Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC分化为神经前体细胞<ol><li>发展和扩大在人胚胎干细胞人iPS细胞（HES）培养基（ 表1）共培养上的小鼠胚胎成纤维细胞（MEF）饲养层，直到大（但汇合）菌落准备神经分化经由胚状体（EB）的中间（ 图2）。常规hiPSC培养条件是公别处16,17描述简单地说，生长在HES媒体人iPS细胞在MEF饲养层，直到融合，然后通过酶胶原酶（1mg / ml的DMEM中），并扩大约为1：3，每7天。 </li><li>在无菌水中塑料表面制备聚-L-鸟氨酸/层粘连蛋白包被的板，涂层板用10μg/ ml的聚-L-鸟氨酸（50μg/ ml的玻璃表面）并在室温下孵育3-24小时。用无菌水，然后涂上5克/ ml的层粘连蛋白洗至少一次在无菌PBS在RT 3-24小时。 注意：如果包裹在塑料，板材可贮存长达六个月-20 C。 </li><li>在第1天，酶解除从板一样大菌落中的DMEM / F12利用1mg / ml的胶原酶IV汇合人iPS细胞（通常生长在MEF中，或人iPS细胞在限定培养基如TESR 18上生长的基质胶）。 注：继37ºC1-2小时的潜伏期，殖民地将会漂浮在菜。 </li><li>轻轻洗hiPSC菌落（通过沉降，离心分离不）1-2x用DMEM / F12，重悬在2毫升/孔的N 2 / B27培养基（ 表1），并转移到非粘附6孔培养皿（结合3-井成1- -嗯）19。 注：一夜之间，菌落形成浮动球团称为“胚体”（EBS）。预计大量的细胞死亡。 </li><li>第2天，倾斜板，并允许胚解决。小心地取出媒体和DMEM / F12洗一次，清除杂物。饲料用N2 / B27媒体辅以双SMAD抑制剂（0.1M LDN193189和10M SB431542）12 </SUP&gt;。 </li><li>关于天3-7，neuralization将发生在双SMAD抑制的上下文中检查胚是圆的，但不是囊。订阅的EB每隔一天用补充有0.1M LDN193189和10M SB431542 N 2 / B27培养基。 </li><li>在7-14天，下列的EB电镀聚L-鸟氨酸/层粘连蛋白涂层板，检查使用明显微镜神经花环开始在几天之内出现。（定性为圆簇神经上皮细胞apico - 基底极性的; 图1）。继续喂粘附的EB每隔一天用N 2 / B27培养基补充有0.1M LDN193189，10M SB431542和1μg/ ml的层粘连蛋白。 </li></ol>神经玫瑰花奖2丰收注：我们建议的神经花环使用神经玫瑰花选择试剂20或类似的选择试剂酶收获。虽然神经花环可以手动挑入6孔聚-L-鸟氨酸/层粘连蛋白包被的平板，THI方法论需要大量的训练来掌握，而且，依赖于用户的技术，可能需要第二轮采摘20天，以进一步丰富了筹备和消耗非神经细胞类型。 <ol><li>对于在第14天的酶的选择神经花环，从附着的EB抽吸媒体和加入1 ml，每孔神经花环选择试剂的一个6孔板。在37ºC1小时。 </li><li>随着P1000的Pipetman，轻轻地从每孔除去酶。加入1毫升每孔的DMEM / F12的洗。 </li><li>与P1000，收集1毫升DMEM / F12，并迅速排出DMEM / F12回入井，从而分离从板块的花环。收集到玫瑰花猎鹰管。 </li><li>吸取的另一个1毫升DMEM / F12，并迅速排出到同一以及分离剩余花环。 （不要磨碎，尽量不要分手花环聚集）。收集神经花环成相同的猎鹰管。 </li><li>重复步骤需要2.4。如果花环不容易分离，添加钼重新酶和重复上述过程（步骤2.1-2.4） </li><li>旋转细胞在300×g离心3分钟。吸洗，并重新暂停2毫升NPC媒体（ 表1）花环。转移的细胞（1：1）到聚-L-鸟氨酸/层粘连蛋白包被的6孔板中。 </li><li>从15-21天，神经花环将扩大;饲料花环每隔一天与NPC媒体。 </li><li>评价神经花环的质量，并确保扁平成纤维细胞样细胞不培养内扩大。 注意：如果不坚持花环，全国文化有时可以通过人工采摘打捞上来，只选择最好的采摘玫瑰花成的Accutase。分离15分钟，在37ºC。颗粒，洗净，板NPC媒体到24孔聚L-鸟氨酸/层粘连蛋白涂层板。 </li></ol> 3.扩大神经祖细胞注：hiPSC的NPC可以生长在任Matrigel-或聚-L-鸟氨酸/层粘连蛋白包被的板。我们通常使用Matrige升感光板，因为它们可以更快速地制备并以更低的成本。 <ol><li>为了制备基质胶包被的板，快速解冻，重悬基质胶的1mg的冷冻等分试样以24毫升冷的DMEM / F12，并立即分发两个六孔板2毫升至每个孔中。孵育至少1小时，在37ºC。 注：基质胶仅需要被吸入，而不是洗涤，在使用前立即。 </li><li>饲料的NPC每隔一天，并以极高的密度保持或他们就会自发地分化为神经元。 注：虽然更成熟的NPC一般分为1：每周4，极低的通道的NPC可以被分割1：2的次数，每两周一次。 </li><li>分裂，第一抽吸媒体和加入1毫升温的Accutase（1X）每6孔板井。在37ºC10-15分钟。 </li><li>分离的细胞轻轻转移到15毫升管含有DMEM / F12用尽可能少的机械应力成为可能。不要滴定细胞，而酶。颗粒细胞spinni在1000×g离心纳克5分钟。 </li><li>吸洗，并在〜1毫升NPC媒体重新挂起的NPC每原井的6孔。 注：期望6孔培养皿的汇合阱具有5-10百万个细胞;这可以通过用血细胞计数之前重新电镀计数细胞的10升等分来确认。 </li><li>下面再悬浮，重制版的NPC作为NPC的，神经元或神经球。以保持的NPC，板每一个6孔板的大约1-2百万个细胞。的神经球的形成效率可以实验和细胞系之间有所不同，但一般发生最好之间200000和百万细胞每一个非粘附6孔板的井接种;如果神经球的聚集发生，降低细胞接种的数量。分化为神经元，板约200000每一个6孔板的细胞中，与神经元的媒体替换鼻咽癌介质。 </li><li>免疫组化验证每个NPC建立行。一旦有足够的多孔材料一直EXPANDED，标签的NPC与抗体NESTIN和SOX2。验证NPC线也应该分化成神经元4-6周，并标有抗体III微管蛋白和MAP2AB。 <ol><li>固定的细胞在PBS中的4％多聚甲醛在4℃下10分钟。透的NPC在RT在1.0％的Triton的PBS中15分钟，然后在5％驴血清阻滞与0.1％的Triton在RT 30分钟。 </li><li>孵育在5％驴血清初级抗体用0.1％的Triton，于4℃过夜。 注意：我们推荐以下抗体：羊抗SOX2，1：200;小鼠抗人巢蛋白，1：200;兔抗βIII微管蛋白，1：500;小鼠抗βIII微管蛋白，1：500;小鼠抗MAP2AB，1：200。 （ 图2）。 </li><li>以下用PBS洗涤，孵育二级抗体的细胞与合适的共轭次级抗体山羊，小鼠或兔1：300，在5％驴血清和0.1％的Triton为1-2小时，在室温。为了显现细胞核，染色的细胞用0.5微克/毫升的DAPI（4&#39;，6-二脒基-2-苯基吲哚），然后装入带的Vectashield。 </li></ol></li></ol> 4.全国人大转导<ol><li>使用spinfection（细胞培养板离心中病毒的存在下，在1000×g离心1小时），以增加转染的细胞21的百分比。抽吸培养基并替换为相关过表达（或控制）慢病毒（或反转录病毒），滴定感染的所需复数（MOI）（通常为1-10），稀释在鼻咽癌介质。使用每一个6孔板的孔1.5ml体积（或其他类型的组织培养板中的适当缩放体积）。旋在1000×g离心37ºC1小时在平板离心机。 注意：理想情况下，在1-2天内分裂转导的NPC与慢病毒或逆转录病毒载体。是否使用商业或实验室制备的病毒，也可以是有帮助的通过连续稀释至适当滴度的病毒的批次提前。 （ 图3）。 </li><li>为了减少CEL细胞性死亡，在spinfection 8小时更换介质。 注：病毒整合和转基因的表达应该是在24小时内报告基因的荧光检测。 （如果病毒缺乏荧光报道，这是更难以评估;成功的转染可以最好地通过免疫印迹，免疫组织化学或定量PCR对表达产物进行验证。）全部表达可能需要3-7天;可能需要重复spinfections与其他病毒以提高转染的细胞的百分比。复发性spinfections每天可以发生，但不必如此频繁。理想情况下，如果使用的是荧光报道，&gt; 80％的细胞应被标记。 </li></ol> 5.神经球迁移实验注：神经球形式自发，以下的NPC的酶解（通过等同于在鼻咽癌膨胀中使用的方式 - 步骤3.3-3.6），如果细胞是在悬浮液中鼻咽癌培养基中培养。 <ol><li>简单地说，长大分离在鼻咽癌媒体48小时在非粘附板ð的NPC，以便产生神经球。 （ 图4）。 </li><li>为神经球迁移，采用冷鼻咽癌媒体，而不是培养基，在96孔板中，以神经球采摘前1-2小时制备的新鲜基质胶板。不要从油井吸基底膜。 </li><li>最初公布的胚胎干细胞衍生的神经球22，手工挑NPC来源的神经球在显微镜下，在全国人民代表大会媒体清洗一次，以去除细胞碎片之后。传送一个神经球到基质胶包被的96孔板的每个孔中。 </li><li>添加额外的0.5毫克的Matrigel，稀释于冷鼻咽癌媒体，向神经球在每个96孔板中。 </li><li>手动居中每个单独的神经球在使用吸管尖阱的中间。 注意：挑大小相似的神经球，以减少可变性的结果是很重要的。 </li><li>允许迁移发生48小时，然后修复细胞瓦特第i个4％多聚甲醛和染色与期望的免疫组织化学标记。 </li><li>如果放射状迁移要确定自己完全采用4倍显微镜物镜，照片神经球。排除任何神经球接触所述井的边缘或从分析的第二神经球。 注意：如果发生的时间超过48小时的迁移，采用了4倍物镜所得径向迁移可能会过大，以图像。 </li><li>测量从用NIH ImageJ的软件（ 图5）的每个神经球平均迁移。这可以使用任何一种下面描述的分析方法来完成： <ol><li>放射状迁移： <ol><li>手动追踪使用徒手选择工具然后使用测量（CTRL + M）函数来计算得到的形状的区域中的最远迁移细胞的边缘。前测量的面积确保区域在分析选项卡下设置测量窗口进行检查。使用面积测量与方程为一个圆（A =πR2）的面积的值来确定所述半径（外）。 </li><li>跟踪原始神经球的边缘，测量面积，并计算半径（内）以相同的方式。计算总径向迁移作为外半径和内半径之间的差异。 </li></ol></li><li>最大的细胞迁移： <ol><li>测量采用直线选择工具其次是测量（按Ctrl + M）功能从内神经球的边缘移动的五个细胞最远的距离。 </li></ol></li></ol></li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+in+human+neural+progenitor+cells.">L1 retrotransposition in human neural progenitor cells.</a> Nature. 460, 1127-1131 (2009).</li><li>Xia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells+to+model+spinocerebellar+ataxia+type+2+in+vitro.">Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro.</a> Journal of molecular neuroscience : MN. 51, 237-248 (2013).</li><li>Aasen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26, 1276-1284 (2008).</li><li>Delaloy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-9+coordinates+proliferation+and+migration+of+human+embryonic+stem+cell-derived+neural+progenitors.">MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors.</a> Cell Stem Cell. 6, 323-335 (2010).</li><li>Kriks, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Maroof, A. 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The authors have nothing to disclose.

