This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

1.材料和培养基的制备<ol><li>为SCZ的解剖和离解，包住大脑基质，双刃刀片，并用铝箔钳子，然后通过高压消毒他们。 </li><li>制备50毫升冷PBS缓冲液洗涤全鼠脑。 </li><li>设置一个解剖显微镜和制备用于脑（蒸压剪刀和镊子）的解剖所需的外科手术工具和SCZ（1ml注射器，地下30针，脑基质，和细镊子）的分离。 </li><li> N2培养基的制备: <ol><li>制备的F-12 / DMEM（+ L谷氨酰胺，+碳酸氢钠）有2％B27，1％N2补充和1％青霉素 - 链霉素介质。 注:生长培养基由N2培养基和生长因子（20毫微克/毫升纯化的表皮生长因子（EGF）和20毫微克/毫升碱性成纤维细胞生长因子（bFGF2））。 </li></ol></li><li>制备消化缓冲液（40单位/ ml木瓜蛋白酶，2.4单位/ ml dispas二号和2％青霉素 - 链霉素的PBS），用于组织消化。 </li><li>喷涂板和盖玻片的制备: <ol><li>制备聚-L-鸟氨酸（PLO; 0.01％）和层粘连蛋白（溶于DH 2 O 10微克/毫升）。为维护SCZ-aNSCs作为单层或用于免疫染色18毫米盖玻片外套6孔板，用PLO孵育他们过夜，在4℃。然后用DH 2洗3次O.允许板和盖玻片，最后清洗后晾干。 </li><li>接着，孵育用层粘连蛋白将板在4℃下过夜。然后，洗3次DH 2 O. 注意:不要干层粘连蛋白，这会影响细胞附着。 注:涂布溶液可重复使用3次。 </li></ol></li></ol> 2.分离和成人SCZ解离<ol><li>在此之前的文化准备，置于冰上大脑基质和双刃剃须刀。 注意:不要冷冻大脑矩阵，因为BR艾因可以附加到它。 </li><li>通过CO 2窒息或颈椎脱位牺牲一个鼠标（8周龄）。 <ol><li>喷洒70％乙醇后切断与锋利的剪刀头。以固定头部，保持头部两侧紧密。用剪刀剪开皮肤在一尾，喙方向的中线。这促进了彻底清除从头骨的皮肤。 </li><li>删除头骨（后方拉姆​​达）第一尾椎部位，然后插入颅骨和大脑之间的镊子在背中线位置。抢左（或右）顶骨，小心地把它剥开。重复此过程用于去除其他的顶骨。 注意:视神经和除去从大脑脑膜的断线使其更容易从头骨分离大脑。 </li><li>脑转移到冷的PBS缓冲液（25毫升）和冲洗两次以除去过量的血液。 </li></ol></li><li>将脑入冰和马脑矩阵科冠状切口，以获得1mm厚的片。传送包含位于大脑的冷PBS中在35毫米的塑料陪替氏培养皿后部的SCZ区域脑切片（1毫米）。 注意:为了获得冠状切片的平行平面，保证大脑裂缝被放置在大脑基质的中线;关键是要避免在部分不必要的变化。 注:在2-3毫米后到前囟获取大脑切片;这允许来自SVZ的SCZ的分离。 </li><li>以下的低倍率解剖显微镜，显微解剖从皮层和海马与弯曲30G针6,9的白质区域SCZ。然后，取出上述SCZ皮层的区域。广场切片解剖SCZ地区变成冰35毫米的塑料培养皿中无冷PBS。 注意:包含成熟神经元的额外区域的污染可能会影响aNSCs和神经球形成b的可行性ecause他们在接受ANSC培养条件下的细胞死亡。 </li><li>使用弯曲为30G针头，立即砍解剖组织切成小块。 注意:如果需要解剖SCZ组织较长时间，砍前浸泡在冷PBS解剖组织。 </li><li>重悬用1ml消化缓冲液的切碎组织，将组织转移到含2ml消化缓冲液的15ml试管中，并在37℃水浴中孵育它为30分钟。 注:摇管每隔10分钟拌匀。 </li></ol> 3. Subcallosal区衍生的成体神经干细胞培养<ol><li>挖掘管温和解离经消化的组织，然后离心管中，在145×g离心5分钟。弃去上清液，再悬浮消化用1mL预热N2培养基的组织洗出消化缓冲液，并轻轻吸取样品溶液最多使用P1000吸管5次。 注:过捣碎用窄枪头可以降低细胞活力和随后的生长。 </li><li>离心管中，在145×g离心5分钟。丢弃上清液后，再悬浮在1ml N2培养基的细胞沉淀。 </li><li>制备1毫升N2培养基的一个非涂布6孔板并加入1毫升悬浮的细胞，以使2毫升的最终体积。 </li><li>添加EGF（20毫微克/毫升）和bFGF（20毫微克/毫升）到每个孔中。用手轻轻摇动6孔培养皿与铺板的细胞以混合添加的生长因子。保持6孔板在37℃和5％CO 2培养箱。 </li><li>每天补充EGF（20毫微克/毫升）和bFGF（20毫微克/毫升），以每孔8天。每第三天，添加200μl的N2培养基的维持培养基的近似的2ml体积。 </li></ol> 4.神经干细胞传代神经球和单层培养<ol><li>收集神经球，并将它们转移到一个新的15 mL锥形管。 注:神经球（直径&gt; 50微米）的数量PE- [R以及从一个单一的小鼠大脑的SCZ为64.3±7.31，这是小于SVZ的（190.5±6.33）9。 </li><li>孵化用0.5ml解离缓冲液的5分钟的神经球在37℃水浴中向神经球解离成单细胞。初级神经球可以解离成单细胞，并保持在几个传代为神经球或单层培养物。 注:扩展SCZ-aNSCs作为单层优于神经球因为SCZ-aNSCs可以在神经球培养格式进行传代以单层培养格式&gt; 10倍，但&lt;5次。 注意:SCZ-aNSCs显示强聚集，并且很难将其解离成单细胞。因此，不推荐常规细胞扩增神经球文化的格式。 </li><li>轻轻吸取样品溶液上下P1000的吸移管低于5倍和离心管中，在145×g离心5分钟。弃上清，重新苏花神经球用1毫升N2介质。 注:在一个狭窄的枪头捣碎会降低细胞活力和随后的生长。 </li><li>计数细胞，使1:细胞悬液（10微升）和0.4％台盼蓝溶液的1:1混合物，然后计数活细胞上血细胞计数的数目。用PLO /层粘连蛋白涂覆6孔板之后，板上的细胞在用2ml每个N2培养基的孔2.5×10 5个细胞/ ml。 注意:在细胞密度的变化可以影响其条件和分化潜能。 </li><li>保持与每日治疗的生长因子的SCZ-aNSCs（2毫升，20纳克/毫升）5天，然后将它们通过它们。 </li></ol> 5.分化Subcallosal区衍生的成体神经干细胞<ol><li>板aNSCs到PLO /层粘连蛋白包被的18毫米盖玻片用1×10 5个细胞/ ml的1ml N 2用生长因子（20毫微克/毫升）的SCZ-aNSCs的分化。 </li><li>下一天，当细胞被牢固地附着在盖玻片，用N 2交换生长培养基以除去生长因子。 </li><li> 6天之后，洗分化的细胞用1毫升的PBS，除去细胞碎片并修复它们的免疫染色。 注意:BrdU的可掺入增殖细胞的新合成的DNA。因此，如果需要的话，的BrdU（10微克/毫升）可加入细胞固定前活细胞。 </li></ol> 6.免疫染色成体神经干细胞和分化祖<ol><li>对于免疫染色，洗涤细胞，用PBS和用4％PFA固定它们在室温下20分钟。 注意:PFA有剧毒;避免与皮肤和眼睛接触。 </li><li>去除4％PFA，漂洗固定的细胞用PBS 3次，然后将其存储在4℃Cuntil它们所需的免疫染色。 </li><li>孵育盖玻片细胞用封闭溶液（3％牛血清白蛋白和0.1％的Triton X-100在1×PBS），用于在室温下至少30分钟。 </li><li>制备新鲜封闭溶液的第一抗体，并在4℃下过夜孵育样品。 <ol><li>免疫染色aNSCs <ol><li>染色使用抗巢蛋白和抗BrdU抗体aNSCs。固定前，添加BrdU的20微克到aNSCs孵育他们2小时。执行用2N HCl进行20分钟，在37℃Cbefore进行封闭步骤的变性步骤。 </li></ol></li><li>免疫染色的祖细胞分化: <ol><li>使用抗O4，抗BIII微管蛋白和抗胶质细胞原纤维酸性蛋白（GFAP）的抗体来标记分化祖细胞。 </li></ol></li></ol></li><li>用PBS洗涤3次试样并用缀合至荧光染料（1:500）的第二抗体孵育它们在室温下30分钟封闭溶液。然后，用PBS洗涤样品3次。 注:匹配主的那台主机使用的二抗抗体。 Hoechest33343（1:2,000）用于核染色。 </li><li>安装溶液添加到载玻片，并与安装继续。在多个波长观察和图像上的共焦显微镜的样品:FITC（488纳米），Cy3标记（543纳米），Cy5的（647纳米），和Hoechest33343（405纳米）。 </li></ol>

