这项工作是支持的NIH授予R01AR054593，和R01AR054593-S1徐。

1。组织培养板的制备<ol><li>外套的各孔具有足够的聚-D-赖氨酸（PDL），以覆盖的井的底部。允许板在通风橱中干燥。 </li><li>井干后，用无菌水冲洗，并吸。允许板在通风橱中干燥。 </li><li>当干燥时，纤连蛋白涂层（BioWhittaker公司水溶解于在37℃下过夜，在6毫升的浓度为1毫克）。 </li><li>添加非公务员合约介质[95毫升DMEM/F12，1毫升佩恩/链霉素（P / S），1毫升N2，B272毫升，100μl的巯基乙醇（2ME; 50mM的股票），碱性成纤维细胞生长因子（20 ng / ml的培养基），胰岛素样生长因子-1（20纳克/毫升培养基）和EGF（20 ng / ml的培养基中）]之前，纤连蛋白的板在干</li></ol> 2。提取人的头皮上的毛囊细胞<ol><li>在启动的过程中分离的非公务员合约之前，需要准备各自的媒体和试剂（ 见表1）。 </li><li>新鲜成人头皮换装程序或fe河谷头皮组织，收集含有青霉素 - 链霉素的PBS洗涤。 </li><li>皮肤，然后转移到50ml试管，并与中性蛋白酶（10毫克/毫升）过夜培养在DMEM中，在4℃下在37°C培养2-4小时也有效。皮片应该是一个最大宽度为1厘米，以使酶渗透。 </li><li>皮肤进入一个消毒的培养皿中转移，拉每根毛发从皮肤上抓头发轴靠近皮肤表面，紧紧拉住顺利。毛囊显示形态的任一生长期（ 图1A）或休止期（ 图1B）。 </li><li>孤立卵泡片段孵育15-20分钟，在室温下振荡定期在0.05％胰蛋白酶-EDTA（Invitrogen）中，添加有10％FBS的DMEM4毫升停止反应。滤泡上皮细胞被胰蛋白酶消化，并通过40微米的过滤器过滤，以获得含有不同大小和形状的细胞的单细胞悬液。 </li><li>纺在200×g离心5分钟，弃去上清液小心，重新悬浮于1ml PBS含有2％血清（FBS）。 </li><li>另外，弹拨的毛囊可以放置在文化，没有胰蛋白酶消化长出的头发在原地领域。 </li></ol> 3。使用流式细胞仪检测细胞分选分离毛囊非公务员合约员工<ol><li>毛囊的单细胞悬浮液的胰蛋白酶消化，并通过以下方式获得标记的抗CD271（APC共轭）号/ HNK1（FITC标记的）或CD271 / alpha4整合（PE共轭的）在黑暗中在冰上40分钟。 </li><li>在室温下在200×g离心5分钟离心，吸出上清液。 </li><li>在PBS中含有2％血清（FBS），重悬细胞之前，排序，PI被添加到栅极，满分的死细胞。 </li><li>用流式细胞仪（FACS）进行细胞分选。收集CD271 +，HNK1 +双阳性细胞或CD271 +，alpha4整合+双阳性细胞（ 图2）。 </li><li>排序后ING，CD271 +，HNK1 +双阳性细胞或CD271 +，alpha4整合+双阳性细胞培养在超低的附件板的非公务员合约雇员的介质中</li></ol> 4。主要非公务员合约员工文化<ol><li>文化脱离滤泡细胞或FACS排序的细胞在超低的附件板中的非公务员合约雇员的介质，改变介质的每一天。 </li><li>每天在显微镜下检查细胞培养。非公务员合约员工将开始形成浮动小团聚几天后，和良好的领域，在2-5周的文化，根据年龄的捐助者（ 图3A）。副产物会出现在几天内在隆起区域时，整个毛囊的非公务员合约雇员的介质中培养良好的领域在几个星期内原位 （ 图3B）。 </li></ol> 5。扩展的非公务员合约员工<ol><li>用PBS洗涤细胞一次。 </li><li>加入预温（37°C，关键）accutase和孵化，在37°C 5-10分钟周期性振荡，使确定的神经干细胞球，解离成单细胞（附着培养非公务员合约，孵育与accutase细胞在37℃下5分钟，以使确保毛囊干细胞的分离）。 </li><li>洗净离心细胞两次在DMEM/F12培养基在室温下在200×g离心5分钟以除去任何剩余accutase溶液。 </li><li>与非公务员合约雇员的培养基重新悬浮细胞。 </li><li>对于浮动文化，​​将细胞在超低的附件板。对于贴壁培养，将细胞预处理细胞培养板。 </li></ol> 6。代表性的成果非公务员合约培养基是足以维持在未分化的状态，而不需要为饲养细胞hNCSCs。角质形成细胞不能增殖，并在介质中逐渐死亡。某些小圆形细胞增生，形成小聚集在暂停后3〜5天。这些漂浮聚集慢慢增加我n大小，生成的三维球体状的结构，我们称之为毛球（ 图3A）。如果涂层板中培养，非公务员合约员工将附着在表面，不形成任何领域。附加的非公务员合约员工成长的速度比悬浮液中。球体形成或连接的干细胞表达非公务员合约标记。当整个毛囊文化，球体形成的凸起区域对应的区域（ 图3B）。 <img alt="图1" src="/files/ftp_upload/3194/3194fig1.jpg" /> 图1。弹拨生长期（A）和休止期（B）毛囊。 <img alt="图2" src="/files/ftp_upload/3194/3194fig2.jpg" /> 图2。代表性的图像流式细胞仪分析的非公务员合约员工。左图：门控使用的抗CD271和抗alphe的的整合素细胞。右面板：使用反CD271和抗HNK1的抗体，细胞门控。 <img alt="图3" src="/files/ftp_upload/3194/3194fig3.jpg" /> 图3。形态的头发球。（B）（A）形态的，浮毛球，左侧面板：低功耗，右图：高功率。在隆起区域，左侧面板：在休止期的头发球凸出的毛球原位形态，右侧面板：在隆起区域的生长期毛囊的头发球。