我们已经描述了通过该分化人iPS细胞的NPC成，神经细胞类型，其中hiPSC衍生的神经元的基因标记的显著分数是保守，并且可以作为一个代理可能造成疾病的发病机制8的发育途径的方法， 11。此外，正如我们已经详细介绍，NPC的是一个稳健的复制和转导容易神经元群，我们相信这可能是用于疾病易感性的分子和生化研究。 虽然我们已经详细方法分化和培养前脑?...

神经元由我们1 体外分化自人类诱导多能干细胞（人iPS细胞）和其他2,3的基因表达的研究表明，神经元hiPSC类似于胎儿，而不是成人脑组织。目前，hiPSC为基础的模型可能更适合于易感性的研究，而不是最近的功能，神经系统疾病。我们以前曾报道，精神分裂症hiPSC衍生的神经元的基因标记的显著馏分保守精神分裂症hiPSC源性神经祖细胞（NPC的），这表明的NPC可能是一种有用的细胞类型用于研究分子途径促成精神分裂症1 。我们和其他人已经报道异常迁移，增加的氧化应激和活性氧，灵敏度到亚阈值的环境压力和在精神分裂症受损线粒体功能hiPSC的NPC 1,4-6，以及减小神经Çonnectivity和精神分裂症hiPSC神经元突触5,7-10功能。如果分子因素在精神分裂症hiPSC的NPC有助于异常迁移和/或氧化胁迫也背后精神分裂症hiPSC衍生的神经元的减少神经连接性，NPC的可能是一个强大的和高度复制神经元群，用以研究负责疾病的机制。此外，由于可以迅速地产生大量的细胞，并且不需要等待数周或数个月的神经元成熟，鼻咽癌基测定法适用于较大的患者群的研究，并且更适合于高通量筛选。我们相信，hiPSC的NPC可以作为可能造成疾病的发病机制的发育途径的代理，如已被证明在疾病等不同精神分裂症1和亨廷顿氏病11。 从人iPS细胞，在最初的神经分化的NPCduction由双SMAD抑制（0.1mM的LDN193189和10毫SB431542）12完成。通过拮抗骨形态发生蛋白和TGF与这些小分子信令，内胚层和中胚层规范被阻塞，加速神经元分化，并导致的可见神经花环形成电镀的一个星期内。神经形成发生在这一过程的早期，在神经花环形成，随后立即期间大概。在没有其他线索，这些原始神经细胞承担前端脑样的命运13。紧随其后的神经花环形成，并在整个NPC不断扩大，前脑的NPC与FGF2 8,14培养。它们具有双重谱系的潜力，并且可以分化为70-80％III微管蛋白阳性的神经元和20％-30％胶质纤维酸性蛋白（GFAP）阳性的星形胶质细胞（ 图1）的神经种群。大多数脑hiPSC神经元是VGLUT1阳性等等都是推测谷氨酸，尽管神经元的大约30％是GAD67阳性（GABA能）8。 NPC的例行传代在体外的十倍以上，同时保持一致分化型材，和没有积累核型异常。组报道传代的NPC超过40倍15，然而，我们发现，超过10通道，NPC的显示增加倾向星形胶质细胞分化。 NPC的良好耐受多次冷冻 - 解冻，并且可以转换通过简单地在培养非粘附板成长为神经球。的NPC用病毒载体转导的效率，从而使遗传扰动的分子和细胞后果快速评估，并易于扩展，以产生足够的材料用于生化研究。另外，因为病毒载体容许鲁棒过度表达和/或与疾病相关的基因敲除，在对照或患者来源neur人的细胞，可以使用这个平台来测试对这些操作的遗传背景的影响。虽然不适合突触或基于活动的检测需要成熟的神经元，NPC的可能是来自患者的神经细胞的许多简单的分子或生化分析一个实际的选择。

