笔者想感谢艾萨克·桑布拉诺博士及其工作人员在曼彻斯特眼科医院眼库的人供体眼组织的供应，以及生命科学组织学机构为H＆E染色图像学院的皮特·沃克先生。特别感谢罗杰·梅多斯先生对他的帮助与显微镜。 SJC是一个医学研究理事会的接受者（MRC）职业发展奖学金（MR / K024418 / 1），作者也承认，从MRC（G0900538和K004441）等近期的研究经费，争取视力（1866年）和黄斑社会。

从曼彻斯特皇家眼科医院眼库下去除角膜移植获得人体眼组织同意用于研究目的。利用研究人体组织是按照人体组织法案（2004）的指导方针，并与大学研究伦理委员会的批准（参考11305）。 1.丰富全布鲁赫的膜<ol><li>围绕清洁工作空间和解剖显微镜用1％消毒剂下方（体积/体积）中，然后用70％（体积/体积）乙醇擦拭。 </li><li>确保以下的手:一种新的一次性手术刀，两人育细胞刮刀（组织培养中常用）三对镊子（此处列出的A，B和C，见材料清单）。 </li><li>将眼地球到一次性培养皿，保持盖以备后用。使用新的一次性手术刀，除去残留的视神经，这可能会干扰建立一个扁平月UNT的组织。其次，提出四点切口等间隔创建四个象限，如果在进行第3部分（黄斑布鲁赫的膜浓缩）不通过黄斑区切。 <ol><li>确保该切口穿过的眼组织的整个深度延伸 - 从最里面的视网膜神经上（NSR），通过脉络膜和巩膜（眼睛的白色）。继续切口周围的眼罩视神经的保证金眼球的方式。这将允许眼罩被变平（参见图1B）。 </li></ol></li><li>保持使用镊子甲扁平眼罩的一个象限（防止脉络膜分离），可使用较大的镊子乙夹住玻璃体和底层NSR和拉出此组织离视网膜色素上皮细胞（RPE）开始在外围，移向中心打开的眼罩，然后到相对侧。一般，但并不总是既玻璃体和NSR分离为一体。使用scalpel至切除锚定在视盘任何残留NSR。 </li><li>同样，在出任组织的一个象限的地方使用镊子A，用无菌细胞刮刀从眼罩的整个内表面轻轻打跑RPE单层。这些细胞容易分离，并且可以被看作是深褐色颜料团块。用PBS冲洗从眼罩轻轻洗去脱落的视网膜色素上皮细胞。 </li><li>接下来，使用镊子A到所有4个象限的眼睛轻轻剥离覆暗红色的组织（包括两个Bruch膜和脉络膜）。将切除的组织到一个干净的菜。 </li><li>用一个细胞刮刀的扁平边缘以保持切除的组​​织到位，使用第二个刮刀轻轻刮去组织。打开组织上，重复，去掉尽可能多的多余的材料越好。最终，半透明膜仍将:这是粗略地丰富了布鲁赫的膜。至少20X放大率的使用解剖显微镜的是至关重要的帮助去除脉络布鲁赫的膜和充实。 </li><li>使用巴斯德吸管，简要地冲洗Bruch膜用超纯水将所述膜至1.5 ml微量离心管中之前，以除去残余脉络膜和血液。 </li><li>暂停富集Bruch膜在约1 ml的超纯水和涡流简要（约15秒），以帮助溶解任何污染的细胞物质（ 参见图2）。离心样品在500×g离心30秒以沉淀膜​​，吸上清。 注:其余富集Bruch膜现在准备溶解以后或存储（如在第2下面描述）在-80℃下以供使用。 </li></ol> 2.全溶富Bruch膜和Western Blot分析<ol><li>浸泡在500μl8M的乌尔组织提取丰富布鲁赫的膜蛋白EA 6小时在RT在1.5ml微离心管（图3A）。 </li><li>以10,000×g离心10分钟，离心溶解样品以沉淀任何残留的细胞外物质。除去上清液，并将其转移到3500沓MWCO透析膜，然后将样品在1升的PBS中在4℃至透析16小时。 </li><li>对于每个样品，加入40微升蛋白质分离珠和旋转样品/珠混合物（20rpm下在旋转混合器）在RT 30分钟。 </li><li>离心样品以10,000×g离心5分钟以沉淀珠。除去上清液和溶解蛋白质结合珠粒于30μl2×上样缓冲液（65.8毫Tris-盐酸，pH为6.8，2.1％SDS中，26.3％（重量/体积）甘油，0.01％溴酚蓝，3％β巯基乙醇）。 </li><li>孵育样品在100℃下进行10分钟，然后离心以10,000×g离心5分钟以沉淀剩余的珠。 </li><li>加载包含所释放的蛋白质上，以一个预制上清液的SDS-页面梯度凝胶（见材料清单）的蛋白质分离。 </li><li>可视化使用共同的染色方案，例如，考马斯蓝分离的蛋白条带。 <ol><li>简言之，加入20毫升亮蓝染色（0.1％亮蓝（重量/体积），50％甲醇，10％冰乙酸，40％水）的凝胶，并孵育30分钟，在室温温和搅拌。 </li><li>丢弃染色液并用去离子水洗涤染色的凝胶直至背景染色被充分减小，以使染色蛋白带可视化。 </li><li>或者，转印分离蛋白带至硝酸纤维素为西方分析，如下所述。 </li></ol></li><li>转移分离的蛋白质条带至硝化纤维膜，在80毫安使用在转移缓冲液半干转印装置（25毫摩尔Tris，192 mM的甘氨酸，10％[体积/体积]甲醇）2.5小时。 </li><li>阻挡膜在10毫升PBS中，10％（重量/体积）奶粉和0.02％（重量/体积）BSA的16小时，在4℃日之前Ë另外的蛋白质检测所需的主要抗体。 注意:如这里的示例， 图3C示出了溶解Bruch膜的代表性免疫印迹从三个独立供体探查补体级联，因子H样蛋白1（FHL-1）的一个调节器。 </li></ol>黄斑布鲁赫的膜3富集<ol><li>按照步骤1.1 - 1.3如前所述。 </li><li>虽然NSR仍然就位，定位中心凹黄色着色，代表该中央黄斑区（ 见图 4）。使用无菌6毫米活检穿孔器，并将其放置在黄斑区上方，坚决削减黄斑。 <ol><li>保持活检打孔的地方，做一个单独的切口与眼罩的边缘手术刀活检凸模刃口边缘，然后使用镊子B，剥离周围的眼球材料，实现了清洁活检。 </li></ol></li><li>使用镊子C，删除组织来源于清洁培养皿盖的冲头和地点6毫米光盘。放置在解剖显微镜下的培养皿的盖子和黄斑一拳。 </li><li>拆下细NSR盘（检查是否有黄色中心凹染色来推断活检的准确度）。使用无菌细胞刮刀轻轻取出视网膜色素上皮细胞。 </li><li> 通过使用镊子A的尖端巩膜固定黄斑光盘，慢慢剥去脉络膜和使用镊子C.同样Bruch膜复合物，使用细胞刮棒从Bruch膜除去不需要的脉络膜和血液物质。 </li><li>放置黄斑Bruch膜至1.5 ml微量离心管中，并暂停在1ml超纯水中。短暂涡旋（约15秒），以帮助溶解任何污染的细胞物质。离心样品在500×g离心30秒以沉淀膜​​，吸上清。 注意:富集Bruch膜从黄斑分离现在可用于蛋白质组的使用alysis用户首选消化协议。 </li></ol>