<ol>
	<li>Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Existence+of+slow-cycling+limbal+epithelial+basal+cells+that+can+be+preferentially+stimulated+to+proliferate:+implications+on+epithelial+stem+cells.">Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.</a> Cell. 57, 201-209 (1989).</li><li>Cotsarelis, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-retaining+cells+reside+in+the+bulge+area+of+pilosebaceous+unit:+implications+for+follicular+stem+cells,+hair+cycle,+and+skin+carcinogenesis.">Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.</a> Cell. 61, 1329-1337 (1990).</li><li>Tanimura, S., 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=Hair+follicle+stem+cells+provide+a+functional+niche+for+melanocyte+stem+cells.">Hair follicle stem cells provide a functional niche for melanocyte stem cells.</a> Cell Stem Cell. 8, 177-187 (2011).</li><li>Yu, 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=Isolation+of+a+novel+population+of+multipotent+adult+stem+cells+from+human+hair+follicles.">Isolation of a novel population of multipotent adult stem cells from human hair follicles.</a> Am. J. Pathol. 168, 1879-1888 (2006).</li><li>Yu, H., Kumar, S. M., Kossenkov, A. V., Showe, L., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J. Invest. Dermatol. 130, 1227-1236 (2010).</li><li>Wong, C. E., 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=Neural+crest-derived+cells+with+stem+cell+features+can+be+traced+back+to+multiple+lineages+in+the+adult+skin.">Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.</a> J. Cell Biol. 175, 1005-1015 (2006).</li><li>Sieber-Blum, M., Grim, M., Hu, Y. F., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Amoh, 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=Implanted+hair+follicle+stem+cells+form+Schwann+cells+that+support+repair+of+severed+peripheral+nerves.">Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17734-17738 (2005).</li><li>Koliatsos, V. E., Xu, L., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell+grafts+as+therapies+for+motor+neuron+disease.">Human stem cell grafts as therapies for motor neuron disease.</a> Expert Opin. Biol. Ther. 8, 137-141 (2008).</li><li>McKenzie, I. A., Biernaskie, J., Toma, J. G., Midha, R., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+precursors+generate+myelinating+Schwann+cells+for+the+injured+and+dysmyelinated+nervous+system.">Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system.</a> J. Neurosci. 26, 6651-6660 (2006).</li><li>LeDouarin, N. M., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neural Crest. , (1999).</li><li>Tumbar, 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=Defining+the+epithelial+stem+cell+niche+in+skin.">Defining the epithelial stem cell niche in skin.</a> Science. 303, 359-363 (2004).</li><li>Roh, 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=Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles.">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In Vitro Cell Dev. Biol. Anim. 44, 236-244 (2008).</li></ol>

