Name: human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation
Uploaded: 2015-06-17T13:00:00
Description: Watch this Scientific Journal Video about Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation at JoVE.com

We thank M. Kapitza, Margit Henry, Tamara Rotshteyn, Susan Rohani and Cornelia B&#246;ttinger for excellent technical support. This work was supported by grants from the German Research Foundation (RTG 1331) and the German Ministry for Research (BMBF).

使用人胚胎干细胞系（人类胚胎干细胞）H9进行以下协议。此细胞系常规地在有丝分裂失活的小鼠胚胎成纤维细胞（MEFs）在人类胚胎干细胞的培养基补充有bFGF和然后培养干细胞培养基上6厘米涂有基底膜基质如基质胶，培养皿除掉的MEF的培养。从&gt; 80％汇合板的H9细胞用于进一步的通道。 H9细胞对基底膜基质平板上培养被用于EB的形成。在以下协议中提及的所有程序使用标准?...

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传统方法毒理试验涉及广泛的动物研究中从而使测试昂贵和费时。此外，由于种间差异的临床前动物安全性研究并不总是有效的预测与人类有关的潜在药物毒性作用。虽然非人类灵长类动物是最可预测的，还是强烈的伦理和socioeconomical需求正在迅速被现代社会提高发展中国家在体外试验敏感和强大 有关人类的安全系统。 人类胚胎干细胞的独特能力分化成所有的?...

人类胚胎干细胞（hESC细胞）的分化成各种细胞的能力打开了体外毒性试验1中 ，疾病建模和再生医学2的新时代。干细胞被赋予的能力，自我复制，以保持其多能状态，并分化成特定细胞3,4。的人类胚胎干细胞的特性（容量来区分所有主要的细胞类型），也发现在其它人多能干细胞，如人类诱导多能干细胞（hiPSC）或通过核移植5产生的细胞。例如，许多不同的hESC细胞系已分化为神经元6，肾细胞7，神经嵴细胞8，心肌9-12，或肝细胞样细胞13,14。此外，人类胚胎干细胞可自发地分化成胚状体（EB）19,20所有三种胚层15-18的细胞。 Ë阿尔利胚胎发育是通过与已捕获在mRNA水平通过使用微阵列技术15转录的不同胚层的多种基因的差异表达调控。这些努力使建立基于人类胚胎干细胞/ hiPSC和转录组分析（综述见21,22）器官特异性毒性模型。这些车型有优势的传统使用实验动物的毒理学研究，如使用实验动物并不总是预测人类安全的临床前研究。在患者中遇到的药物诱导的毒性往往与人类和实验动物之间是不同的代谢或信号传导过程。的物种差异防止了可靠的早期检测发育毒性在人类，例如药物如沙利度胺23,24和己烯雌酚25,26从市场上撤回由于致畸。塔利domide并没有表现出在大鼠或小鼠任何发育毒性。环境化学品如甲基汞27导致相对于神经系统中的各种物种产前发育毒性，但人的表现已经很难在动物模型。为了解决种属特异性问题的问题，科学家们根据不同的项目工作的基础上的干细胞一样重新保护，ESNATS，侦探等所从事的不同型号的发展胚胎毒性，神经毒性，心脏毒性，肝毒性和肾毒性涉嫌使用对人体有毒物质影响人类。根据欧洲联盟项目的胚胎干细胞为基础的新型替代测试策略（ESNATS）“五个测试系统已经建立。一个测试系统的所谓UKK（UniversitätsķlinikumķOLN）测试系统部分捕捉人类早期胚胎发育。在这个System人类胚胎H9细胞分化中15和胚层特定的签名已被抓获转录使用Affymetrix芯片平台简介三个胚层（外胚层，内胚层和中胚层）。像沙利度胺28，丙戊酸，甲基汞16,17，或阿糖胞苷15各个发育毒物在该系统已经过测试，并且具体毒物基因签名已被获得。在第二测试系统，即所谓的UKN1（U。高校ķonsta N个Z的）测试系统1中，H9细胞分化到神经外胚层祖细胞（NEP）6天。这是通过高表达的神经基因标记如PAX6和OTX2证实。在分化6天，NEP细胞已经暴露于发育神经毒物如丙戊酸钠，甲基汞。毒物特异性解除管制的转录概况已obtai通过使用Affymetrix芯片平台定义16,29以及。 对于21 世纪的毒理学的新远景设想，测试系统不仅产生的表型的描述，如组织病理学体内 ，或转录变化在长期毒物孵化结束。它，而表明试验提供机械信息3，而该信息可以被映射到所谓的不利后果的途径（AOP），提供了一个科学原理为危险影响30。为了提供这种信息，应用的测试系统必须高度控制质量31，作为例如通过稳健标准操作程序记载。此外，依赖于时间的变化需要被映射为高的分辨率。这就要求测试系统同步变化32。这里所描述的UKN1和UKK测试系统进行了优化这些需求。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320082</td>
			<td>Dulbecco&#39;s Modified Eagle Medium:Nutrient Mixture F-12</td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td>Knockout Serum Replacement</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td>GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td>MEM Nonessential Amino Acids Solution</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-0144</td>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, without calcium, without magnesium</td>
		</tr>
		<tr>
			<td>mTeSR medium</td>
			<td>Stemcell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>V bottom plate</td>
			<td>VWR</td>
			<td>734-0483</td>
			<td>Plate,Microwell,V BTTM,96 Well,Sterile 1 * 50 ST</td>
		</tr>
		<tr>
			<td>Vbottom plate lid</td>
			<td>VWR</td>
			<td>634-0011</td>
			<td>Lid, Microtitre plates, Cond. Ring 1 * 50 ST</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Penicillin- Streptomycin, Liquid</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Life Technologies</td>
			<td>15230-089.</td>
			<td>Sterile Distilled Water</td>
		</tr>
		<tr>
			<td>Human FGF-2 (bFGF)</td>
			<td>Millipore</td>
			<td>GF003AF-100UG</td>
			<td>Fibroblast Growth Factor basic, human recombinant, animal-free</td>
		</tr>
		<tr>
			<td>Filter 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td>Stericup-GP, 0.22 &#956;m, polyethersulfone, 250 ml, radio- sterilized</td>
		</tr>
		<tr>
			<td>StemPro EZPassageTM Disposablte</td>
			<td>Invitrogen</td>
			<td>23181010</td>
			<td></td>
		</tr>
		<tr>
			<td>BD MatrigelTM, hESC qualified Matrix</td>
			<td>Stemcell Technologies</td>
			<td>354277</td>
			<td>5 ml vial</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D-2650</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAlater Stabilization Solution</td>
			<td>Life Technologies</td>
			<td>AM7020</td>
			<td>It stabilizes and protect the RNA integrity in unfrozen samples.</td>
		</tr>
		<tr>
			<td>70 &#956;m Cell Strainer</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td>Cell strainer with 70 &#956;m Nylon mesh</td>
		</tr>
		<tr>
			<td>35 &#956;m Lid cell strainer, 5 ml tube</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td>5 ml polystyrene round bottom test tube, with a cell strainer cap (35 &#956;m)</td>
		</tr>
		<tr>
			<td>50 ml sterile Polypropylene tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>50 ml Polypropylene tube with conical bottom, Sterile</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td>CELLSTAR Filter Cap Cell Culture 75 cm2 Flasks</td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Life Technologies</td>
			<td>10296010</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well optical bottom plates</td>
			<td>Thermo Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Blue</td>
			<td>Promega</td>
			<td>G8081</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>PAA</td>
			<td>L11-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Apotransferin</td>
			<td>Sigma-Aldrich</td>
			<td>T-2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemicals</td>
			<td>LS002104</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Tocris Bioscience</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAA</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-2</td>
			<td>R&#38;D Systems</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco Invitrogen</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco Invitrogen</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I-6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco Invitrogen</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>R&#38;D Systems</td>
			<td>719-NG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom AG</td>
			<td>L1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesteron</td>
			<td>Sigma-Aldrich</td>
			<td>P7556</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P-5780</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Tocris Biosciences</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris Biosciences</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bio-Rad</td>
			<td>161-0416</td>
			<td></td>
		</tr>
		<tr>
			<td>Selenium</td>
			<td>Sigma-Aldrich</td>
			<td>S-5261</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco Invitrogen</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>List of Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>QIAGEN</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneChip Hybridization, Wash, and Stain Kit</td>
			<td>Affymetrix</td>
			<td>900721, 22, 23</td>
			<td>This kit provides all reagents required for hybridization wash and staining of microarrays.</td>
		</tr>
		<tr>
			<td>Rnase-Free DNase Set</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">List of equipment.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>IX71</td>
		</tr>
		<tr>
			<td>Genechip Hybridisation Oven - 645</td>
			<td>Affymetrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Genechip Fluidics Station-450</td>
			<td>Affymetrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Affymetrix Gene-Chip Scanner-3000-7 G</td>
			<td>Affymetrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax M5</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>List of softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Affymetrix GCOS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Partek Genomic Suite 6.25</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Online tools for Functional annotation 
			DAVID 
			Onto-tools Intelligent Systems and Bioinformatics Laboratory</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation