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The authors declare that they have no competing financial interests.

这里，我们描述Bruch膜的富集进行进一步的分析，允许随后的分析，包括Western印迹11，质谱和Bruch膜的利用改性尤斯灌流室11,14的扩散性质。关键步骤包括:a）眼的内表面的透彻刮削和洗涤以除去所有的RPE细胞b）该底层脉络膜的重复和仔细刮削而不会引起布鲁赫瓦解和的切除布鲁赫c）如超纯水洗涤以确保残余细胞的裂解。此外，如果富集Bruch膜是将要用于蛋白组学分析，蛋白质提取由尿素处理是最有效的在打破该弹性组织相比，其它方法如机械均化。丰富Bruch膜的组织学显示脉络膜的ECM不会被删除。这是恩存在意外作为pentilaminar Bruch膜的外层是由脉络膜毛细血管内皮细胞基底膜形成。事实上，它可能是有利的脉络膜毛细血管的ECM保持为发生变化这在AMD的包括末端补复杂/膜攻击复合物（MAC）15的沉积。应当指出的是，对于Bruch膜的详细的组织学分析，其结构将能更好地保留，如果切片在巩膜，脉络膜，和Bruch膜的存在。事实上， 在图2中呈现的组织学完全是意在展示富集从本手稿中描述的技术产生的程度。 黄斑布鲁赫富集需要小心剥离眼罩，确保NSR和尤其中央凹不受损坏;否则鉴定并因此隔离黄斑是不可能的。该Tissu酒店的完整性e是同样重要的是这个过程，它因此，建议使用下48小时验尸时间样本。需要富集的程度捐助者之间的变化，并且可受供体年龄，验时间，以及任何眼睛病理的存在。利用丰富的术语是故意在确认该技术的限制，这是那个&#39;净化&#39;或Bruch膜的使用这种方法&#39;隔离&#39;根本是不可能的。事实上，由于脉络膜的ECM并入Bruch膜有完全分离是不可行的。 理解的各眼组织的生化改变是在了解眼睛疾病进展至关重要。 Bruch膜的这种独特的丰富有利于这种认识;正在进行的研究是用质谱在AMD的不同阶段，研究丰富了黄斑布鲁赫的膜蛋白质组并在样本从受试者不同基因型始发改善的AMD的分子病理学的理解。已经在这里描述的技术已成功地用于鉴定FHL-1作为在上富集Bruch膜Bruch膜11，并进一步研究的主要补体调节器可能导致更多的新的见解的AMD的分子病理学。

在眼科研究中使用的验尸人眼组织对眼睛疾病的发病机制的认识的宝贵资源。人类供体眼分析作出了挑剔的支撑年龄相关性黄斑变性（AMD），这是失明的在西方世界1主要原因机制作出了重要贡献。从全球来看，AMD占所有失明的约8.7％，并与人口日益老龄化，预计影响到1.96亿人，到2020年2，AMD会导致中央视力的丧失，对病人的独立性和生活质量产生深远的影响3。早期AMD的特征在于玻璃疣的形成和它可以发展为地图样萎缩（有时被称为"干"AMD）或脉络膜新生血管形成（也称为新血管或"湿"的AMD）。虽然抗VEGF注射可稳定或改善视力的新生血管性AMD是我不是治愈性，并且在目前，还没有治疗存在地图样萎缩。 虽然某些遗传变型已被示出修改的AMD风险4 - 8，很少有关于这些变体如何影响黄斑在生化水平。在AMD发病的重要站点是Bruch膜。这种结构和各种研究中的玻璃膜疣的形式已经和周围Bruch膜在AMD 9提供补体激活的证据。布鲁赫膜是细胞外基质，从脉络膜分离的视网膜色素上皮（RPE）细胞的片材，并且大约4微米厚。布鲁赫膜并入脉络膜，一层包含有孔的毛细管和外部此脉络膜是含有粗的血管10（图1A）层。 布鲁赫的膜是额外的一个pentilaminar片蜂窝基质（ECM），以及这种方法详述如何隔离Bruch膜，随着底层脉络膜毛细血管的ECM（以下称富含Bruch膜），这在很大程度上是脱细胞，无红血细胞。富布鲁赫的膜，然后用于免疫印迹和组织学实验。此外，方法用于从眼睛的黄斑区域富集Bruch膜仅仅被描述，这将是特别感兴趣的是那些研究的AMD的生物化学方面的。