<ol><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+and+population+analyses+demonstrate+that+an+EGF-responsive+mammalian+embryonic+CNS+precursor+is+a+stem+cell%5BTitle%5D)+AND+%22Developmental+biology%22%5BJournal%5D)">Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.</a> Developmental biology. 175, 1-3 (1996).</li>
<li>Babu, H., Cheung, G., Kettenmann, H., Palmer, T. D., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enriched+monolayer+precursor+cell+cultures+from+micro-dissected+adult+mouse+dentate+gyrus+yield+functional+granule+cell-like+neurons%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Enriched monolayer precursor cell cultures from micro-dissected adult mouse dentate gyrus yield functional granule cell-like neurons.</a> PloS one. 2, e388(2007).</li>
<li>Alvarez-Buylla, A., Garcia-Verdugo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenesis+in+adult+subventricular+zone%5BTitle%5D)+AND+%22The+Journal+of+neuroscience+%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Neurogenesis in adult subventricular zone.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 629-634 (2002).</li>
<li>Kempermann, G., Song, H., Gage, F. H. Neurogenesis in the Adult Hippocampus. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKempermann%2C+G+%22Cold+Spring+Harbor+perspectives+in+biology%22">Cold Spring Harbor perspectives in biology</a>. 7, a018812(2015).</li>
<li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+neural+stem+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Mammalian neural stem cells.</a> Science. 287, 1433-1438 (2000).</li>
<li>Seri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Composition+and+organization+of+the+SCZ%3A+a+large+germinal+layer+containing+neural+stem+cells+in+the+adult+mammalian+brain%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Composition and organization of the SCZ: a large germinal layer containing neural stem cells in the adult mammalian brain.</a> Cereb Cortex. 16 Suppl 1, i103-i111 (2006).</li>
<li>Kim, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+the+spontaneous+production+but+massive+programmed+cell+death+of+new+neurons+in+the+subcallosal+zone+of+the+postnatal+mouse+brain%5BTitle%5D)+AND+%22The+European+journal+of+neuroscience%22%5BJournal%5D)">Evidence for the spontaneous production but massive programmed cell death of new neurons in the subcallosal zone of the postnatal mouse brain.</a> The European journal of neuroscience. 33, 599-611 (2011).</li>
<li>Kim, H. J., Kim, J. Y., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-dependent+changes+in+the+subcallosal+zone+neurogenesis+of+mice%5BTitle%5D)+AND+%22Neurochemistry+international%22%5BJournal%5D)">Age-dependent changes in the subcallosal zone neurogenesis of mice.</a> Neurochemistry international. 61, 879-884 (2012).</li>
<li>Kim, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promotion+of+Cortical+Neurogenesis+from+the+Neural+Stem+Cells+in+the+Adult+Mouse+Subcallosal+Zone%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Promotion of Cortical Neurogenesis from the Neural Stem Cells in the Adult Mouse Subcallosal Zone.</a> Stem Cells. , (2015).</li>
<li>Nunes, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+isolation+of+multipotential+neural+progenitor+cells+from+the+subcortical+white+matter+of+the+adult+human+brain%5BTitle%5D)+AND+%22Nature+medicine%22%5BJournal%5D)">Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain.</a> Nature medicine. 9, 439-447 (2003).</li>
<li>Gould, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+widespread+is+adult+neurogenesis+in+mammals%3F%5BTitle%5D)+AND+%22Nature+reviews.+Neuroscience%22%5BJournal%5D)">How widespread is adult neurogenesis in mammals?</a> Nature reviews. Neuroscience. 8, 481-488 (2007).</li>
