这项研究是由美国国立卫生研究院授予R01 HL119984到AH支持

1.培养小鼠胚胎成纤维细胞的（MEF中） <ol><li>制备MEF培养基，Dulbecco改良的Eagle氏培养基（DMEM，高葡萄糖），补充有15％胎牛血清（FBS）。 </li><li>外套百毫米细胞培养皿以在室温下（RT）30分钟0.5％的明胶溶液。 </li><li>计数使用流式细胞仪的MEF。除去明胶溶液，并立即倒入MEF培养基预先加热至37℃。迅速解冻丝裂霉素C处理的MEF的小瓶在37℃水浴中2分钟，然后播种每100mm明胶包被的培养皿2.8×10 6的MEF。如果使用其它尺寸的菜肴相应调整细胞数。孵育的MEF，在37℃，5％CO 2过夜。 </li><li>变化对第二天的媒介。培养2-3天，直至MEF层汇合。 </li></ol> 2.鼠标ESC文化<ol><li>制备ESC培养基，Iscove氏改良的Dulbecco氏培养基（IMDM），补充了15％FBS和103 U / mL的白血病抑制因子（LIF），0.1mM非必需氨基酸，55毫摩尔2-巯基乙醇，青霉素（100U / ml），链霉素（100微克/毫升），庆大霉素（200微克/毫升），和0.2％支原体抗生素。 </li><li>从在步骤1中制备的盘除去MEF培养基，并用37℃下预热的ESC培养基更换。 </li><li>除霜的ESC小瓶和种子细胞在MEF层的顶部。孵育在37℃，5％CO 2，直至胚胎干细胞达到汇合。 </li><li>编写包含的MEF融合单层新的细胞培养皿。到通道中的胚胎干细胞，用PBS洗一次，并用0.25％胰蛋白酶/在37℃，5％CO 2分离EDTA 2-5分钟。加入新鲜IMDM有15％FBS的分离细胞停止胰蛋白酶消化，在160×g下在室温下转移细胞悬液至15mL管中，离心5分钟。 </li><li>去除上清，重悬胚胎干细胞用新鲜的IMDM和通道中的细胞对每个新的MEF涂覆的培养皿的五分之一;孵育直至细胞达到汇合（通常4-5天）。 </li></ol> 3.撤回明胶包被的平板的MEF和胚胎干细胞培养<ol><li>一旦ECSS达到汇合，用0.25％胰蛋白酶/ EDTA分离它们，将MEF如在步骤2.4，重悬在新鲜的IMDM细胞，转移到非粘性细菌培养皿，并在37℃，5％CO孵育40分钟2。 </li><li>小心转移含有的MEF培养基的胚胎干细胞和一个新的明胶包被的平板使用5毫升吸管，避免反复吹打。留在培养皿中的细胞是MEF中，由于胚胎干细胞不坚持。 </li><li>通道的每3-4天的胚胎干细胞。较少见的传代可以减少ESC多能性。不超过60％汇合，因为它可能有利于分化。 </li><li>重复步骤3.2-3.3三次或更多，根据需要，直到MEF中不再检测通过细胞培养显微镜使用20倍的目标，以确保MEF中不存在。 </li><li>验证ESC多能性通过检查ESC培养以查看是否细胞形成致密的集落与典型ESC的多边形形态（ 图1A）。通过定量反转录酶聚合酶链式反应（qRT-PCR）17，17的免疫印迹，或核心的多能性转录免疫荧光试验细胞干性因子17（ 图2）。 </li></ol> 4. EB形成<ol><li>准备以10mM DMSO全反式 - RA贮备液，并等分入1.5mL的光保护的微量离心管中。解决的办法是在-80℃下长达两周稳定。避光保存它。 </li><li>胰蛋白酶化胚胎干细胞如在步骤2.4; replate在无LIF新鲜IMDM，作为单细胞悬浮液。 </li><li>计数使用血球细胞，并在IMDM与0.5μMRA制备5×10 5细胞/ mL的悬浮液。 </li><li>板100，每100毫米培养皿20-μL滴用8通道移液器和200μL的提示，转化菜，和填充用PBS倒盖，以防止干燥悬滴。避光含有RA-培养基。 </li><li>培养的EB在悬滴，在37℃，5％CO 2，4天。 </li></ol> 5. 2D EB文化<ol><li>外套12毫米圆形玻璃盖玻片上，用30微克/毫升纤连蛋白在室温下30分钟。放置每个盖玻片的24孔板的孔中，并且每孔加入1个毫升IMDM。 </li><li>收获3天生长由一个用200μL移液器吸头悬滴的EB一个和种子他们在纤连蛋白包被的盖玻片上，20级的EB每孔，使用相同的移液管尖端。 注意:因为他们更好地附着在盖玻片3天的EB超过4天的EB的优选。 </li><li>继续用IMDM-15％FBS培养无LIF。媒体更换，每3-4天。根据需要收集蛋白质分泌分析所使用的平台。 </li></ol> 6.分泌蛋白的检测<ol><li>转移EB中介的500微升微米至10kDa截止离心过滤器，旋在20000×g离心60分钟，4℃。 </li><li>检查剩余的离心过滤器内的每次15-20分钟，以防止过度丰富的媒介。停止离心当剩余介质的体积已经下降到25微升。 </li><li>反相滤波器，并在4℃以160×g下旋转时，按照制造商的说明书后收集剩余的浓缩介质。 </li><li>通过用特异性抗体进行免疫印迹17检测分泌的蛋白质。 </li></ol> 7. 3D EB文化<ol><li>收集在如步骤5.2中所述悬滴培养4天的EB，并将其放置在1.5毫升离心管（每管30个EBS）。 </li><li>以下所述3D胶原培养试剂盒的（见材料/设备表 ）中的说明制备胶原凝胶溶液。稀释的胶原溶液和5倍DMEM（试剂盒成分）适当卷;添加n个eutralization溶液（试剂盒成分）和井立即混合，并保持此冰上。 </li><li>吸取冷冻胶原溶液到含有的EB的1.5mL管中的适当体积，并轻轻地用1个毫升移液管尖端转移至6孔板的孔中。避免气泡。 </li><li>立即将板转移到37℃，5％CO 2，60分钟以引发聚合胶原。 </li><li>覆盖用IMDM含EB-板。 </li><li>更换培养基每3-4天。 </li></ol> 8. EB解离<ol><li>由一个收集一天4的EB（来自步骤4）之一，用200μL移液器吸头，并将它们传送到有15％FBS的非粘合含IMDM细菌培养皿;培养在37℃，持续4天以上5％CO 2。检查将EB每天两次，以确保他们不重视的底部;轻轻摇动培养皿防止EB附接至底部。 </li><li>离心机中以185×g下将EB在5分钟RT，在台式离心机。 </li><li>取出上清液。将沉淀不应超过在每个管100μL。 </li><li>添加到沉淀的EB 1毫升0.25％的I型每管胶原酶，补加在PBS中的20％FBS。 </li><li>孵育1个小时EB-胶原酶混合物在37℃，5％CO 2，轻轻吹打它每隔20分钟，用1ml移液管尖端。 </li><li>洗涤细胞，用PBS轻轻三次。如果细胞聚集体存在，使用细胞滤过器以100μm的网格，以除去它们。 </li><li> Replate在IMDM明胶包被60mm的培养皿的细胞。然后孵育在37℃，5％CO 2。 </li></ol> 9.转染分离性EB的<ol><li>对于qRT-PCR的和免疫印迹，涂布6孔板用0.5％明胶。 </li><li>对于免疫荧光，涂层的玻璃盖玻片上，用30微克/毫升纤连蛋白在室温下30分钟。放置每个盖玻片的24孔板的孔中。加入IMDM。 </li><li>种子4.75×10 5或1×10 5 </SUP&gt;解离的EB细胞（步骤8.4-8.7）每孔中的5,6或24孔板中，分别。 </li><li>转染前生长的细胞至70％汇合。 </li><li>由非脂质体脂类干细胞最佳试剂17转染细胞（见材料/设备 表 ），按照制造商的说明进行操作。 注意:我们使用的试剂通常达到50％的转染效率（参见代表性结果 ）。 </li><li>分析通过qRT-PCR或通过免疫印迹法分别转染后17 2〜3天，将样品。 </li></ol>通过免疫荧光EB分化10.分析<ol><li>洗涤2D的EB或解离的EB 3D用PBS并用4％多聚甲醛固定在PBS中在RT下30分钟。洗涤固定的细胞用PBS 3倍，以除去漂浮的细胞碎片。 注意:多聚甲醛是一种对皮肤和眼睛有刺激性;操作时务必小心。 </li&gt; <li>加入1％的Triton X-100的PBS透化20分钟将细胞在RT，然后用PBS洗3次。 </li><li>除去PBS并用PBS中5％BSA于30分钟阻断。 </li><li>制备在1％BSA的第一抗体稀释/ PBS补充有0.03％的Triton X-100。代替由初级抗体溶液中的封闭溶液。孵育3小时，在室温，或过夜，在4℃，然后洗细胞3次，用PBS。 </li><li>根据制造商的说明书在PBS申请第二抗体。在室温下孵育1个小时，然后洗涤细胞，用PBS 3倍。 </li><li>安装在玻璃载玻片上的盖玻片与光学显微镜的抗褪色平台。 </li></ol>

