CACW得到医学研究理事会博士生的支持。 GMF部分由医学研究委员会新研究者奖（MR / N000609 / 1）和Tenovus苏格兰研究资助部分支持。

小分子激酶抑制剂库的整理<ol><li>来自可公开获得的激酶抑制剂收集物的分页抑制剂文库， 例如来自哈佛医学院的基于综合网络的细胞标记库（LINCS），228种有效和选择性激酶抑制剂的靶向收集物（http://lincs.hms.harvard.edu） ，和蛋白激酶抑制剂组（PKIS），由工业研究人员（https://www.ebi.ac.uk/chembldb/extra/PKIS）组装的376化合物收集品13 。或者，从市售化合物组装定制的激酶抑制剂收集物。 注意:在我们的实验室，我们汇集了一系列72种有效和选择性的工具激酶抑制剂，涵盖51种主要靶向激酶，所有这些都可从商业来源获得。 </li><li>将激酶抑制剂装配到96孔板中，以实现多种浓度的最佳高通量处理（使用1，0.1 and 0.01mM）。包括在含有DMSO的每个平板对照孔和参考对照抑制剂PD173074（FGFRi）和Ruxolitinib（JAKi）中，其分别促进幼稚和引发的多能态。在电子表格或类似软件中注释。 </li></ol> 2. mESC培养条件和筛选程序<ol><li>通过向10厘米平板中加入5毫升0.1％（w / v）明胶制备0.1％明胶包被的10厘米培养皿，并在室温下孵育5分钟。吸出明胶，让板干燥2分钟。 </li><li>在含有100ng / mL GST-LIF，10％胎牛血清和5％敲除血清的标准mESC培养基（参见材料表 ）中，在37℃5％CO 2的0.1％明胶包被的10cm培养皿上培养任何标准mESC系替代。每天更换培养基，通过mESCs，每隔约80％汇合，稀释1:5-1:10。 </li><li>通过mESCs，吸出培养基并用5 mL o洗涤f磷酸盐缓冲盐水（PBS）。 </li><li>加入每平板mESCs 1 mL胰蛋白酶EDTA（0.05％胰蛋白酶，0.02％EDTA），37℃孵育10 min。 </li><li>将胰蛋白酶处理的细胞重悬于4mL的mESC培养基中，并以1,200rpm离心5分钟。 </li><li>在5mL的mESC培养基中彻底重悬细胞沉淀，上下移液以产生单细胞悬液。 </li><li>通过将10μL细胞悬浮液和10μL台盼蓝（0.4％）组合计数细胞。放入细胞计数室或使用血细胞计数器和光学显微镜。 </li><li>将3×10 3个 mESC种子加入到0.1％明胶包被的96孔板中，最终体积为100μL的培养基，使用多通道移液管。 </li><li>使用多通道移液器，以1:100稀释（1μL抑制剂加入100μL培养基）中应用激酶抑制剂。轻轻移液培养基以混合抑制剂和细胞悬浮液，然后使细胞在组织培养罩中沉降1小时以确保相等分布acro电镀表面。培养细胞48h，不改变培养基。 注意:1 / 0.1 / 0.01mM的储备板将分别给出10/1 /0.1μM的最终抑制剂浓度。我们建议以1μM的最终浓度启动荧光屏，以确保有效抑制主要靶激酶，同时最大限度地减少目标效应。 </li></ol>激酶抑制剂筛选分析<ol><li>使用多通道吸气器和移液管在200μLPBS中洗涤96孔mESC平板。 </li><li>在150μL裂解缓冲液（20mM Tris pH7.4,150mM NaCl，1mM EDTA，1％NP-40（v / v），0.5％脱氧胆酸钠（w / v），10mMβ-甘油磷酸钠，10mM焦磷酸钠，1mM NaF，2mM Na 3 VO 4 ，完全蛋白酶抑制剂鸡尾酒片）。 </li><li>通过在V底96孔板中以1,500×g离心30分钟澄清提取物。 </li><li>将100μL上清液固定在硝基上lulose膜使用96孔真空斑点印迹歧管。 </li><li>干燥膜，并用40毫升Ponceau S染色，以确保一致的转印。 </li><li>用TBST洗涤膜，并在TBST / 3％（w / v）牛奶中封闭。 </li><li>以1:1,000（v / v）稀释于TBST / 3％（w / v）奶粉中的Nanog和Dnmt3b抗体孵育过夜。 </li><li>在TBST中以3×10分钟洗涤膜，并在TBST / 3％（w / v）牛奶中稀释1:10（v / v）的30mL二级抗体中孵育。 </li><li>开发使用数字免疫印迹成像系统。 注意:如果可能，使用定量荧光免疫印迹检测Nanog和Dnmt3b。然而，通过荧光免疫印迹对Dnmt3b的背景信号太高。因此，使用800nm荧光抗兔（Nanog）和抗小鼠HRP（Dnmt3b）二抗。 </li></ol> 4.筛选数据分析和抑制剂作为真菌多能调节剂的验证<ol><li>使用免疫印迹分析软件量化Nanog和Dnmt3b信号，并将值导入电子表格或类似软件。与DMSO对照相比，计算所有样品的Nanog:Dnmt3b比例。此外，确定总Nanog和Dnmt3b信号，以确保激酶抑制剂通过改变mESC活力不影响Nanog:Dnmt3b比例。 </li><li>使用排列的Nanog:Dnmt3b比率来鉴别促进幼稚多能性（Nanog:Dnmt3b比高于对照）和引发多能性（Nanog:Dnmt3b比低于对照）的抑制剂。将截止值设置为与对照值的2倍偏差，并过滤掉产生总Nanog + Dnmt3b信号的激酶抑制剂50％的DMSO对照值，以产生稳定初始（高Nanog:Dnmt3b比）和引发（低Nanog:Dnmt3b比）多能态的激酶抑制剂的高置信列表。 </li><li>通过在2mL培养基中每6孔以2×10 5个细胞接种mESC来验证激酶抑制剂，并在1μM下加入激酶抑制剂48小时，而不改变培养基。 </li><li> Lyse mESCs在裂解缓冲液中，通过常规SDS-PAGE免疫印迹分析Nanog和Dnmt3b表达。 </li><li>通过SDS-PAGE免疫印迹进一步验证初始多能性标记Klf2和Klf4以及Oct4，以确保48小时抑制剂治疗后保持多能性。 </li></ol>

