这项工作是支持的补助，以JLRR：自闭症，尼​​娜爱尔兰，韦斯顿避风港基金会，NIMH R01 MH081880和NIMH R37 MH049428。 PRW是由来自台湾的国家科学委员会的奖学金支持。 SFS是由F32（MH103003）的支持。

伦理学声明：以下程序已通过我们的机构和动物的协议。确保获得批准，涉及生存手术开始实验前的所有程序，并确认所有的协议都是最新的。 1.慢病毒准备（可选步骤） <ol><li>拆分HEK293T细胞的3个10 CM平板的每个慢病毒被制成，以10ml的DMEM / 10％FBS的存在下，并生长至〜60-70％汇合。生长的细胞在培养箱中在37℃，5％的CO 2。 </li><li>转染以下DNA质粒（10微克总）转换成使用任何转染试剂各板：6.4微克CAG-FLEX-GFP的慢病毒载体（DNA载体序列在表1中提供），1.2微克pMD2.G（编码VSV-G），1.2微克PRSV-REV，和1.2微克pMDLg / pRRE。三个慢病毒包装载体是可商购的（表 注意：这种特殊的方法利用第三代lentivir人的载体，但第二代媒介和相关质粒也将正常工作。 </li><li>准备包含BSL2认证罩内10％的漂白粉溶液灭活的联系，或包含任何慢病毒解决方案或设备废料容器。接下来，转染到漂白粉溶液后，彻底去除媒体4-6小时，更换新媒体的同一卷，并允许细胞生长。 </li><li>后4天，收集培养基入50ml锥形管中并离心以1000×g离心15分钟以沉淀任何细胞碎片。接着，过滤所述上清液通过0.45μm的膜。任何低蛋白结合过滤器，如PVDF，效果很好。注意：将固体和液体废弃物病毒进入漂白粉溶液。 </li><li>负载30毫升过滤的上清液到超速离心管并进入超速离心机。离心机以100,000×g离心2.5小时，在4℃。打开离心管中BSL2罩和上清到10％的漂白粉。添加〜100微升的PBS到PEllet，这将产生1×10 ^ 7-8传染性单位/ ml的滴度。分装在次日并且储存在-80ºC。慢病毒等份是稳定的，在-80℃下至少6个月。 </li><li>重要的考虑因素：许多机构现在有病毒的核心。获得浓缩的病毒用1×10 7感染单位/ ml的滴度最低，以获得最佳的结果。 </li></ol> 2.供体小鼠的MGE解剖<ol><li>首先，建立了利益的Cre表达线，无论是野生型（WT）鼠标或鼠标，将表达Cre重组酶介导的重组后的记者之间的定时交配。 注：很多的Cre-驱动线转基因和维护，并孕育在半合状态。因此，只有一半的胚胎将包含酶Cre转基因。 DNA可从胚胎迅速准备一个短的PCR来检测酶Cre等位基因。所述酶Cre +胚胎然后可收获，使得所期望的基因型的仅MGE组织被使用。 Alternatively，一个依赖于Cre的报道，可以使用，以选择胚胎MGE解剖和移植。 </li><li>计算这一天将对应于E13.5。如果动物配对前的晚上，插头的一天是0.5。为了定时孕鼠或WT女性，P1幼崽，以时间为出生后（P）1小鼠当天的供体组织将E13.5。另外，在房子配对供体动物的前一星期设置这些交配。 注意：产生两个E13.5和P1胚胎的前策略/幼仔为同一天往往很难做到可靠。 </li></ol> 3.准备的媒体，工具​​和设备。 <ol><li>制备的试剂和工具的MGE细胞制剂（ 见表2）。 <ol><li>制备一个清洁的表面，并把清洁的镊子，剪刀以及一个显微刀或细镊子（用于精确组织解剖）的表面上。 </li><li>制备Hank氏平衡盐溶液（HBSS）中，f或剥离，并Dulbeco改进的Eagle培养基（DMEM）中，用10％胎牛surem（FBS），用于转导。 </li><li>预温育20-30毫升的DMEM / 10％FBS在37℃组织培养箱进行约1小时。该盖应松开，以允许介质气体交换和pH平衡。 注：媒体可以预孵育刚开始解剖前准备。 </li></ol></li></ol> 4. MGE细胞的制备<ol><li>根据批准的动物协议牺牲孕小鼠并取出胚胎成含有冰冷的HBSS 10cm的培养皿。 </li><li>从使用解剖范围每个胚胎取出脑（做一次一个，留下的胚胎的其余部分在冰冷的HBSS）中，然后进行切出的MGE组织。 注意：下面详细，并在图1中 ，也展示了如何删除MGE关键步骤。另外， 图2是示出电影的整个解剖流程。 <ol><li>定位大脑腹侧朝上（ 图1A，A“）。注意：它也还可以有背侧朝上。切开大脑中的一半沿矢状平面上以生成两片。一个例子切成对半的脑示（ 图1B，B&#39;）中 ，请注意，背侧（上覆皮质）和脑盖腹侧方面和包围该神经节隆起（GE）的组织。 </li><li>接着，用优势手，保持或者非常细的镊子或stabknife（ 表2），而固定带由非优势手保持镊子的半球。 （半球的内侧方面应朝上）。展开背，腹组织钳和刺一刀，露出底层的GE组织（ 图1C和图解图1C“）。 注意：删除从解剖培养皿一些媒体可以更容易稳定，而PE的组织rforming的MGE清扫。 </li><li>使直线切割与stabknife或细镊子分离专家咨询小组，LGE和室间隔组织（见例如削减记为红色虚线， 图1D和D&#39;）。这些切口可以以任何顺序进行。 注：想象一下，MGE的&#39;盒子&#39;被切出的周围组织。这样做有助于重复做类似的削减每个解剖，从而减少可变性的组织解剖。 </li><li>最后，打开MGE在其一侧并修剪掉所述底部（什么是脑的外侧面）。这消除了MGE的地幔区（丢弃在图1E和1E&#39;）从MGE的其余部分。 MGE其余方面主要包含脑室区和MGE，其中包含几乎所有的MGE祖细胞的脑室下区部分。当您的组织。 </li></ol></li><li>重要的考虑因素：添加硅氧烷凝胶至培养皿CA的底部ñ作为一个很好的剥离面，因为它可以保护镊子的细腻技巧，并提供了一​​个柔软的表面钉扎组织用钳子。 </li></ol> 4.3其他策略MGE解剖。 <ol><li>削减使用镊子似剪刀，迅速取出脑胚胎的皮肤。作为胚胎规定在其一侧，保持所述组织在头部与用于锚定组织的钳子的基极。首先，切割前腹侧到大脑和后部腹侧到大脑，然后连接所述两个切口沿着胚胎的一侧。然后抓住皮肤的护翼并在大脑的顶部拉出。大脑然后可以迅速解剖远离剩余的组织。 </li><li>可替代地，锚定皮质背后的全脑的后部的方面。同时，掌握每个半球的皮层的最背侧尾方面和撕裂皮层远离锚定组织中的前方向。以这种方式，使用Cortices可以被删除，双侧神经节隆起都显露出来，仍然附着于组织保持内侧 - 外侧解剖学的其余部分。 </li><li>切换到刺一刀，使周围的MGE精确的削减对每个半球。使用一个单独的干净的一套镊子收集MGE或一大笔小费吸管（ 即 ，P1000）。 注意：保持组织收集远离所有其他剖分材料，作为当MGE收集一些媒体不可避免地转移。 </li><li>收集MGE和把组织成含有DMEM / 10％FBS（500微升的体积是足以收集MGEs）1.5ml的收集管。重复其他半球的地方在同一个收集管。保持样品在冰上，直到所有组织收集。 </li></ol> 5.慢病毒标签和移植<ol><li>移动MGE组织的管成BSL2认证的发动机罩和取出介质。 注意：MGE组织是大到足以被肉眼可见的，并会沉降到管允许成为冷介质容易且轻轻除去的底部。 </li><li>添加〜500微升的DMEM / 10％FBS的培养基中，该预孵育在37℃组织培养孵化器。接下来，添加凝聚胺以促进传导到每个管中以8微克/毫升的最终浓度。磨碎的MGE组织以建立一个单一的细胞悬浮液，使用的是P1000的枪头。最后，添加〜15-20微升浓缩慢病毒向每个管中。 </li><li>重要的考虑因素：10-12倍甲研磨足以从单个胚胎分手MGE组织，但也可需要更triturations如果多个MGEs汇集在一起​​。尝试不同的研磨条件来决定什么效果最好。 </li><li>盖紧每个试管，颠倒混合，比地方所有的管子在37℃的孵化器。孵育细胞中的慢病毒为至少30分钟和最多1小时。更长的时间导致德折痕细胞活力。反转管每隔10分钟，直到时​​间到了。 </li><li>孵育慢病毒后，从孵化器和离心机在〜700×g离心试管3分钟以沉淀细胞。在BSL2罩，除去上清液并丢弃到10％的漂白剂。接着，加入1 ml的DMEM / 10％FBS和磨碎沉淀2-3次洗涤，然后再离心以相同的速度，以沉淀细胞。重复此洗涤步骤2-3多次，以除去过量的病毒。 注意：执行离心步骤在4℃下，然而，降低的生存力没有被观察到，当短的自旋都在室温进行。 </li><li>最后一次洗涤后，除去尽可能多的介质尽可能并把各管在冰上。由于在管的侧面残留介质，〜2-3微升介质将最终由移植过程的开始时间覆盖细胞沉淀。 </li></ol> 6.移植和验证<ol><li>掌握主机P1的幼仔，并准备程序区域通过喷洒下来，用70％的乙醇。以在操作过程中保持无菌，建议在一个专用清洁过程室执行这些过程。另外，使用高压灭菌手术工具消毒表面，该幼仔将在接触与使用70％的乙醇。执行移植和有代表性的程序区所需代表性的设备的一个例子示于图3。 </li></ol> 注意：虽然此步骤是注射小体积，并且不会在外科手术过程，将需要一个切口，开放性伤口或缝合线，它仍然是建议一个新微量用于每个鼠被注入。的微量是热拉和斜面上被喷上70％的乙醇被存储在密封容器中的表面时进行灭菌。 <ol><li>产生微量具有直径在前端40-80微米之间。以下这些方面将产生最佳结果。热灭菌微量时拉出并斜面上的表面和存储微量​​在密封容器中之前喷以70％的乙醇。 注意：可替代地，如果获得所需要创建这些针状物（ 表2）的装置是限制性的，可以使用任何其他注射装置。但是，这些可能不那么适合作为由于不同直径的微量。 </li><li>制定矿物油成1ml注射器，并用一个30 1/2 G针，完全填满玻璃吸管与矿物油。接下来，安装活塞到立体定位装置，然后装上玻璃吸管的立体装置和负载到活塞。使用液压驱动器，移动大约一半柱塞进入玻璃吸管，除去矿物油泄漏出来，用干净干净的纸巾。 <ol><li>可替代地使用注射器，淹没在矿物油中的移液管的后端和填充通过毛细管作用。