<table border="0" cellpadding="0" cellspacing="0" width="1174">
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			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#11330</td>
			<td>for HES media</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Glutamax</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#35050</td>
			<td style="width:500px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">NEAA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#11140</td>
			<td style="width:500px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#21985-023</td>
			<td style="width:500px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">FGF2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0074</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">#450-02</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td style="width:216px;">&#160;#05832</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Accutase</td>
			<td style="width:171px;">Innovative Cell Technologies</td>
			<td style="width:216px;">AT-104</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#17104019</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">CF1 mEFs</td>
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			<td style="width:216px;">#PMEF-CF</td>
			<td style="width:500px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

神经花环可以形态被识别，使用明场显微镜，由它们的特征外观圆形簇神经上皮细胞apico -基底极性（ 图1）的。虽然筹备通常培养在非常高的细胞密度，紧接传代，略呈锥体形体躯和双极突起结构是可见的（ 图1D）。验证的NPC表达巢蛋白和Sox2在大多数细胞中，虽然III微管蛋白染色也是可见在所有鼻咽癌群体，这表明的NPC的一些比例都在不断的培养物（ 图1F）...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

指南生成和使用hiPSC派生的NPC的神经系统疾病的研究

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

a-guide-to-generating-using-hipsc-derived-npcs-for-study-neurological

Research

JoVE Journal

Medicine

18.1K 次观看.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

视频: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

利用经颅磁刺激，以研究人类的神经肌肉系统

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

一个表型养生为用于研究衰老的人类疾病基因牵连转基因小鼠

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

建立中风病研究小组算法并进行急诊科基于仿真的培训 - 实用指南

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

自动放射化学合成[<sup&gt; 18</sup&gt; F] 3F4AP：一种用于成像脱髓鞘疾病的新型PET示踪剂

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

利用视网膜成像研究痴呆

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...