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作者声明本研究没有产生利益冲突。

在这里，我们展示了一种广泛可用的方法来探索激酶信号通路在调节初始化多能转换中的作用。这解决了ESC字段中的一个关键问题。虽然高通量基因组学和转录组学方法常规用于鉴定初始和引发多能状态的关键转录调控因子，但是阐明多能性信号传导网络已被证明是具有挑战性的。我们现在提供一种灵活的策略，采用化学抑制剂来鉴定在mESCs中控制初始化多能转化的激酶。这取决于大多数研究细胞信号传导和ESC生物学实验室的简单设备，试剂和材料。对于这个平台的成功至关重要的是我们开发了一个强大的测定来量化初始和引发多能态之间的转变。建立该测定需要对细胞平台进行重大的迭代修饰抑制剂孵育时间和免疫印迹条件。 小分子方法在治疗性ESC应用15,16中得到合理使用。小分子筛选的主要优点是设计和/或修饰工具化合物以有效的细胞摄取。转染效率并不局限于此，不需要使用慢病毒等危险递送系统。此外，抑制剂经常抑制激酶家族内的多种同种型（ 例如 Fgfr1-4，Jak1-3），其克服阻碍基因/基因组干扰技术如RNAi和CRISPR / Cas9的功能冗余。由于学术和药物药物发现计划中提供了更有效和更有选择性的工具化合物，因此研究较少且知之甚少的激酶将被推向ESC研究的前沿。因此，我们建议这样高的粗略筛选方法将为核心ESC监管网络开辟新的研究途径。 该技术的一个小小的限制是即使是最有效和选择性的小分子抑制剂也可以在体内接触和抑制多种不相关的激酶。然而，日益全面的激酶抑制剂分析数据可以容易地鉴定激酶抑制剂的功能靶点（http://lincs.hms.harvard.edu/kinomescan; http://www.kinase-screen.mrc.ac.uk/kinase -inhibitors）。最后，原则上我们的平台可以应用于询问可获得高质量免疫印迹抗体的任何小分子收集和/或细胞测定。这将允许研究多能调节系统的多能调节和促进鉴定改变不同细胞过程的小分子抑制剂。具体来说，我们设想应用新兴的小分子收集物，如epigene由结构基因组学联盟（http://www.thesgc.org/chemical-probes/epigenetics）开发的tic探针有潜力揭示更多新型多能转录调控因子。