为了防止气泡在pipettE，维持注射器正压，直到完全脱离玻璃吸管。 注：其他的设备可以用来成功地移植MGE细胞14。任何设备，可提供一个容积超过或接近100 NL作品以及在此过程中少。 </li></ol></li><li>接下来，使用消毒纸巾，或任何吸收材料，具有扭曲成一个细点的角落，微妙去除上面的MGE细胞沉淀多余的介质。这一步浓缩的细胞密度，以便较小的注射体积就可以实现。接下来，使用低容积（P2优先）吸管慢慢地拟定细胞沉淀和移动细胞悬液到疏水表面之前磨碎1-2次。量应略低于1微升。移动针进入细胞悬浮液的尖端，并使用液压驱动，绘制出细胞悬浮到吸管。 </li><li>麻醉通过低温一个P1小狗（包装在一个乳胶手套，并雪藏了〜2-4分钟）。检查麻醉与有害刺激效果。捏脚趾之间的皮肤用细镊子：如果捏了一把小狗应该是反应迟钝。接着，将小狗到一个模具，它是注射装置之下，然后使由拉回皮肤上的头部，并与标准实验室胶带固定拉紧皮肤。 注：虽然低温是对新生小鼠，并导致低死亡率非常有效，程序必须要快，因为幼崽将开始在10分钟中恢复。此外，在冰上4分钟是什么是可以容忍的上限。试图只诱导低温一次，如果需要用于注射的时间较长。但是，如果小狗的早期复苏，首先让小狗再次引起体温过低前完全恢复。此外，仅诱导低温一个更多的时间来执行注射。幼崽将在低温5-10分钟恢复。这可以通过将垃圾，在妈妈的幼崽，或者将小狗在一个温暖的面传热提供便利E要恢复。 </li><li>使用立体定位装置，定位玻璃吸管的尖端的表面上的小狗的头部，在垂直于头部的表面上。当吸移管是在与头部接触，认为这是“0”。接着，通过皮肤和颅骨插入皮层的表面推移液管，然后插入并停止之前缩回移液器〜2倍。为了获得最佳的细胞靶向到新皮质，注射时执行微量是在0.1毫米的深度。然而，这应该由用户进行优化（见下面的注释）。 注意：当该深度可靠地与上述设备的目标更深新皮层的层，几个深度应测试经验确定。此外，一系列的深度不同rostro-尾鳍和内 - 外位置应当进行测试，以确定是什么深度是最佳的在每个站点。另外，在初始穿刺应迅速，因为缓慢渗透到大脑皮层减弱的能力到cleanly穿透所述组织。尝试不同的速度时，首先启动该程序，找出最。此外，用于测试的坐标，没有真正的替代使用标记的细胞，如染料已经靠不住来表示的位置。 </li><li>一旦针在位置，旋转液压装置来推进所述柱塞进入玻璃吸管，推细胞悬浮液的设定容积进皮层。注入每个站点50-70 NLS。重复此过程在3-6不同站点在不同的延髓 - 尾的水平，如果目标是获得细胞的整个皮层一个宽的分布。 注意：100 NL或更高的卷可能导致损伤和导致注射细胞未能有效地迁移出注射部位的大的团块。因此，较小的进样体积（〜70 NL或更少）是最优的，在更多的网站，如果时间允许。 </li><li>当注射完成后，从舞台上拆下小狗并标记（趾裁剪是一种可靠的方式来识别PUPS更高版本）。一旦小狗已经恢复，并能够将其自己的（这种情况发生时从舞台中取出后几分钟内），把它带回了妈妈和垃圾。 注：由于这是一种微创手术，没有手术后的程序一旦小狗可自由移动和温暖。然而，幼仔应检查不仅程序，但也翌日，以确保后它们具有健康状况恶化的迹象。 </li><li>开始的下一个小狗的过程之前，喷向下面积，用70％的乙醇，以保持无菌的工作​​区。允许注入的幼仔开发然后评估所述组织在适当的发育阶段（多个）。 </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=Interneurons+from+embryonic+development+to+cell-based+therapy.">Interneurons from embryonic development to cell-based therapy.</a> Science. 344 (6180), 1240622 (2014).</li><li>Hunt, R. F., Girskis, K. M., Rubenstein, J. L., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+progenitors+grafted+into+the+adult+epileptic+brain+control+seizures+and+abnormal.">GABA progenitors grafted into the adult epileptic brain control seizures and abnormal.</a> Nature Neuroscience. 16 (6), 692-697 (2013).</li><li>Braz, J. M., 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=Forebrain+GABAergic+Neuron+Precursors+Integrate+into+Adult+Spinal+Cord+and+Reduce+Injury-Induced+Neuropathic+Pain.">Forebrain GABAergic Neuron Precursors Integrate into Adult Spinal Cord and Reduce Injury-Induced Neuropathic Pain.