<table border="0">
	<tbody>
		<tr>
			<td>Auxillary objective 0.5x</td>
			<td>GT-Vision Ltd</td>
			<td>3638</td>
			<td>Useful (but not essential) for lower magnification imaging of whole eye flat-mount</td>
		</tr>
		<tr>
			<td>Biopsy Punch 6mm</td>
			<td>Oncall Medical Supplies</td>
			<td>SCH-33-36</td>
			<td>6mm Biopsy Punches</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma - Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma - Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td>For membrane enrichment</td>
		</tr>
		<tr>
			<td>CyroPure Cyrovials</td>
			<td>Sarstedt</td>
			<td>72.38</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpels</td>
			<td>Fisher Scientific</td>
			<td>12387999</td>
			<td>Sterile, individually wrapped Swann-Morton scalpels.</td>
		</tr>
		<tr>
			<td>Dual Gooseneck LED spot lights on 30cm arms</td>
			<td>GT-Vision Ltd</td>
			<td>0153</td>
			<td>Flexible light source essential for dissection and imaging</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma - Aldrich</td>
			<td>D8537</td>
			<td>Used to prevent macula drying out and during membrane enrichment.</td>
		</tr>
		<tr>
			<td>(A) Forceps</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4280</td>
			<td>(A) Fine (100mm) and straight</td>
		</tr>
		<tr>
			<td>(B) Forceps</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4272</td>
			<td>(B) Broad and blunt (150mm). Useful for lifting large portions of sample</td>
		</tr>
		<tr>
			<td>(C) Forceps</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4323</td>
			<td>(C) Straight fine points</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma - Aldrich</td>
			<td>G1345</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma - Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>GXCAM 5 digital colour camera &#38; GT-Vision software</td>
			<td>GT-Vision Ltd</td>
			<td>1122</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma - Aldrich</td>
			<td>M/4000/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels</td>
			<td>Life Technologies</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sterilin</td>
			<td>101VR20</td>
			<td>90mm, used as a dish for samples</td>
		</tr>
		<tr>
			<td>Protein isolation beads (Strataclean resin)</td>
			<td>Agilent</td>
			<td>400714-61</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereozoom microscope GXMXTL3T (magnification range 7x-45x)</td>
			<td>GT-Vision Ltd</td>
			<td>5595</td>
			<td>Stereozoom microscope attached to boom stand for ease of use during dissection</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma - Aldrich</td>
			<td>T3253</td>
			<td></td>
		</tr>
	</tbody>
</table>

enrichment of bruch's membrane from human donor eyes

年龄相关性黄斑变性（AMD）是在发达国家视力障碍的主要原因。该疾病本身表现由中心视网膜的，称为黄斑的破坏，从而导致中心视力的丧失。早期AMD的特征在于小，淡黄病变称为软脉络膜小疣，可以进步的存在到晚期AMD如地理萎缩（干性AMD）或新血管形成（湿性AMD）。虽然临床变化很好的描述，并赋予AMD风险遗传影响的认识越来越以往任何时候都更加细致，一个领域缺乏重大进展是遗传风险的生物化学后果的认识。这部分是由于难以理解Bruch膜，非常薄的细胞外基质，它作为材料的从血液供给一个生物过滤器和视网膜色素上皮（RPE）细胞单层驻留的一个支架的生物化学。玻璃膜疣˚F内Bruch膜和它们的存在ORM破坏营养流向RPE细胞。仅通过调查Bruch膜的蛋白质组成，而事实上其他蛋白质与它如何交互，可以研究人员希望解开支撑玻璃膜疣形成，发展的AMD和随后的视力丧失的生化机制。本文详细方法用于富集要么整个Bruch膜，或者仅仅从黄斑区，因此，它可用于下游生化分析，并提供了这是如何已经改变Bruch膜生物化学的理解实施例。