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作者宣称他们没有竞争或经济利益

在这个协议中，我们提出一个相对简单的和方便的方法来研究的鼠胚胎干细胞的神经分化。在先前的协议中，RA在第2天或EB悬滴8的第4天或由悬浮培养7加入到培养基中，分别或后立即EB悬滴聚集21。在我们设计的方案，RA较早加入。尽管较早引进RA的到的EB通过悬浮培养形成的，这个方案产生的神经分化标记8更高的表达。 <p c...

胚胎干细胞从胚泡内细胞团起源。这些细胞是多能的， 即它们具有分化成起源的生物体的任何细胞类型的能力。 ESC 体外分化为广泛兴趣，作为调查的发育途径和机制的实验系统。它提供了一个强有力的和灵活的模型系统来测试的细胞和组织功能障碍修正新的治疗方法。 EB的早期胚胎发育过程中重演细胞分化的许多方面。具体地，可以当胚胎致死性使得难以确定胚胎缺陷1,2的蜂窝式的基础上使用的EB。的EB可以通过悬滴或液体悬浮液技术3形成。前者的优点是，以产生一致的大小和密度的EB中，从而有利于实验的再现性的能力。 4识别的基底膜成分。 RA是诱导神经分化5,6维生素A的一小的亲脂性代谢物。高浓度的RA的促进神经基因表达和在EB形成7,8阻遏中胚层的基因表达。 RA是由维生素A的氧化通过任一醇或视黄醇脱氢酶，随后醛氧化成最终产品通过醛脱氢酶9醛生产。神经分化需要从细胞质RA的运输<html>由蜂窝RA-结合蛋白2（CRABP2）细胞核。在细胞核中，RA结合由一个RAR-RXR异二聚体10的其同源受体复合物。这导致招聘转录共激活因子和转录9,11的开始。此外，RA促进磷酸化的（活性）SMAD1的降解，从而拮抗BMP和Smad信号12。除了这些活动，RA增加Pax6的表达，支持神经分化13的转录因子。 RA信令由沉默调节蛋白1（Sirt1的），核烟酰胺腺嘌呤二核苷酸（NAD +）调制-即deacetylates CRABP2，以其易位干扰到细胞核依赖性酶，并因此与RA结合RAR-RXR异二聚体14,15 16。 e_content"&gt;我们在设计这里所述的RA处理的EB协议的目标是为了便于调节ESC分化成神经元前体细胞中的信号传导途径的体外分析，以优化神经分化，其中一个这种协议的优点之一是便利细胞功能的免疫荧光。3D的EB的分析不能很好地穿透抗体和难以成像。EB解离成在特定的时间点的2D单层期间神经分化通过共聚焦显微镜促进免疫标记和细胞的成像。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MEFs</td><td>EMD Millipore</td><td>PMEF-CF</td><td>ESC feeder layer</td></tr><tr><td>ESC</td><td>EMD Millipore</td><td>CMTI-2</td><td></td></tr><tr><td>Cell culture dish (60 mm)</td><td>Eppendorf</td><td>30701119</td><td>Cell culture</td></tr><tr><td>Cell culture dish (100 mm)</td><td>Falcon</td><td>353003</td><td>Cell culture</td></tr><tr><td>Petri dish (100 mm)</td><td>Corning</td><td>351029</td><td>Hanging drops</td></tr><tr><td>24-well plate</td><td>Greiner Bio-One</td><td>662160</td><td>2D EBs</td></tr><tr><td>6-well plate</td><td>Eppendorf</td><td>30720113</td><td>Transfection</td></tr><tr><td>Dark 1.5 mL centrifuge tube</td><td>Celltreat Scientific Products</td><td>229437</td><td>RA stock solution</td></tr><tr><td>Microscope cover-glass</td><td>Fisherbrand</td><td>12-545-80</td><td>Circular, 12 mm diameter</td></tr><tr><td>Superfrost-plus microscope slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>3D collagen culture kit</td><td>EMD Millipore</td><td>ECM675</td><td>3D culture</td></tr><tr><td>Effectene Transfection Reagent</td><td>Qiagen</td><td>301427</td><td>Stem cell transfection</td></tr><tr><td>Microcon&amp;nbsp;Centrifugal Filters (10 kDa)</td><td>EMD Millipore</td><td>MRCPRT010</td><td>Protein concentration</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-709F</td><td>MEFs culture</td></tr><tr><td>IMDM</td><td>Gibco</td><td>12440-046</td><td>ESCs culture</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>EMD Millipore</td><td>ES-009-B</td><td>ESCs culture</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G2625</td><td>Dish coating</td></tr><tr><td>LIF</td><td>R&amp;amp;D Systems</td><td>8878-LF-025</td><td>To maintain ESC pluripotency</td></tr><tr><td>MEM Non-Essential Amino Acids Solutions</td><td>Gibco</td><td>11140050</td><td>Cell culture</td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>Cell culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>Cell culture</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750060</td><td>Cell culture</td></tr><tr><td>MycoZap Plus-PR</td><td>Lonza</td><td>VZA-2022</td><td>Cell culture</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-072</td><td>Cell culture</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>All-trans-retinoic acid</td><td>Sigma-Aldrich</td><td>R2625-50MG</td><td>Induction of neural differentiation</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7030-50G</td><td>Blocking and antibody dilution&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-100ML</td><td>Cell membrane permeabilization</td></tr><tr><td>Cell strainer</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Prolong Gold anti-fade reagent with DAPI</td><td>Life Tech.</td><td>P36931</td><td>Mounting reagent</td></tr><tr><td>16% Paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Cell fixation</td></tr><tr><td>Fibronectin</td><td>R&amp;amp;D Systems</td><td>1030-FN</td><td>Dish coating</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington Biochem. Corp</td><td>LS004196</td><td>EB dissociation</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nestin (Rat-401)</td><td>Santa Cruz Biotech</td><td>sc-33677</td><td>Detection of neural differentiation</td></tr><tr><td>Oct4</td><td>Santa Cruz Biotech</td><td>sc-5279</td><td>Detection of neural differentiation</td></tr><tr><td>Nanog</td><td>Bethyl Laboratories</td><td>A300-398A</td><td>Detection of neural differentiation</td></tr><tr><td>Sox2</td><td>Cell Signaling</td><td>3579</td><td>Detection of neural differentiation</td></tr><tr><td>Tubulin b3 (AA10)</td><td>Santa Cruz Biotech</td><td>sc-80016</td><td>Detection of neural differentiation</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-Mouse-Alexa555</td><td>Life Tech.</td><td>A31570</td><td>Immunofluorescence</td></tr><tr><td>Donkey anti-mouse-Alexa488&amp;nbsp;</td><td>Life Tech.</td><td>A21202</td><td>Immunofluorescence</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Wide-field microscope</td><td>Nikon</td><td>Eclipse TS100</td><td>Cell culture imaging</td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>C2</td><td>Immunofluorescence imaging</td></tr></tbody></table>

analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies

从胚泡（通常在一天E3.5）的内质分离的小鼠胚胎干细胞（ESC），可以用作体外模型系统用于研究早期胚胎发育。在不存在白血病抑制因子（LIF）的，胚胎干细胞分化默认成神经前体细胞。它们可以积累成三维（3D）的球形聚集体称为胚状体（EB），由于其相似性的早期胚胎中。的EB可在纤连蛋白包被的盖玻片接种，在那里它们通过生长的二维（2D）扩展展开，或在3D胶原基质在那里继续成长为球状体，并分化成三个胚层注入:内胚层，中胚层和外胚层。三维胶原文化更加接近地模拟体内环境比2D日圆。所述2D EB培养通过免疫荧光和免疫印迹来跟踪分化促进分析。我们已经制定了两步神经不同点重刑协议。在第一步骤中，的EB通过悬滴技术生成的，并且，同时，被诱导通过暴露于视黄酸（RA）进行区分。在第二步骤中，在不存在RA的2D或3D格式神经分化前进。

OCT4，NANOG，SOX2和是赋予ESC自我更新和多能性的核心转录因子。我们应用上述协议ESC的神经分化从野生型和从那里SYX，一个基因编码的RhoA特定交换因子SYX，被破坏基因改造的小鼠的菌株进行比较。我们曾在18血管牵连三亚。我们注意到，在EB的行为差异从三亚+ / +和三亚汇总- / -胚胎干细胞，并且进行了测试，如?...

Watch this Scientific Journal Video about Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies at JoVE.com

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

我们描述了使用小鼠胚胎干细胞生成二维或三维的胚状体的技术。然后，我们解释如何以诱导胚状体细胞视黄酸，以及如何的神经分化由祖细胞标志物的免疫荧光和免疫印迹来分析其分化状态。

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

analysis-retinoic-acid-induced-neural-differentiation-mouse-embryonic

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.9K 次观看.  Sidney Kimmel Medical College, Thomas Jefferson University. 我们描述了使用小鼠胚胎干细胞生成二维或三维的胚状体的技术。然后，我们解释如何以诱导胚状体细胞视黄酸，以及如何的神经分化由祖细胞标志物的免疫荧光和免疫印迹来分析其分化状态。 

视频: Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

科研

发育生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Source: Ababon, M. R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indicating RA activity in the embryonic cells.

在共培养物中使用报告细胞研究小鼠胚胎细胞产生视黄酸

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

一种从小鼠胚胎干细胞生成神经祖细胞的技术

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Advanced Neuronal Models

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

Source: Leszczy&#324;ski, P., et al.,&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/58225">Neurogenesis Using P19 Embryonal Carcinoma Cells</a> J. Vis. Exp. (2019)
This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.