</a> Neuron. 74, 663-675 (2012).</li><li>Vogt, D., 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=Lhx6+Directly+Regulates+Arx+and+CXCR7+to+Determine+Cortical+Interneuron+Fate+and+Laminar+Position.">Lhx6 Directly Regulates Arx and CXCR7 to Determine Cortical Interneuron Fate and Laminar Position.</a> Neuron. 82 (2), 350-364 (2014).</li><li>Alvarez-Dolado, M., 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=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J. Neurosci. 26 (4), 7380-7389 (2006).</li><li>Du, T., Xu, Q., Ocbina, P. J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKX2.1+specifies+cortical+interneuron+fate+by+activating+Lhx6.">NKX2.1 specifies cortical interneuron fate by activating Lhx6.</a> Development. 135 (8), 1559-1567 (2008).</li><li>Arguello, A., 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=Dapper+Antagonist+of+Catenin-1+Cooperates+with+Dishevelled-1+during+Postsynaptic+Development+in+Mouse+Forebrain+GABAergic+Interneurons.">Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons.</a> PLoS ONE. 8 (6), e67679 (2013).</li><li>Lee, A., 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=Pyramidal+neurons+in+prefrontal+cortex+receive+subtype-specific+forms+of+excitation+and+inhibition.">Pyramidal neurons in prefrontal cortex receive subtype-specific forms of excitation and inhibition.</a> Neuron. 81 (1), 61-68 (2014).</li><li>Chen, Y. J. J., 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=Use+of+&#8220;MGE+Enhancers&#8221;+for+Labeling+and+Selection+of+Embryonic+Stem+Cell-Derived+Medial+Ganglionic+Eminence+(MGE)+Progenitors+and+Neurons.">Use of &#8220;MGE Enhancers&#8221; for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons.</a> PLoS ONE. 8 (5), e61956 (2013).</li><li>Pattabiraman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+enhancers+active+in+protodomains+of+the+developing+cerebral+cortex.">Transcriptional regulation of enhancers active in protodomains of the developing cerebral cortex.</a> Neuron. 82 (5), 989-1003 (2014).</li></ol>