胚胎干细胞（ESCs）具有自我更新或分化成成体内任何细胞类型的能力，这种现象称为多能性1 。最近的证据表明，存在发育不同的多能性状态，称为"初始"和"初始化"多能性2 。初始胚胎干细胞代表与植入前胚胎相似的发育状态3 。相比之下，引发多能的胚胎干细胞准备退出多能性并分化成专门的胚胎谱系4,5 。 原始的多能性状态以不同的基因调控网络为特征。最初的多能性的特征在于表达关键的多能转录因子，如Nanog，Krueppel样转录因子（Klfs），Rex2和Esrrb <supclass ="xref"&gt; 6。在小鼠ESCs（mESCs）中，引发多能性的特征在于初始标记物的表达减少，特异性基因表达特征包括从头DNA甲基转移酶Dnmt3b 7 。 在体内 ，引发多能的植入后表皮细胞干细胞（EpiSCs） 4,5还附加表达Epiblast标记Fgf5和谱系引发标志物如Brachyury 8 。 mESCs提供了一种易于理解的模型，用于探索在体外控制初始化多能转录的机制 。当在白血病抑制因子（LIF）和胎牛血清（FBS）中培养时，mESCs在初始和引发的多能态之间经历动态过渡9,10 。 LIF-Jak-Stat3信号传导功能，促进幼稚基因调控网络11 </而通过成纤维细胞生长因子4（Fgf4）Erk1 / 2通路的自分泌信号驱动转变到引发状态12 。然而，系统地评估蛋白激酶在指定明显多能状态中的作用仍然是一个挑战。 在这里，我们描述了一个定量和可扩展的平台，通过该平台为初始化多能转换的激酶调节剂进行目标小分子筛选。我们使用简单的mESC培养条件和标准试剂，材料和设备来发现和验证激酶抑制剂，迄今为止尚未得到稳定的初始多能性的能力。此外，我们还讨论了该技术的潜在扩展应用，包括筛选其他小分子系列，例如靶向表观遗传调节因子的新兴抑制剂。

