这手稿是致力于到文澜王博士的记忆2011年05月26日，通过了。我们感谢道格拉斯A·克尔博士慷慨提供的HBG3 MES在这项研究中使用的细胞。这项工作是由内穆尔，赠款（2 RR016472-10）根据国家研究资源中心（NCRR），和1 COBRE补助金从NCRR（5 P20的RR020173-05），以支持中心的INBRE方案儿童，美国特拉华州威尔明顿，在阿尔弗雷德一，杜邦医院的儿科研究。

1。步骤1：小鼠胚胎干细胞（MES）文化（定时：3天） 1。电镀主小鼠胚胎成纤维细胞（PMEF） <ol><li>大衣4个100毫米组织培养皿中，用明胶PMEF粘附。加入8毫升0.1％的明胶溶液（干细胞），每道菜，并在室温下30分钟的孵育。 </li><li>去除多余的明胶从菜肴。不要冲洗碗碟。 </li><li>稀包含约一小瓶丝裂霉素C灭活PMEF（Millipore公司）。 5×10 6细胞（配方见表1），到PMEF媒体40毫升到4个100毫米组织培养皿板。 PMEF提供一个mES细胞的饲养层。 </li><li> PMEF应镀加入MES细胞培养前至少一天。 PMEF可使用1周后镀。 </li></ol> 2。电镀MES细胞监察MES细胞分化成运动神经元的效率，我们使用HBG3，下控制运动神经元特异性启动子HB9表达EGFP转基因小鼠的胚胎干细胞系。 <ol><li>除霜1小瓶的HBG3 mES细胞在37℃水浴中（约1×10 7，由道格拉斯·克尔博士，美国约翰霍普金斯大学提供）。加入5毫升和15毫升管ES介质（配方见表1）降速为5分钟800转的细胞。 </li><li>吸出上清液，重悬MES细胞的ES媒体20毫升新鲜新增的白血病抑制因子（LIF），在终浓度为10毫微克/毫升。注：应添加LIF的一天的使用，并须保持在未分化状态的细胞MES。 </li><li>从每道菜的PMEF 10的毫升PMEF媒体和添加10毫升的HBG3胚胎干细胞悬液，每道菜。 </li><li> 24小时后，电镀，MES细胞形成小菌落，大小类似箭头表示在图1B殖民地。 72小时后，电镀，coloniES尺寸的增加，许多直径约100微米（ 图1B，用箭头表示）。这些细胞分化的准备。媒体应每天更换，以维持细胞的增殖。 </li></ol> 2。第2步：神经诱导（定时：2天） 诱导分化成运动神经元的MES细胞，mES细胞需要分开从PMEF和悬挂的环境中培养。明胶涂层烧瓶用于分离mES细胞PMEF。 <ol><li>当MES殖民地准备神经诱导（分化第0天），涂0.1％明胶（18毫升/瓶），在室温为30分钟2 T150烧瓶，然后去除多余的明胶和用1X PBS三倍。注：每100毫米的菜文化需要一个T150-明胶涂层烧瓶分离PMEF。 </li><li>小心取出，通过抽吸的介质，确保不打跑松散的连接菌落。添加10毫升PBS简要LY然后愿望删除完成清洗步骤。 </li><li>分开从PMEF的MES细胞，加入0.25％胰蛋白酶/ EDTA（3毫升/碟，干细胞技术），并在37°C孵育3-5分钟，直到干细胞和PMEF脱离板。无LIF和吸管细胞加入15毫升新鲜的ES媒体向上和向下突破殖民地（约15-20倍）。然后，添加0.1％的明胶涂层T150烧瓶，孵育30分钟，在37°C。孵化后，PMEF应重视糊化表面，而胚胎干细胞应漂浮。收集到50毫升管浮动mES细胞。 </li><li>降速在10毫升的神经分化培养基（配方见表1），在800 rpm和重悬细胞。 </li><li>使用到100毫米的细菌或暂停培养皿1血细胞计数板约2×10 6 mES细胞的细胞计数。这些板块允许悬浮培养的生长，促进形成胚状体（EBS）。 </li><li>上分化TION第1天，MES细胞形成的小浮动总量（EBS），光学显微镜下可见。任何结转PMEF附加菜。悬浮细胞和媒体转移到了15​​毫升管和低速离心3分钟（500转）。仔细吸干媒体，添加10毫升新鲜的神经分化培养基颗粒胚，然后板细胞中一种新的细菌菜。在除了补益媒体，这一步删除任何PMEF，从之前的步骤进行。 </li><li>分化第2天，EBS是随时可以诱导成运动神经元。 </li></ol> 3。第三步：运动神经元规格（定时：5天） <ol><li>在分化第2天，漩涡培养皿和15毫升管转移到媒体和EBS。让胚比重（约10分钟），或在低速离心3分钟（500转）定居。 </li><li>在10毫升（国防部）中型运动神经元分化的培养基认真吸重悬胚（见配方<strong&gt;表1）。在一个新的细菌菜盘EBS和媒体每天为5天。 </li><li>在分化第7天，EBS应该在直径约150 - 200微米，并应对此表示强烈的绿色荧光信号（ 图1C）。这些胚准备涂层菜或盖玻片上poly-DL-ornithine/laminin分离和镀金。 </li></ol> 4。第4步：制备Poly-DL-ornithine/laminin涂层盖玻片（定时：2天） 前两天游离胚（即在分化第5天），准备poly-DL-ornithine/laminin-coated盖玻片。 <ol><li>地方12毫米的圆形盖玻片的24孔板底部（华纳仪器）。溶解聚-DL-鸟氨酸（Sigma-Aldrich公司），25毫升25毫克无菌双蒸水（D 2·H 2 O）的获得10倍的股票解决方案（1毫克/毫升）。当准备使用，使H 2 O 2与D 1/10稀释的聚-DL-鸟氨酸获得concent口粮100微克/毫升。加入0.5毫升，每4°C间良好的24孔板孵育过夜。 </li><li>翌日（分化第6天），吸聚-DL-鸟氨酸，让组织文化罩板和盖玻片空气干燥。 D 2 H 2 O三次冲洗液体。让我们为一个小时，空气干燥。 </li><li> 1毫克小鼠层粘连蛋白（Millipore公司）溶于5毫升冰冷的1×PBS 100X原液（200微克/毫升）。当准备使用，使在冰冷的1X PBS（粘连2微克/毫升）稀释1/100。添加0.5毫升/ 24孔板孵育4℃过夜。播种前细胞，多余的粘连应该被删除，并两次用1X PBS冲洗井。盖玻片可以存储在国防部中等（0.5毫升/孔）。 </li></ol> 5。第5步：轴突伸长率（执行时间：2天） <ol><li>在分化的第7天，收集到15毫升管EBS和允许胚定居（约3分钟）。抽吸媒体和重新-S在PBS uspend胚。允许胚定居，然后吸PBS。重复此步骤3次。加入1毫升到EBS Accumax（Millipore公司），轻轻混匀，静置室温5分钟。然后吸过剩Accumax涵盖了EBS。 </li><li>游离EBS，添加3毫升国防部介质。吸取了上下20次使用1毫升的Eppendorf微量（蓝色提示）扰乱胚。孵育室温2分钟。悬浮细胞与细胞过滤帽（屋宇署猎鹰）转移到12x75毫米管，获得单细胞悬液。重复此剩余的团块，并保留通过过滤器，以丰富的单细胞产量细胞的分离步骤。最后的单细胞悬液，将6毫升。 </li><li>轻轻混匀这个单细胞悬液，用血球计数细胞。稀释细胞密度2×10 5细胞每毫升入国防部培养基（辅以10毫微克/毫升每个脑源性神经营养因子GDNF的，睫状神经营养因子，和NT-3）。 24孔板含宝加入细胞悬液（0.5毫升/）ly-DL-ornithine/laminin涂层盖玻片（事先准备好的如上所述）。分化的细胞扩大培养后2天（ 图1D）长突起。 </li></ol> 6。代表结果 图1A显示了一个协议的轮廓。在第1步，MES细胞培养PMEF LIF的补充，以防止自发分化。在一般情况下，未分化的MES殖民地是圆而紧凑，并有明确界定的边缘。殖民地是不能彼此接触。通常情况下，应mES细胞分裂1:4的比例，每2〜3天。然而，每个人的细胞会以不同的速率增长，必须凭经验确定分流比。在图1B箭头指示后，在3天的PMEF饲养层培养mES细胞的典型外观。这些殖民地是大（直径在50-100微米），但仍保持轮和界定边缘第在这个阶段的殖民地分化或亚文化。在图1B箭头显示一个小的MES殖民地您通常电镀后24小时。电镀后4天，MES细胞变得杂草丛生。他们表现出一个扁平的外观和损失定义的边界，这表明他们已经开始分化。这种细胞分化成神经细胞的最佳。 mES细胞分化成运动神经元，mES细胞需要培养悬浮条件允许的EB形成。在第2步，胚胎干细胞被转移到无饲养层，以促进EB形成一种文化表面。在这一步，他们培养Noggin的，碱性成纤维细胞生长因子，FGF-8辅以2天直接向神经系细胞的神经分化中等。小EBS可以分化第1天在显微镜下观察。他们应漂浮在培养基。也可以在第1天发现结转PMEF分化，必须拆除。坚持这些细胞被淘汰时胚代到一个新的细菌菜细菌菜肴。因此，分化2天，很少或根本没有PMEF应看到，在培养皿中。在第3步，继续EBS转移到新的菜肴，应确保去除任何剩余PMEF的。胚培养运动神经元分化（国防部）与RA补充媒体和凹陷分化成运动神经元细胞为5天。在培养过程中，EBS继续扩大规模，并可以通过肉眼看到，在分化第3天〜4次。 图1C显示EBS的分化第7天的微观外观。不同的是未分化的细胞，EBS显示较强的荧光，由于GFP的表达。这些胚是最佳的分化和分离的准备。流式细胞仪检测表明，51％±0.8％游离胚细胞分化成绿色荧光蛋白表达细胞5。在步骤5中，日文化ESE电机神经元GDNF的，睫状神经营养因子，脑源性神经营养因子，延长长期性轴索预测和NT3结果存在。 图1D显示分化细胞分离胚电镀后第2天的外观。注意镀细胞的细胞体延伸的长突起。免疫荧光染色显示，GFP +细胞表达泛神经元标记（神经丝蛋白中链）和两个运动神经元的特异性标志物，（胰岛-1和胆碱乙酰转移酶， 图2）。 <img alt="图1" src="/files/ftp_upload/3813/3813fig1.jpg" /> 图1。mES细胞分化成运动神经元（从武等图片修改。5）。 a）计划mES细胞运动神经元的分化协议。二）MES的未分化细胞形成的成纤维细胞饲养层的顶部的圆形菌落。他们有弱的绿色荧光蛋白表达。 c）MES细胞分开饲养层和cultu在低附着菜红，形成胚。在第一天的分化过程中，细胞暴露于50毫微克/毫升Noggin的20毫微克/毫升碱性成纤维细胞生长因子，20 ng / ml的FGF-8。随后，他们被诱导分化为1μm维甲酸和1μM的5天的刺猬激动剂凹陷。 MES的分化细胞中表达GFP荧光强。 d）经过7天的分化，胚分离poly-DL-ornithine/laminin涂层板镀。分化的细胞延长2天中文化后的很长的神经过程。比例尺= 200微米。百万=电机的神经元。 B，C和D是亮场图像和乙&#39;，C&#39;和D&#39;是相应的荧光图像。 <img alt="图2" src="/files/ftp_upload/3813/3813fig2.jpg" /> 图2 MES细胞衍生的运动神经元表征。免疫荧光染色神经丝蛋白中链NF-M（红色），聊天（红色），胰岛-1（红色）与GFP（绿色）expr的重合分裂国家。的DAPI（蓝色）被用来确定原子核。比例尺= 50微米。