The authors declare that they have no competing financial interests.

从胚胎神经节隆起（GES）为基础的细胞治疗使用皮质GABA能中间神经元前体正显示出的承诺了很多条件，12 -1 4。需要精确的分子技术进行跟踪，并表示在特定的中间神经元亚组感兴趣的基因。在这里，我们提供了一个详细的协议，用于在移植前用慢病毒标记胚胎MGE细胞并展示如何这种技术可以用来表达的体内分析中的具体的皮层中间神经元亚群的兴趣基因。 本?...

GABA能皮质的interneurons包括在哺乳动物大脑皮质的神经元〜20-30％，而其余的对应兴奋性，谷氨酸主要神经元。的interneurons在电生理特性，轴突和树突形态和突触目标1高度多样化，并在兴奋/抑制色调失衡假设是背后的神经/精神神经疾病，包括孤独症，精神分裂症和癫痫2部分的表型。本文所描述的协议的总体目标是提供对移植的体内分析之前有效地遗传修饰GABA能皮层中间神经元祖细胞的方法。 皮质GABA能中间是出生在内侧和尾神经节隆起（MGE和CGE，分别）3,4，以及视前区5。皮质中间神经元祖细胞经过长距离迁移切线后面通过放射状迁移，达到他们的最终目标。抵达目的地，这些皮质的interneurons必须正确集成到现有网络中的神经元，每个独特的中间神经元亚组会以特定的方式有助于皮质电路。四个主要子组，可以区分由分子标记：MGE衍生的促生长素抑制素（SST）+和小清蛋白（PV）+亚组，和CGE衍生血管活性肠肽（VIP）+和颤+; SST -亚组6。不同皮层中间神经元亚群都在MGE和CGE 7,8胚胎发育过程中所生了不同的时间。这些和其他皮质GABA能中间神经元标记物已被用于产生特异的Cre驱动线许多这些亚组9-11的。 MGE祖细胞的移植已成为一个潜在的基于细胞的疗法来治疗可通过不平衡引起的疾病励磁/抑制12 - 24。这些治疗益处可能部分是由于MGE祖细胞（分散，分化和整合入宿主脑），或可能是因为许多关节周围的体细胞抑制性的PV +细胞是从MGE衍生的独特能力。 MGE细胞也可以快速和有效地转导的移植15之前慢病毒，允许被遗传修饰在体外的细胞在体内进行研究。开发这种方法的原理是克服障碍在研究GABA能中间神经元皮质发育和成熟。特别是，移植的MGE允许研究人员研究突变的细胞在体内的发展，当突变小鼠将否则在早期时间点死亡。此外，通过引入移植前感兴趣的基因上的突变体的表型的特定基因的作用，可以在一个EFF评估icient方式。 在这里，我们提供了一个详细的协议转导的MGE细胞移植前的慢病毒。此外，我们显示如何这种技术可以适用于从皮层中间神经元前体的一组异质性表达在特定中间神经元亚群的目的基因，采用依赖于Cre的表达慢病毒和可用Cre的驱动鼠标线的组合。此外，该协议引入的技术和研究人员的一个平台，以基因修饰GABA能中间神经元的皮质前体的体内研究以独特的方式。这种技术比其他现有方法的一个优点是，移植的MGE细胞将分散远离注射部位。此外，与焦病毒注射后的MGE细胞分散它们的形态是更容易评估。这种方法可用于研究引入感兴趣基因引入野生型或突变体细胞中，引入的细胞类型特异性的效果记者评估形态，或潜在学习的疾病等位基因体内的效果。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and equipment for MGE cell transplantation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>301/2 gauge needle</td>
			<td>305106</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td colspan="2">Drummond scientific company</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td colspan="2">Polysciences, Inc.</td>
		</tr>
		<tr>
			<td>Stereo microscope with boomstand **</td>
			<td>MZ6</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital just for mice stereotaxic instrument **</td>
			<td>51725D</td>
			<td colspan="2">Stoelting company</td>
		</tr>
		<tr>
			<td>Single-axis oil hydraulic fine micromanipulator **</td>
			<td>MO-10</td>
			<td>Narishige</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond coated rotary beveler</td>
			<td>n.a.</td>
			<td colspan="2">made in house</td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td colspan="2">Kopf instruments</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td colspan="2">Any drug store or pharmacy</td>
		</tr>
		<tr>
			<td colspan="2">Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Optional Reagents (for making Lentiviruses)</td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td colspan="2">Fisher Scientific</td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td colspan="2">Beckman Coulter&#174;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (1.5 ml size)</td>
			<td>11668019</td>
			<td colspan="2">Invitrogen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other commercially available supplies</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid encoding gag/pol</td>
			<td>12251</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev plasmid encoding Rev</td>
			<td>12253</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid encoding VSVG</td>
			<td>12259</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-Flex-GFP</td>
			<td>28304</td>
			<td>Addgene</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABA能皮质的interneurons，从胚胎内侧和尾神经节隆起（MGE和CGE）得出，在功能和形态多样。进展已在理解的不同的皮层中间神经元亚群的作用，但是，也有可能向不同类型的GABA能细胞的发育和成熟仍有许多机制来制定。此外，改变的GABA能信令可以有助于孤独症，精神分裂症和癫痫的表型。特异性表达Cre-驱动线已经开始包裹出独特的中间神经元亚群的功能。尽管在小鼠模型中的进步，它往往是难以有效地研究GABA能皮层中间神经元祖细胞与分子的方法在体内进行 。用于研究这些细胞的细胞中自主编程的一个重要的技术是移植的MGE细胞向宿主皮层。这些移植的细胞广泛迁移，分化，一第二功能集成。此外，MGE细胞可以有效地转导的慢病毒立即移植前，允许多种分子的方法。下面我们详细的协议移植体内分析之前，有效地转导MGE细胞，利用现有的Cre-驱动线和依赖于Cre的表达载体。这种做法是有利的，因为它结合了精确的遗传操作与这些细胞的分散移植后，允许在体内更大的细胞类型特异性的分辨率的能力。