<li>Gage, F. H., Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+stem+cells%3A+generating+and+regenerating+the+brain%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neural stem cells: generating and regenerating the brain.</a> Neuron. 80, 588-601 (2013).</li>
<li>Shaker, M. R., Kim, J. Y., Kim, H., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+secondary+neural+tube-derived+embryonic+neural+stem+cells+in+vitro%5BTitle%5D)+AND+%22Stem+cells+and+development%22%5BJournal%5D)">Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.</a> Stem cells and development. 24, 1171-1181 (2015).</li>
<li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strengths+and+limitations+of+the+neurosphere+culture+system%5BTitle%5D)+AND+%22Molecular+neurobiology%22%5BJournal%5D)">Strengths and limitations of the neurosphere culture system.</a> Molecular neurobiology. 34, 153-161 (2006).</li>
<li>Suslov, O. N., Kukekov, V. G., Ignatova, T. N., Steindler, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+stem+cell+heterogeneity+demonstrated+by+molecular+phenotyping+of+clonal+neurospheres%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14506-14511 (2002).</li>
<li>Richards, L. J., Kilpatrick, T. J., Bartlett, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+generation+of+neuronal+cells+from+the+adult+mouse+brain%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">De novo generation of neuronal cells from the adult mouse brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 89, 8591-8595 (1992).</li>
<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li>
<li>Gritti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+and+fibroblast+growth+factors+behave+as+mitogenic+regulators+for+a+single+multipotent+stem+cell-like+population+from+the+subventricular+region+of+the+adult+mouse+forebrain%5BTitle%5D)+AND+%22The+Journal+of+neuroscience+%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 19, 3287-3297 (1999).</li>
<li>Wachs, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+efficacy+of+clonal+growth+and+expansion+of+adult+neural+stem+cells%5BTitle%5D)+AND+%22Laboratory+investigation%3B+a+journal+of+technical+methods+and+pathology%22%5BJournal%5D)">High efficacy of clonal growth and expansion of adult neural stem cells.</a> Laboratory investigation; a journal of technical methods and pathology. 83, 949-962 (2003).</li>
<li>Xiong, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+time+for+passaging+neurospheres+based+on+primary+neural+stem+cell+cultures%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Optimal time for passaging neurospheres based on primary neural stem cell cultures.</a> Cytotechnology. 63, 621-631 (2011).</li>
<li>Lee, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+Matrigel-based+culture+for+expansion+of+neural+stem+cells%5BTitle%5D)+AND+%22Animal+Cells+and+Systems%22%5BJournal%5D)">Optimization of Matrigel-based culture for expansion of neural stem cells.</a> Animal Cells and Systems. 19, 175-180 (2015).</li>
<li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse%5BTitle%5D)+AND+%22Nature+protocols%22%5BJournal%5D)">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nature protocols. 7, 2005-2012 (2012).</li>
<li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One+mouse%2C+two+cultures%3A+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice%5BTitle%5D)+AND+%22JoVE+%28Journal+of+Visualized+Experiments%29%22%5BJournal%5D)">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> JoVE (Journal of Visualized Experiments). , e51225(2014).</li>
</ol>

