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>

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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。当非神经干细胞从邻近组织，如大脑皮层，进行培养以SCZ-aNSCs，发生灾难性死亡，其可行性和SCZ-的NSCs 16-18,22的球体形成产生不利影响。尾鳍SCZ（2-3毫米后到前囟门）被确认为产生不同的SCZ-aNSCs的最佳区域。其次，approp在收获和传代步骤riate酶处理是用于实现细胞的高产率的关键。分散酶II和木瓜蛋白酶为用于分离aNSCs比胰蛋白酶更有效。对于解离缓冲液代替胰蛋白酶传代细胞的分离增强自己的生存能力19。机械解离和研磨用吸管应该很小。第三，细胞过滤网在原代培养系统一般用于组织消化后，除去细胞碎片。然而，由于SCZ-aNSCs的定位，是白质酶消化和机械研磨断裂后获得这些细胞。期间用细胞过滤网过滤，将细胞的大量将丢失。因此，在不使用细胞过滤培养SCZ-aNSCs是一种更好的方式来获得细胞的高产。 同时兼具神经球文化和单层培养可应用于维持神经干细胞时，神经的一个限制球体文化是单一的神经干细胞分离后拆分可以随机聚合。聚合结果在不同尺寸的神经球。当神经球的大小达到一定的临界值时，神经球生长作为多相结构，由于缺乏营养素，生长因子，和氧气在芯20上 。此外，大尺寸的神经球不容易与解离缓冲液解离，并需要有广泛的机械研磨较长酶处理时间，从而导致较低的细胞生存力。因此，单层培养系统，建议通过多个通道保持SCZ-aNSCs。在单层培养系统，aNSCs被稳定地保持为神经干细胞（B型）无自发分化成特定细胞如祖细胞（类型C和A）（ 图3A）。 SCZ-aNSCs传代长时间，&lt;5代的神经干格式&gt; 10代作为单层。这是利弊istent与先前的结果，这表明，单层培养系统保持的NSCs 在长期培养21 体外 。然而，增殖速度降低，和死亡的细胞的部分增加了5代。扩展传代会影响多能，并增加染色体畸变22神经元分化。因此，为了避免扩展的传代效果，建议使用早期传代（&lt;通道5）细胞增殖和分化的分析。虽然SCZ-aNSCs的自我更新潜力是类似于SVZ-aNSCs，较少的神经元分化示于SCZ-aNSCs 9。 用于从成人脑的神经区域，包括SVZ和齿状回（DG）分离aNSCs方法，已经建立了22。虽然这样的协议，促进了分离和体外aNSCs的培养，有几个限制到获得高数细胞。许多协议利用脑组织斩波斩波过程期间可能引起的脑组织的损失。从神经源性地区隔离aNSCs另一种方法使用手术刀采用冠状切口穿过大脑。这之后是或者DG沿着纵向裂缝23的SVZ的显微解剖。其他脑区的存在可引起其它细胞类型而污染ANSC培养，这可能会影响在体外细胞的活力。与目前的协议，许多不同的样品可以在一次实验中进行管理，包括控制与各种实验组。此外，使用脑基质切片优于脑斩波工具，因为它允许NSCs的来自不同脑区具有高细胞产量的实现。它也使从同一脑的不同区域aNSCs的比较。 内源性神经干细胞移植和工程一直considereð为干细胞疗法可能的策略。对于这一点， 在体外约aNSCs的特性的研究也应全面的探讨。因此，建立一个良好的特点体外培养体系将成为促进神经干细胞的应用很有帮助。在这种培养体系，SCZ-aNSCs的干细胞性质维护良好，通过在适当条件下的自我更新和多谱系分化为证。因此，该培养系统可以用于SCZ-aNSCs的扩大为生物学研究和治疗应用。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</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>Growth factor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</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>Dissociattion buffer</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>Tool</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 (1mm)</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 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</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>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</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>Coating materials</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>Primary antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>Abcam</td>
			<td>ab2430</td>
			<td>rabbit (1:1000)</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 (1:2000)</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>rat (1:1000)</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>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td rowspan="12">1:500</td>
		</tr>
		<tr>
			<td>anti-mouse cy3</td>