<ol>
	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+motoneurons+derived+from+mouse+embryonic+stem+cells.">Functional properties of motoneurons derived from mouse embryonic stem cells.</a> J. Neurosci. 24, 7848-7858 (2004).</li><li>Wichterle, H., Peljto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+to+spinal+motor+neurons.">Differentiation of mouse embryonic stem cells to spinal motor neurons.</a> Curr. Protoc. Stem Cell. Biol. Chapter 1, Unit 1H.1.1-Unit 1H.1.9 (2008).</li><li>Kiris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem Cell Res. 6, 195-205 (2011).</li><li>Wu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+assessment+of+a+cell+model+of+spinal+muscular+atrophy.">Proteomic assessment of a cell model of spinal muscular atrophy.</a> BMC Neurosci. 12, 25 (2011).</li><li>McMahon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin-mediated+antagonism+of+BMP+signaling+is+required+for+growth+and+patterning+of+the+neural+tube+and+somite.">Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite.</a> Genes Dev. 12, 1438-1452 (1998).</li><li>Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+posterior+neural+tissue+is+induced+by+FGF+in+the+chick+embryo.">Early posterior neural tissue is induced by FGF in the chick embryo.</a> Development. 125, 473-484 (1998).</li><li>Launay, C., Fromentoux, V., Shi, D. L., Boucaut, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+FGF+receptor+blocks+neural+induction+by+endogenous+Xenopus+inducers.">A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.</a> Development. 122, 869-880 (1996).</li><li>Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purmorphamine+activates+the+Hedgehog+pathway+by+targeting+Smoothened.">Purmorphamine activates the Hedgehog pathway by targeting Smoothened.</a> Nat. Chem. Biol. 2, 29-30 (2006).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14071-14076 (2002).</li><li>Lee, S. M., Danielian, P. S., Fritzsch, B., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+FGF8+signaling+from+the+midbrain-hindbrain+junction+regulates+growth+and+polarity+in+the+developing+midbrain.">Evidence that FGF8 signaling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.</a> Development. 124, 959-969 (1997).</li></ol>