高效的协议来区分人多能干细胞，以组合各种组织与组学技术开辟了新的视野为潜在药物的体外毒性测试。为了提供这种试验坚实的科学基础，这将是非常重要的获得发展的时间进程和底层的监管机制，系统生物学方法的定量信息。两个实验都因此被调整为在这里这些要求。在UKK测试系统中，人胚胎干细胞（hESC细胞）（或其它多能细胞）都留给自发分化为胚状体14天，以允许产生所有三个胚层的细胞。该系统概括的人类早期胚胎发育的关键步骤，并且它可以预测人特异性早期胚胎毒性/致畸性，如果细胞分化过程中暴露于化学品。所述UKN1测试系统是基于人类胚胎干细胞分化为一个populat神经外胚层祖离子（NEP）细胞6天。该系统概括早期神经发育，并预测早期发育神经毒性和化学品引发的表观遗传变化。两个系统，在与转录微阵列研究结合，适合于识别的毒性的生物标志物。此外，它们也可以组合使用以产生用于系统生物学分析的输入数据。这些测试系统具有比传统的毒理学研究需要大量的动物的优点。该测试系统可能有助于减少药物开发和化学安全性评价的费用。他们的组合揭示尤其是对可能影响具体化合物的神​​经发育光。

在UKK测试系统甲基汞暴露与H9的EB进行的细胞毒性测定，得到的IC 10值（10％减少活力的）甲基汞的细胞毒性（ 图1）。我们还进行了微阵列基础（Affymetrix公司的平台）的生物标志物研究。的H9的EB已暴露于甲基汞（0.25和1μM）14天。在第14天，将样品已收集使用Trizol和分离RNA。使用人类基因组U133加2.0阵列芯片进行转录谱。的数据进行了分析以将Partek基因组套?...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation at JoVE.com

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

该协议描述了基于人胚胎干细胞和转录研究2 在体外发育毒性试验系统（UKK和UKN1）。测试系统预测人类发育毒性危险，并可能有助于减少动物研究中，成本和所需的化学安全测试的时间。

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 ...

Research

JoVE Journal

Developmental Biology

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

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

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 ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve-like State with Improved Multilineage Differentiation Potency

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...