<table><tbody><tr><td>&lt;strong&gt;mESC media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CCE mESCs</td><td>ATCC</td><td>ATCC&amp;nbsp;SCRC-1023</td><td></td></tr><tr><td>DMEM Media</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td>Final: 2 mM</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>Final: 50 U/ml</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>Final: 0.1 mM</td></tr><tr><td>Leukemia Inhibitory Factor (GST fusion)</td><td>MRC-PPU reagents and services</td><td></td><td>Final: 100 ng/mL</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Thermo</td><td>PMC9484</td><td>Final: 100 ng/mL</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco</td><td>11140050</td><td>Final: 0.1 mM</td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360070</td><td>Final: 1 mM</td></tr><tr><td>KnockOut Serum Replacement</td><td>Invitrogen</td><td>10828-028</td><td>Final: 5%</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>Fisher</td><td>10703464</td><td>Final: 10%</td></tr><tr><td>&lt;strong&gt;Name&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;TC materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo</td><td>25300054&amp;nbsp;</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>1890</td><td></td></tr><tr><td>Nunc MicroWell 96-Well Microplates</td><td>Thermo</td><td>156545</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190136</td><td></td></tr><tr><td>V-bottom 96 well plates</td><td>Greiner Bio-one</td><td>651261</td><td></td></tr><tr><td>&lt;strong&gt;Name&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;Lysis Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris buffer</td><td>VWR</td><td>103157P</td><td></td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>97061</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E4884</td><td></td></tr><tr><td>1% NP-40</td><td>Sigma</td><td>9016-45-9</td><td></td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma</td><td>D6750-100g</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma</td><td>G9422</td><td></td></tr><tr><td>Sodium Pyrophosphate</td><td>Sigma</td><td>13472-36-1</td><td></td></tr><tr><td>Sodium Fluoride</td><td>Sigma</td><td>450022</td><td></td></tr><tr><td>Sodium Orthovanadate</td><td>Sigma</td><td>450243</td><td></td></tr><tr><td>Roche Complete Protease Inhibitor Cocktail Tablets</td><td>Sigma</td><td>CO-RO</td><td></td></tr><tr><td>&lt;strong&gt;Name&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;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tween</td><td>Sigma</td><td>P1379-1L</td><td></td></tr><tr><td>Milk Powder</td><td>Marvel</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&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;Western Blotting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Protran BA 85 Nitrocellulose 0.45 &amp;micor;M&amp;nbsp; Pore size (20 cm x 20 cm Sheets) (25 Sheet)</td><td>GE</td><td>10401191</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P7170-1L</td><td></td></tr><tr><td>&lt;strong&gt;Name&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;Laboratory Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Minifold I System</td><td>GE</td><td>10447850</td><td></td></tr><tr><td>ChemiDoc imager</td><td>BioRad</td><td>1708280&amp;nbsp;</td><td></td></tr><tr><td>LiCor Odyssey Imager</td><td>LiCor</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&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;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse Nanog</td><td>Reprocell</td><td>RCAB001P</td><td></td></tr><tr><td>Anti-Dnmt3b</td><td>Imgenex</td><td>IMG-184A</td><td></td></tr><tr><td>IRDye 800CW Donkey anti-Rabbit</td><td>Licor</td><td>926-32213</td><td></td></tr><tr><td>Anti-mouse-HRP</td><td>Cell Signalling Technology</td><td>7076S</td><td></td></tr><tr><td>Anti-Klf2</td><td>Millipore</td><td>09-820</td><td></td></tr><tr><td>Anti-Klf4</td><td>R&amp;amp;D Systems</td><td>AF3158</td><td></td></tr><tr><td>Anti-Oct4</td><td>Santa Cruz</td><td>sc-5279</td><td></td></tr></tbody></table>

a simple method to identify kinases that regulate embryonic stem cell pluripotency by high-throughput inhibitor screening

胚胎干细胞（ESC）可以自我更新或分化成所有的细胞类型，这种现象称为多能性。已经描述了不同的多能性状态，称为"天真"和"初始化"多能性。控制初始化过渡的机制了解甚少。特别是，尽管提供了高质量的工具化合物探索激酶功能的可用性越来越多，但我们对于指定初始和初始多能性状态的蛋白激酶仍然知之甚少。在这里，我们描述了一个可扩展的平台，用于对小鼠胚胎干细胞幼虫初始化多能转化激酶调节剂进行靶向小分子筛选。这种方法利用简单的细胞培养条件和标准试剂，材料和设备来发现和验证激酶抑制剂，迄今为止对多能性的不受欢迎的影响。我们讨论该技术的潜在应用，包括筛选其他小分子诸如越来越复杂的激酶抑制剂和表观遗传工具化合物的新兴文库的集合。