上面描述的协议以产生富集Bruch膜导致Bruch膜，脉络膜的ECM的分离和除去大部分细胞/细胞碎片包括所有的视网膜色素上皮（图2）的。富集Bruch膜在水中的洗涤是在裂解任何剩余脉络膜细胞的关键步骤。杀脉络膜期间施加的压力出现压缩一些的少数剩余细胞核成被定义为Bruch膜（图2B）的区域。 富集Bruch膜的增溶允许其蛋白质含量的下游分析。蛋白质条带从溶解富集Bruch膜使用4分离 - 12％梯度SDS-PAGE凝胶，而没有用在自己的权利，以确定蛋白质含量，的确证明这种技术，以减少污染的血液蛋白质，AR的能力Ë非特异性结合（ 图3B）。在这方面，从蛋白质凝胶运行一个显着的缺席是人血清白蛋白的一个相当大的频段（〜66 kDa的），其可以以其他方式进行后续免疫印迹更难以解释:富集Bruch膜的组织学分析显示于图2还支撑本。实际上，作为工作部分先前描述11，溶解布鲁赫从一系列单独供体的膜进行探测的一个通过Western印迹（图3C），使用特异性抗体的蛋白质。在这里所示的情况下，抗体（称为OX23）12针对先天免疫的155 kDa的调节器，被称为补体因子H（FH）中，使用和证实从三个独立的较低分子量带的Bruch膜样品中的存在供体（ 图3C）。随后这原来是因子H样蛋白1（FHL-1）中，49 kDa蛋白与FH和来自相同基因13的剪接变异产生的。 FHL-1似乎是存在于Bruch膜11主要补体调节剂。 <img alt="图1" src="/files/ftp_upload/53382/53382fig1.jpg" /> 图1.解剖Bruch膜的人眼和充实。（A）Bruch膜是一种细胞外基质屏障。脉络膜包含一层有孔毛细血管连续合并到布鲁赫膜（称为脉络膜），并且这些分离的脉络膜的其余部分（含大血管）从所述视网膜色素上皮细胞（RPE）细胞单层，其生理上支承感光细胞在视网膜神经上皮层（NSR）。（B）的四个切口进入人眼杯是由允许它被打开，以创建平面安装。该玻璃体可以与使用镊子除去。 NSR的可能脱落连同玻璃体，但也可以利用镊子除去。甲细胞刮刀用于从Bruch膜除去RPE细胞层，同时它仍然固定在脉络膜和巩膜。在Bruch膜/脉络膜复合物可以剥离远离巩膜，并用新的细胞刮刀，外脉络膜可以去掉。在超纯水最后清洗裂解所有剩余的细胞物质。 <a href="https://www.jove.com/files/ftp_upload/53382/53382fig1large.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/53382/53382fig2.jpg" /> 图2.组织学Bruch膜和前（A） 的脉络。H＆E染色切片及售后服务 （二）从Bruch膜和洗涤超纯WA刮去外脉络膜结构之三，以产生丰富Bruch膜。比例尺代表20微米。 <a href="https://www.jove.com/files/ftp_upload/53382/53382fig2large.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/53382/53382fig3.jpg" /> 图3.增溶作用富集布鲁赫膜对蛋白质含量分析。（A）的蛋白质可以从富集Bruch膜中提取由培养在8M尿素的6小时，在室温，被透析的PBS之前。对于Western分析，从整体Bruch膜透析蛋白可以使用蛋白隔离珠用的Laemmli还原样缓冲液煮沸分离和洗脱。（B）的代表性的考马斯蓝染色的SDS-PAGE凝胶，显示出既纯净和稀释溶解Bruch膜（BM）的使用方法D，形旁切的（A）（C）溶解布鲁赫的膜从3个人捐助者的代表性免疫印迹（泳道1 - 3）。探查H因子样蛋白1（FHL-1） <a href="https://www.jove.com/files/ftp_upload/53382/53382fig3large.jpg" target="_blank">请点击这里查看大图这个数字。</a> <img alt="图4" src="/files/ftp_upload/53382/53382fig4.jpg" /> 图4.分离富集布鲁赫膜从黄斑的。视网膜黄斑区域是一个5毫米面积可由覆中央凹的黄色着色易于识别，而且由于靠近相邻的视盘。使用凹作为导向，放置在平坦安装人眼一个6毫米活检穿孔切除一个6毫米黄斑组织块。除去覆NSR和从切黄斑刮去RPE细胞。剩下的Bruch膜/脉络膜复合物才能使折射率n去皮远离巩膜，彻底刮除，除去脉络，剩下的裂解通过浸没在超纯水中细胞物质。 <a href="https://www.jove.com/files/ftp_upload/53382/53382fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Enrichment of Bruch's Membrane from Human Donor Eyes at JoVE.com

Enrichment of Bruch's Membrane from Human Donor Eyes

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

从人类供体眼富集布鲁赫的膜

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

enrichment-of-bruch-s-membrane-from-human-donor-eyes

Research

JoVE Journal

Neuroscience

12.0K 次观看.  University of Manchester. Identifying proteins specifically associated with Bruch’s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

视频: Enrichment of Bruch's Membrane from Human Donor Eyes

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

髓母细胞瘤干细胞的分离，浓缩，和维修

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

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 ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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

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

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

从人多能神经嵴前体细胞的无饲养推导干细胞

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

从尸检人脑软脑膜捐助外植体培养的推导

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

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 of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...