没有利益冲突的声明。

mES细胞的质量是最关键的参数为运动神经元的高效发电。 mES细胞必须培养，对PMEF防止自发分化。此外LIF的帮助mES细胞保持在未分化状态。由于MES细胞分裂每12至15小时，培养基变得枯竭迅速，必须每天更换。 高效活化Shh的途径是一个关键的参数，需要引起运动神经元的规范。 Shh蛋白（研发体系），Shh信号激动剂，如HH激动剂HhAg1.3（Curis公司），purmorphamine（Calbiochem公司），?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td> PMEF </td><td> Millipore公司</td><td> PMEF - H </td><td>不适用</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF中等 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11965-118 </td><td>不适用</td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 16140-071 </td><td> 10％ </td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司</td><td> 25030-081 </td><td> 1％ </td></tr><tr><td>青霉素/ </td><td> Invitrogen公司</td><td> 15240-063 </td><td> 1％ </td></tr><tr><td></td><td></TD&gt; <td></td><td></td></tr><tr><td> MES中等 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td>干细胞技术</td><td> 36250 </td><td>不适用</td></tr><tr><td> ES细胞合格的胎牛血清</td><td> Invitrogen公司</td><td> 16141-079 </td><td> 15％ </td></tr><tr><td> GlutaMax我</td><td> Invitrogen公司</td><td> 35050-061 </td><td> 1％ </td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司</td><td> 11140-050 </td><td> 1％ </td></tr><tr><td>核苷</td><td> Millipore公司</td><td> ES-008 - D </td><td> 1％ </td></tr><tr><td> β-巯基乙醇</td><td> Millipore公司</td><td> ES-007-E的</td><td> 0.1毫米</td></tr><tr><td>青霉素/ </td><td> Millipore公司</td><td> TMS-AB2型 - C </td><td> 1％ </td></tr><tr><td> LIF的</td><td> Millipore公司</td><td&gt; LIF2010 </td><td> 10纳克/毫升</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 神经分化培养基 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td>干细胞技术</td><td> 36250 </td><td>不适用</td></tr><tr><td> ES的邪教胎牛血清</td><td>干细胞技术</td><td> 06905 </td><td> 15％ </td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司</td><td> 11140-050 </td><td> 1％ </td></tr><tr><td>单硫代甘油</td><td> Sigma-Aldrich公司</td><td>的M-6145 </td><td> 1MM </td></tr><tr><td> Noggin与</td><td> Invitrogen公司</td><td> PHC1506 </td><td> 50毫微克/毫升</td></tr><tr><td> FGF-8 </td><td> Invitrogen公司</td><td> PHG0274 </td><td> 20毫微克/毫升</td></tr><tr><td>碱性成纤维细胞生长因子</td><td> Invitrogen公司</td><td> PHG0024 </td><td> 20毫微克/毫升</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 国防部介质（分化） </td><td></td><td></td><td></td></tr><tr><td> ES邪教的基础培养基- </td><td>干细胞技术</td><td> 5801 </td><td>不适用</td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828-028 </td><td> 10％ </td></tr><tr><td>的N-2的补充</td><td> Invitrogen公司</td><td> 17502-048 </td><td> 1％ </td></tr><tr><td>其补编-B的</td><td>干细胞技术</td><td> 07155 </td><td> 1％ </td></tr><tr><td>抗坏血酸</td><td>干细胞技术</td><td> 07157 </td><td> 1％ </td></tr><tr><td>青霉素/ </td><td> Millipore公司</td><td> TMS-AB2型 - C </td><td> 1％ </td></tr><tr><td> GlutaMax我</td><td>Invitrogen公司</td><td> 35050-061 </td><td> 1％ </td></tr><tr><td> D-葡萄糖</td><td> Sigma-Aldrich公司</td><td> G - 8270 </td><td> D 2 H 2 O 0.15％ </td></tr><tr><td>纤维连接蛋白</td><td>干细胞技术</td><td> 07159 </td><td> 5微克/毫升</td></tr><tr><td>肝素</td><td> Sigma-Aldrich公司</td><td> H3149 </td><td> 20 D 2微克/毫升氢 Ø </td></tr><tr><td> β-巯基乙醇</td><td> Millipore公司</td><td> ES-007-E的</td><td> 0.1毫米</td></tr><tr><td>全反式维甲酸</td><td> Sigma-Aldrich公司</td><td>的R-2625 </td><td> 1 mM </td></tr><tr><td>凹陷</td><td>电解二氧化锰化学品</td><td> 566660 </td><td> 1 mM </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 国防部介质（运动神经元的文化） </td><td></td><td></td><td></td></tr><tr><td> ES邪教的基础培养基- </td><td>干细胞技术</td><td> 5801 </td><td>不适用</td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828-028 </td><td> 10％ </td></tr><tr><td>的N-2的补充</td><td> Invitrogen公司</td><td> 17502-048 </td><td> 1％ </td></tr><tr><td>其补编-B的</td><td>干细胞技术</td><td> 07155 </td><td> 1％ </td></tr><tr><td>抗坏血酸</td><td>干细胞技术</td><td> 07157 </td><td> 1％ </td></tr><tr><td>青霉素/ </td><td> Millipore公司</td><td> TMS-AB2型 - C </td><td> 1％ </td></tr><tr><td> GlutaMax我</td><td> Invitrogen公司</td><td> 35050-061 </td><td> 1％ </td></tr><tr><td> D-葡萄糖</td><td> Sigma-Aldrich公司</td><td> G - 8270 </td><td> D 2 H 2 O 0.15％ </td></tr><tr><td>纤维连接蛋白</td><td> StemCELL技术</td><td> 07159 </td><td> 5微克/毫升</td></tr><tr><td>肝素</td><td> Sigma-Aldrich公司</td><td> H3149 </td><td> 20 D 2微克/毫升氢 Ø </td></tr><tr><td> β-巯基乙醇</td><td> Millipore公司</td><td> ES-007-E的</td><td> 0.1毫米</td></tr><tr><td>脑源性神经营养因子</td><td> R＆D系统</td><td> 248-BD-005/CF </td><td> 10纳克/毫升</td></tr><tr><td>睫状神经营养因子</td><td> R＆D系统</td><td> 257-NY-010/CF </td><td> 10纳克/毫升</td></tr><tr><td> GDNF的</td><td> R＆D系统</td><td> 212-GD-010/CF </td><td> 10纳克/毫升</td></tr><tr><td> NT-3 </td><td> R＆D系统</td><td> 267-N3-005/CF </td><td> 10纳克/毫升</td></tr></tbody></table> ，NA =不适用。 表1。MES细胞培养或分化PMEF和媒体。 <table border="1"><tbody><tr><td> &lt;强&gt;试剂名称</td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td> 0.1％的明胶</td><td>干细胞技术</td><td> 07903 </td><td>不适用</td></tr><tr><td>聚-DL-鸟氨酸</td><td> Sigma-Aldrich公司</td><td> P0421 </td><td>在D 0.1毫克/毫升2 H 2 O </td></tr><tr><td>鼠标粘连</td><td> Millipore公司</td><td> CC095 </td><td> 2微克/毫升的PBS </td></tr><tr><td> 0.25％胰蛋白酶/ EDTA </td><td>干细胞技术</td><td> 07901 </td><td>不适用</td></tr><tr><td> accumax </td><td> Millipore公司</td><td> SCR006 </td><td>不适用</td></tr></tbody></table> ，NA =不适用。 表2。用于涂料和分离试剂。