我们什么都没有透露。

细胞的分离和培养方法的重现性和强大的。我们已经生成的非公务员合约员工从几十个人在广泛的年龄范围。虽然这是最好的，处理组织组织收获后，我们发现头皮组织可以安全地存储在媒体上冰过夜运输对细胞活力的影响最小。 重要的是要与抗生素治疗头皮组织和毛囊分离过程中使用无菌技术，以避免潜在的微生物污染。被丢弃的小改款皮肤或胎儿头皮组织的收益率，数?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330-32</td>
 </tr>
 <tr>
 <td>Heat-inactivated FBS</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 </tr>
 <tr>
 <td>B27 supplement</td>
 <td>Invitrogen</td>
 <td>17504044</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Invitrogen</td>
 <td>17502048</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Invitrogen</td>
 <td>PHG0026</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>R&amp;D system</td>
 <td>236-EG-01M</td>
 </tr>
 <tr>
 <td>IGF-I</td>
 <td>R&amp;D system</td>
 <td>291-G1-050</td>
 </tr>
 <tr>
 <td>0.05% Trypsin/EDTA</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>Invitrogen</td>
 <td>17105041</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15070063</td>
 </tr>
 </tbody> 
</table>
Table 1.

isolation and culture of neural crest stem cells from human hair follicles

毛囊进行终身的增长，头发的周期是一个控制的过程，涉及干细胞的增殖和平静。头发隆起是一个很好的成体干细胞的特征利基。这包含了许多不同类型的干细胞，包括上皮干细胞，黑素细胞的干细胞3和神经嵴类似干细胞4-7段的外根鞘。毛囊代表一个可访问的和不同类型的人类干细胞的丰富来源。我们和其他人隔离的神经嵴干细胞（非公务员合约员工）的成人和胎儿毛囊4,5。这些人类干细胞标记的滞留细胞，通过体外细胞不对称分裂，并能够自我更新。他们表示，未成熟的神经嵴细胞的标记物，但不分化标志物。我们的表达谱的研究表明，他们有着相似的基因表达模式与小鼠皮肤未成熟的神经创建ST细胞。它们表现出的克隆的多能在体外克隆单细胞培养后，可以给引起生肌，黑色素细胞和神经元的细胞谱系。分化的细胞，不仅获得了种系特异性标志物，而且在体外条件下表现出适当的功能。此外，这些非公务员合约员工向间充质谱系分化潜能。分化的神经元细胞能坚持在小鼠大脑和保留神经元的分化。它已经表明，毛囊派生的非公务员合约员工可以帮助神经再生，改善运动功能，这些干细胞在小鼠体内移植后，横断性脊髓损伤8。此外，外周神经干细胞移植和植入的皮肤源性坐骨神经神经前体细胞相邻修复，导致髓鞘10。因此，毛囊/皮肤派生的非公务员合约员工已经显示出可喜的结果为r临床前模型egenerative治疗。 体细胞重编程诱导多能干细胞（iPS细胞）已经显示出巨大的潜力，再生医学。不过，也有iPS细胞，特别是基因/病毒整合和潜在的致瘤性多能干细胞的长期影响仍有许多问题没有得到充分解决。还有许多难以克服的障碍前iPS细胞可用于再生医学。鉴于成体干细胞是已知的，是安全的，它们已被用于临床多年，如骨髓移植。很多病人已经从治疗中获益。自体成人干细胞仍然是优选的用于移植细胞。因此，在人的皮肤/头发毛囊容易获得的，可扩展的成体干细胞是再生医学的重要来源。

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles at JoVE.com

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

本文提出了一种强大的协议从人的头发毛囊的神经嵴干细胞的分离和培养。

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 ...

isolation-culture-neural-crest-stem-cells-from-human-hair

Research

JoVE Journal

Neuroscience

16.8K 次观看.  School of Medicine, University of Pennsylvania. 本文提出了一种强大的协议从人的头发毛囊的神经嵴干细胞的分离和培养。

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

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

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

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 ...

科研

神经科学

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 ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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 ...

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 ...

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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 ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...