胞，神经嵴1是一个不可分割的一部分。表型，神经嵴干细胞（NCSC）的定义，同时表示在他们从神经嵴迁移 2,3,4,5的P75（低仿射神经生长因子受体，LNGFR）和SOX10。这些祖细胞，能分化成平滑肌细胞，嗜铬细胞，神经元和神经胶质细胞，以及黑色素细胞，软骨和骨6,7,8,9。要培养非公务员合约雇员在体外实验中 ，一个特殊的神经嵴干细胞的培养基（NCSCM）是必需的10。 NCSCM最复杂的部分是编制鸡胚提取物（CEE）的代表的非公务员合约雇员，以及其他类型的神经植生长因子的重要来源。旁边的中欧和东欧的其他NCSCM成分均为市售。生产的CCE使用实验室标准的设备，它的高度重视是了解隔离，浸渍，离心，过滤过程中具有挑战性的细节。在这个协议中，我们描述了准确的技术，生产的纯正和高品质的中欧和东欧的最大数额。

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

要在体外培养的神经嵴干细胞（NCSC），一种特殊的介质（NCSCM）是必需的。 NCSCM的重要组成部分，是鸡胚提取物（CEE）。在这里，我们描述准确的技术，生产的纯正和高品质的中欧和东欧的最大数额，包括隔离，浸渍，离心，过滤过程的详细信息。

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

production-chick-embryo-extract-for-cultivation-murine-neural-crest

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.7K 次观看.  University Children's Hospital Essen. 要在体外培养的神经嵴干细胞（NCSC），一种特殊的介质（NCSCM）是必需的。 NCSCM的重要组成部分，是鸡胚提取物（CEE）。在这里，我们描述准确的技术，生产的纯正和高品质的中欧和东欧的最大数额，包括隔离，浸渍，离心，过滤过程的详细信息。

视频: Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

科研

神经科学

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

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.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量:从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

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 ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...