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。 在这里，我们看好应用RA，并在悬滴文化开始诱导神经分化。这种修改允许等于RA暴露于胚胎干细胞时，它们仍然是在一个单一的细胞悬浮液，EB聚合之前。当RA加到聚合的EB，将细胞在EB内部物料很可能感测到较低的RA concentrati上比外层的细胞。在EB聚合开始RA中的应用也是有利的，因为它抑制内胚层和中胚层胚层发育有利于外胚层层7,8的神经分化的。我们证实，RA治疗通过检查的近丰10点17的标记促进神经分化。 还有在这个协议三个关键因素。第一个是最大去除MEF饲养细胞上，实现了高纯度的ESC人口。第二个是RA的光灵敏度:其原液和悬滴必须从光后RA应用进行保护。 RA原液仅两周，在这之后，新的解决方案必须准备是稳定的。第三个考虑因素是RA浓度。在我们的试点实验中，我们发现，在10μMRA延缓细胞生长和亲的浓度duced较小尺寸的EB相比浓度较低，这可能是因为RA可以在高conncentrations 22,23引起的细胞凋亡，24。我们观察到0.5μm的RA浓度产生了比在任一高25个或更低的浓度合式EB的数量较多，且将EB达到的大约200微米的平均直径。较大的EB超过10X物镜的场尺寸和分别为，因此，更难图像。因此，我们选择0.5μM作为最佳RA浓度。 因为它们是冷冻切片太脆分析3D EB的免疫是有问题的。此外，我们发现，冷冻EB部分不坚持好到无涂层的静电处理过的载玻片上。 2D EB文化的制备需要额外的悬滴的聚集，因为有些EB中不能很好地附着在基材上。 EB迪ssociation，这是相对慢的，可以通过它们与胶原酶孵育期间向上和向下在1.5mL微离心管中吸移将EB来加速。 Neurally分化的胚胎干细胞可以用来替换受损的内源性细胞，在帕金森氏病26患者大脑黑质 如失去多巴胺能神经元。虽然人类胚胎干细胞分化的当前技术不需要EB形成（ 同上 ），EB的仍然是在分子水平上神经分化的详细分析的有用工具。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Materials</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&#160;Centrifugal Filters (10 kDa)</td>
			<td>EMD Millipore</td>
			<td>MRCPRT010</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Reagents</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&#38;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&#160;</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&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>R&#38;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>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Primary Antibodies</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>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Secondary Antibodies</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&#160;</td>
			<td>Life Tech.</td>
			<td>A21202</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Instruments</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，如果三亚的神经分化- / -胚胎干细胞相比，他们的三亚+ / +同行更快。 为了比较SYX的初始状态+ / +和SYX - / -胚胎干细胞，我们在每个定量基因型的Oct4，Nanog和SOX2，核心转录因子T的丰度帽子赋予ESC自我更新和多能性。在协议中的步骤10所描述的，胚胎干细胞，通过的Oct4，Sox2和Nanog抗体免疫标记。在SYX + / +和SYX 3个转录因子的丰度- / -胚胎干细胞相似，如通过免疫荧光（ 图2）和免疫印迹17（ 图2）来确定。 在3D胶原基质植入的EB（ 图1B）开始发芽是在具有10X物镜，在植入后第2天的细胞培养物的显微镜可见的细胞扩展。在第6天，5至10豆芽200微米或更小可以在SYX + / +的EB正常观察到的，而在SYX之间- / - EBS，30-50豆芽经常观察到的，其中大部分是长于200μm的，（ 图1C）。 SYX - / -比从SYX + / + 2D的EB（ 图3A）。为了比较来自SYX + / +和SYX延长细胞的神经分化的速率- / -的EB中，我们通过神经干细胞标记物巢蛋白，中间丝调节蛋白19免疫标记他们后2D培养的6天。巢蛋白的丰度为在从SYX延伸细胞显着更高的- / -的EB（ 图3B）。然后，我们比较巢蛋白和微管蛋白β3的丰度（Tubβ3），轴突细胞骨架蛋白20，在从13天的EB 2D解离的细胞。这两种蛋白质均从SYX解离的细胞更丰富- / -的EB比在其SYX + / +对应（ 图3C）。我们通过immunoblo的量化得到类似的结果相同的蛋白17的拟合。 图4示出由组成型绿色荧光蛋白（GFP）转染的细胞-融合活性的RhoA（的RhoA-Q63L），使用干细胞最佳转染试剂（参见材料/设备数据表 ）。 <img alt="图1" src="/files/ftp_upload/55621/55621fig1.jpg" /> 图1:从SYX + / +和SYX萌芽出苗- / - EBS在3D培养。 （A）SYX + / +和SYX - / -胚胎干细胞在生长的菌落簇神经分化的诱导之前。 （B和C）中形成的悬滴与0.5μMRA EB的图像，然后插入到3D胶原基质没有RA，如步骤7.1-7.4中描述。图像由所指示的目标在第6天捕获（刻度尺b<html> ARS = 100微米，A和C，B中为200μm）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55621/55621fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55621/55621fig2.jpg" /> 图2:多能性核心转录因子的存在。代表性图像表示SYX + / +和SYX所指示的核心多潜能标志物的丰度- / -胚胎干细胞（比例尺=50μm）上。见杨等人。 17为免疫标记方法的细节（比例尺= 50微米; DAPI，2-（4-脒基苯基）-1H-吲哚-6-甲脒）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55621/55621fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 古尔3&quot; SRC = "/文件/ ftp_upload / 55621 / 55621fig3.jpg"/&gt; 图3:在EB细胞神经分化标志物的可视化。 2D EB的（A）相位图像中的边缘示出了细胞扩大从SYX更快- / -比从SYX + / +的EB（比例尺= 200微米）。 （B）免疫荧光图像示出了神经分化标志物巢蛋白是从第6天2D SYX扩增细胞更丰富- / -的EB比从其SYX + / +对应（比例尺=50μm）上。 （C）免疫荧光图像示出了神经分化标记物巢蛋白和Tubβ3均从13天3D SYX解离的细胞更丰富- / -的EB比从其SYX + / +对应（比例尺=50μm）上。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55621/55621fig3large.jpg" target="_blank">请点击此处争夺</a>WA更大的版本这个数字。 <img alt="图4" src="/files/ftp_upload/55621/55621fig4.jpg" /> 图4:转染试剂的效率的可视化。融合到GFP的免疫荧光图像，显示由组成型活性的RhoA转染胚胎干细胞的三个重复以说明在协议（比例尺=50μm）上的步骤9.5中使用的试剂的转染效率。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55621/55621fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

科研

发育生物学

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 ...

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 ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...