使用本文提供的程序（ 图1 ），我们筛选了228种有效和选择性激酶抑制剂的LINCS文库，以鉴定调节mESC多能性的那些。以1:100稀释浓度0.1mM的浓度制备文库，并在mESCs中最终筛选浓度为1μM。 48小时后，将mESCs溶解，制备提取物用于Nanog和Dnmt3b表达的定量斑点印迹分析的提取物（ 图2 ，上图 ）。为了简洁起见，其他筛选浓度的结果不在此列出。 Nanog:测定每种激酶抑制剂的Dnmt3b比例，以及应用的化合物和2x截止（ 图2 ，底部）。总的Nanog和Dnmt3b相对于对照的信号覆盖在抑制剂排名上并进行0.5x的切断，以允许显示出显着的mESC毒性的抑制剂的分类。选择稳定初始状态的激酶抑制剂te使用常规Nanog / Dnmt3b免疫印迹进行验证（ 图3 ）。这些抑制剂的主要激酶靶标列于表1 。我们还展示了该屏幕的适用性，以确定稳定起始状态的抑制剂14 。 <img alt="图1" src="/files/ftp_upload/55515/55515fig1.jpg" /> 图1:筛选激酶抑制剂的工作流程，调节初始化过渡。 mESC用终浓度为1μM的228复合激酶抑制剂文库处理。制备裂解物并转移到硝酸纤维素膜上用于免疫斑点印迹分析，并测定Nanog和Dnmt3b水平（图中改编自Williams 等人 14 ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55515/55515fig1large.jpg" target="_blank">请点击这里查看al</a><html>这个数字的arger版本。 <img alt="图2" src="/files/ftp_upload/55515/55515fig2.jpg" /> 图2:初始化多能性筛选分析和抑制物鉴定。 （Top）代表性的Nanog和Dnmt3b免疫印迹图像。 （底部）相对于DMSO对照测定每种激酶抑制剂的Nanog和Dnmt3b值，并确定Nanog:Dnmt3b比率和总相对Nanog + Dnmt3b信号，并且抑制剂与DMSO对照相比排名。发现改变Nanog:Dnmt3b比超过或低于DMSO对照的2倍阈值的抑制剂被确定为初始或初始多能性的驱动因素。将低于DMSO对照的阈值设置为损害mESC存活的分类抑制剂。选择进一步验证的抑制剂被突出显示。 （图改编自Williams 等人 14 ）。s / ftp_upload / 55515 / 55515fig2large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" src="/files/ftp_upload/55515/55515fig3.jpg" /> 图3:确认的选定命中化合物的验证。 mESC用1μMAZD4547（选择性FGFR抑制剂）或从图2中筛选鉴定的指示抑制剂处理。通过标准SDS-PAGE和免疫印迹分析测定的Nanog:Dnmt3b水平。相对Nanog:Dnmt3b和Nanog + Dnmt3b的数值如下表所示，稳定初始多能态的抑制剂以绿色突出显示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55515/55515fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 复合 </td><td colspan="2"> 主要目标 </td><td> 其他目标 </td></tr><tr><td> AV-951 </td><td colspan="2"> VEGFR </td><td></td></tr><tr><td> HG-6-64-01 </td><td colspan="2"> ABL，BRAF，RET，CSF1R，EGFR，EPHA8，FGFR，FLT3，KIT，LOK，MAP4K1，p38b，MUSK，PDGFR，TAOK3，TNNI3K </td><td></td></tr><tr><td> WH-4-023 </td><td colspan="2"> LCK </td><td> SRC </td></tr><tr><td> R406 </td><td colspan="2"> SYK </td><td></td></tr><tr><td> GW-572016 </td><td colspan="2"> HER2 </td><td> EGFR </td></tr><tr><td> KIN001-244 </td><td colspan="2"> PDK-1 </td><td></td></tr><tr><td> WZ-4-145 </td><td colspan="2"> CSF1R，DDR1，EGFR，TIE1，PDGFR2 </td><td></td></tr><tr><td> WZ-7043 </td><td colspan="2"> CSF1R，DDR1，FGFR和TAO1 </td><td></td></tr><tr><td> GW786034 </td><td colspan="2"> VEGFR1 </td><td> VEGFR2，VEGFR3，＃160; </td></tr><tr><td> AZD-1480 </td><td colspan="2"> JAK2 </td><td></td></tr></tbody></table> 表1:选定的命中化合物及其主要激酶靶标。

Watch this Scientific Journal Video about A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening at JoVE.com

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

在这里，我们提出一个定量和可扩展的方案来执行针对性的小分子屏幕，用于初始化多能转换的激酶调节剂。

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

a-simple-method-to-identify-kinases-that-regulate-embryonic-stem-cell

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.4K 次观看.  University of Dundee. 在这里，我们提出一个定量和可扩展的方案来执行针对性的小分子屏幕，用于初始化多能转换的激酶调节剂。 

视频: A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

科研

发育生物学

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

体细胞重编程的动力学测量和实时可视化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

生物化学

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

生物学

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

免疫与感染

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...