			<td>Jackson</td>
			<td>715-165-151</td>
		</tr>
		<tr>
			<td>anti-mouse 647</td>
			<td>Jackson</td>
			<td>715-606-150</td>
		</tr>
		<tr>
			<td>anti-rabbit 488</td>
			<td>Alexa</td>
			<td>A21206</td>
		</tr>
		<tr>
			<td>anti-rabbit cy3</td>
			<td>Jackson</td>
			<td>711-165-152</td>
		</tr>
		<tr>
			<td>anti-rabbit 647</td>
			<td>Jackson</td>
			<td>711-605-152</td>
		</tr>
		<tr>
			<td>anti-goat 488</td>
			<td>Alexa</td>
			<td>A11055</td>
		</tr>
		<tr>
			<td>anti-goat cy3</td>
			<td>Jackson</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>anti-goat 647</td>
			<td>Invitrogen</td>
			<td>A21447</td>
		</tr>
		<tr>
			<td>anti-rat 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
		</tr>
		<tr>
			<td>anti-rat cy3</td>
			<td>Jackson</td>
			<td>712-166-150</td>
		</tr>
		<tr>
			<td>anti-rat 647</td>
			<td>Jackson</td>
			<td>712-605-153</td>
		</tr>
		<tr>
			<td>Immunostaining materials</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使用1mm的脑基质（ 图1A）进行解剖。后用生长因子培养8天，从SCZ衍生aNSCs能够形成神经球，其中细胞可随后保持（ 图1B）。 由于神经干细胞中的神经球的子集可以是SPOntaneously分化13，一个单层培养系统也对维持SCZ-aNSCs的相对同质种群有帮助的。生长因子和分解酶，胰蛋白酶不要轻易渗透到神经球14,15的内心深处。单层培养为神经干细胞的扩张更均匀的条件。从初级神经球，SCZ-aNSCs被消化缓冲处理分离成单个细胞。细胞接种后一天，aNSCs被附着到涂覆的板并表现出细胞增殖（ 图2A）。为了证实它们的增殖的效力，BrdU的加入到介质中。用BrdU温育2小时后，将细胞很容易地用抗BrdU的（为增殖标记物）和抗巢蛋白抗体（用于神经干细胞的标记物）染色，表明SCZ-aNSCs正在积极增殖和维持的关键属性干细胞（ 图2B）。因此，他们没有EXHIBIT为分化的细胞，如表皮生长因子受体（在瞬时放大细胞中表达）和DCX（在神经母细胞表达）（ 图2C）的标志物。 以确认SCZ-aNSCs的多向分化潜能，生长因子是从培养基中移除。 6天后，将细胞用各种制造商为分化的细胞进行免疫染色。为了表现出aNSCs的神经元不同的后代，标记（TUJ1），星形胶质细胞（GFAP）和少突胶质（O4）被雇用;所有这些细胞类型是从SCZ-aNSCs产生（ 图3）。 <img alt="图1" src="/files/ftp_upload/54929/54929fig1.jpg" /> 图 1: 神经球的SCZ地区形成的分离。 （A）程序为SCZ地区从成年小鼠大脑解剖。为了培养SCZ-AN种姓，8周龄小鼠大脑被放置到大脑基质（1mm的间隔）。切片1毫米脑切片，其中包括的SCZ区域（距离前囟2-3毫米）进行解剖（由红色虚线表示）之后。 （ 二 ）神经球的形成。 在体外培养，初级神经球（通道0）后八天，形成并传代。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54929/54929fig2.jpg" /> 图2:SCZ-aNSCs维护作为单层培养。 （A）中播种解剖细胞一天后，SCZ-aNSCs被连接并扩展到一个涂覆培养皿以单层方式（左）。维修后三天，SCZ-aNSCs的数量增加（右）。 （B）用BrdU（红色，用于增殖细胞的标记物），巢蛋白（绿色，用于神经干细胞的标记物），和Hoechest33343（蓝色，对于核标记物）的免疫染色。 （C）的神经干的免疫染色/祖细胞标记物巢蛋白（红色，对于B型神经干细胞的标记物），表皮生长因子受体（绿色，C型瞬态扩增细胞的标记物），和DCX（蓝色，A型的标记神经母细胞）。细胞核用Hoechest33343（白色）复染。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54929/54929fig3.jpg" /> 图3: 从SCZ-aNSCs分化的细胞的免疫染色。与分化标记TUJ1（绿色，未成熟的神经元标记），O4（红，少突胶质细胞标记）和免疫组化GFAP（吆喝流，对星形胶质细胞的标记物）。放大的图像被示为插图。 Hoechest33343（蓝色）用于反染色细胞核。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.0K 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 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 ...

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 ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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.