efficient differentiation of mouse embryonic stem cells into motor neurons

功能运动神经元分化成胚胎干细胞（ES细胞）的直接代表一个有前途的资源，研究疾病机理，筛选新的药物化合物，并制定，如脊肌萎缩症（SMA）和肌萎缩性侧索硬化症的运动神经元疾病的新疗法（ ALS）的。目前许多协议使用维甲酸（RA）和刺猬（SHH）的组合分化成运动神经元1-4小鼠胚胎干细胞（MES）。然而，mES细胞分化成运动神经元的效率只达到中等成功。我们已经开发了一个两步分化协议5，显着提高目前建立的协议相比，分化效率。第一步是Noggin与成纤维细胞生长因子（FGFs）的加入，以加强neuralization过程。 Noggin与骨形态发生蛋白（BMP）受体拮抗剂和被牵连在神经诱导1ccording神经发生和结果形成前神经图案6默认模型。 FGF信号协同诱导神经组织的形成，促进后神经身份7-9 Noggin的行为。在这一步，mES细胞被引两天，以促进对神经谱系分化与Noggin的，碱性成纤维细胞生长因子，FGF-8。第二步是诱发运动神经元的规范。 Noggin与/ FGFs暴露mES细胞培养与RA和Shh的激动剂，理顺激动剂（凹陷），再过5天，以促进运动神经元生成。为了监测乱七八糟的分化成运动神经元，我们使用胚胎干细胞系，从转基因小鼠表达EGFP派生控制下运动神经元特异性启动子HB9 1。使用这个强大的协议，我们实现了51±0.8％的分化效率（N = 3，P &lt;0.01，学生t-检验）5。从免疫的结果染色表明，绿色荧光蛋白+细胞表达运动神经元的特异性标志物，胰岛-1和胆碱乙酰转移酶（CHAT）。我们两步分化协议mES细胞分化成脊髓运动神经元提供了一条有效途径。

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

我们开发了一个新的协议，以提高效率，在体外分化成运动神经元的小鼠胚胎干细胞。分化的胚胎干细胞获得运动神经元功能的神经元和运动神经元采用免疫组化技术标记表达证明。

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

efficient-differentiation-mouse-embryonic-stem-cells-into-motor

Research

JoVE Journal

Biology

22.1K 次观看.  Alfred I. duPont Hospital for Children. 我们开发了一个新的协议，以提高效率，在体外分化成运动神经元的小鼠胚胎干细胞。分化的胚胎干细胞获得运动神经元功能的神经元和运动神经元采用免疫组化技术标记表达证明。

视频: Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. 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 tumorigenicity6-8 (see a schematic in Fig. 1A). 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 problematic9-11. 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 molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...