因为MGE细胞具有迁移并在移植到宿主新皮层16整合的独特能力，它们在体内的研究之前提供用于遗传操作的优良模型系统。这里，我们显示如何能够从E13.5胚胎分离的MGE组织（ 图1和2），然后可以用慢病毒在体外或在移植体内研究前一个快速的方式转导。通过慢病毒标记MGE已经使用了增强驾驶GFP的GABA能神经元15日前已执行。但是，表达能力从异质群...

Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABA能中间神经元皮质祖分散，开发和突触融入移植后的主机皮层。这些细胞可以移植在体内研究转基因GABA能前体的前容易地转导。这里，我们表明病毒标记技术到目标使用现有的Cre线和依赖于Cre的记者特定中间神经元亚群。

病毒介导的标签和内侧神经节隆起移植（MGE）细胞<em&gt;在体内</em&gt;研究

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

viral-mediated-labeling-transplantation-medial-ganglionic-eminence

Research

JoVE Journal

Developmental Biology

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

11.4K 次观看.  University of California San Francisco.  GABA能中间神经元皮质祖分散，开发和突触融入移植后的主机皮层。这些细胞可以移植在体内研究转基因GABA能前体的前容易地转导。这里，我们表明病毒标记技术到目标使用现有的Cre线和依赖于Cre的记者特定中间神经元亚群。 

视频: Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

科研

发育生物学

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and free-swimming medusae. As exemplified in established models such as Hydra and Nematostella, stem cells and/or proliferative cells contribute to the development and regeneration of cnidarian polyps. However, the underlying cellular mechanisms in most jellyfish, particularly at the medusa stage, are largely unclear, and, thus, developing a robust method for identifying specific cell types is critical. This paper describes a protocol for visualizing stem-like proliferating cells in the hydrozoan jellyfish Cladonema pacificum. Cladonema medusae possess branched tentacles that continuously grow and maintain regenerative capacity throughout their adult stage, providing a unique platform with which to study the cellular mechanisms orchestrated by proliferating and/or stem-like cells. Whole-mount fluorescent in situ hybridization (FISH) using a stem cell marker allows for the detection of stem-like cells, while pulse labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), an S phase marker, enables the identification of proliferating cells. Combining both FISH and EdU labeling, we can detect actively proliferating stem-like cells on fixed animals, and this technique can be broadly applied to other animals, including non-model jellyfish species.

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2&#39;-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected.&#160;