The authors declare no conflicts of interest.

本文介绍了详细的方案，从成年小鼠SCZ产生神经干细胞，并保持他们的各种应用。有建立净化和扩大SCZ神经干细胞所需要的体外培养体系三个关键步骤。首先，重要的是要确保该SCZ区域被精确从其他潜在神经原性区域（ 图1B）解剖出。用1毫米间隔脑矩阵获得含有SCZ区域厚和精确的部分，然后被用于从其他皮层区域（ 图1A）的SCZ的显微解剖细针。当非神经干细胞从?...

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table><tbody><tr><td>&lt;strong&gt;Medium components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Gibco</td><td>11320-033</td><td>+L-glutamin, +Sodium bicarbonate</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>&lt;strong&gt;Growth factor&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>EGF</td><td>Gibco</td><td>PHG0313</td><td></td></tr><tr><td>&lt;strong&gt;Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS (10x)</td><td>BIOSOLUTION</td><td>BP007a</td><td>1x dilution</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>&lt;strong&gt;Dissociattion buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>04-942-078-001</td><td></td></tr><tr><td>Papain</td><td>Worthington</td><td>3126</td><td></td></tr><tr><td>Accutase</td><td>ICT</td><td>AT-104</td><td></td></tr><tr><td>&lt;strong&gt;Tool&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fine forceps</td><td>WPI</td><td>555229F</td><td></td></tr><tr><td>Scissors</td><td>Storz</td><td>E3321-C</td><td></td></tr><tr><td>Brain matrix (1 mm)</td><td>RWD</td><td>68707</td><td></td></tr><tr><td>Double-edged razor</td><td>DORCO</td><td>ST-300</td><td></td></tr><tr><td>30 G needle</td><td>SUNGSHIM</td><td>N1300</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 ml tubes</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>50 ml tubes</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>35 mm dish</td><td>SPL</td><td>10035</td><td>Petri dish</td></tr><tr><td>100 mm dish</td><td>SPL</td><td>10090</td><td>Petri dish</td></tr><tr><td>Cover slip (18 mm)</td><td>Deckglaser</td><td>111580</td><td></td></tr><tr><td>12 well dish</td><td>SPL</td><td>32012</td><td>non-coating</td></tr><tr><td>6 well dish</td><td>SPL</td><td>32006</td><td>non-coating</td></tr><tr><td>&lt;strong&gt;Coating materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PLO</td><td>Sigma</td><td>P4957</td><td>0.01%</td></tr><tr><td>Laminin</td><td>Gibco</td><td>23017-015</td><td>10 mg/ml</td></tr><tr><td>&lt;strong&gt;Primary antibodies&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nestin</td><td>Millipore</td><td>MAB353</td><td>mouse (1:1,000)</td></tr><tr><td>EGFR</td><td>Abcam</td><td>ab2430</td><td>rabbit (1:1,000)</td></tr><tr><td>DCX</td><td>Santa Cruz</td><td>SC8066</td><td>goat (1:500)</td></tr><tr><td>Tuj1</td><td>Sigma</td><td>T2200</td><td>rabbit (1:2,000)</td></tr><tr><td>GFAP</td><td>Invitrogen</td><td>13-0300</td><td>rat (1:1,000)</td></tr><tr><td>O4</td><td>Millipore</td><td>MAB345</td><td>mouse (1:500)</td></tr><tr><td>BrdU</td><td>Abcam</td><td>ab6326</td><td>Rat (1:500)</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse 488</td><td>Invitrogen</td><td>A21202</td><td>1:500</td></tr><tr><td>anti-mouse cy3</td><td>Jackson</td><td>715-165-151</td><td></td></tr><tr><td>anti-mouse 647</td><td>Jackson</td><td>715-606-150</td><td></td></tr><tr><td>anti-rabbit 488</td><td>Alexa</td><td>A21206</td><td></td></tr><tr><td>anti-rabbit cy3</td><td>Jackson</td><td>711-165-152</td><td></td></tr><tr><td>anti-rabbit 647</td><td>Jackson</td><td>711-605-152</td><td></td></tr><tr><td>anti-goat 488</td><td>Alexa</td><td>A11055</td><td></td></tr><tr><td>anti-goat cy3</td><td>Jackson</td><td>705-165-147</td><td></td></tr><tr><td>anti-goat 647</td><td>Invitrogen</td><td>A21447</td><td></td></tr><tr><td>anti-rat 488</td><td>Invitrogen</td><td>A21208</td><td></td></tr><tr><td>anti-rat cy3</td><td>Jackson</td><td>712-166-150</td><td></td></tr><tr><td>anti-rat 647</td><td>Jackson</td><td>712-605-153</td><td></td></tr><tr><td>&lt;strong&gt;Immunostaining materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA</td><td>Millipore</td><td>82-100-6</td><td>3% BSA with 0.1% Triton X-100 in PBS</td></tr><tr><td>Triton X-100</td><td>usb</td><td>22686</td><td></td></tr><tr><td>4% PFA</td><td>Biosesang</td><td>P2031</td><td></td></tr><tr><td>Hoechest33342</td><td>Life Technology</td><td>H3570</td><td>Dye for staining nuclei</td></tr></tbody></table>

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.

从未知的神经区域定义aNSCs培养体系是了解这些细胞，并用于开发大脑修复12其潜在的使用至关重要。众所周知，神经干细胞在不同发育阶段，或在不同的区域表现不同3,4。最近，有人报告说，相对于SVZ来源的细胞7-9 SCZ来源的细胞显示出在体内和体外对神经元分化差的电位。因此，能够精确地隔离每个神经区，脑切片包括该SCZ?...

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

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

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 ...

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Research

JoVE Journal

Neuroscience

15.1K 次观看.  Korea University College of Medicine. 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. 

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

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.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

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 ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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&#8208;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.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

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 ...

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 ...

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 ...

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 ...

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 ...

<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 ...

分离和成人神经干细胞的培养从鼠标Subcallosal区

摘要

探索更多视频

成年鼠神经干细胞的分离和扩大使用神经干含量

建立小鼠胚胎神经干细胞培养神经球含量

从人类毛囊神经嵴干细胞的分离和培养的

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

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

胚胎小鼠神经干细胞的分离与培养

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

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

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