Integration of Genomic Biomarkers with the devTOX Human Embryonic Stem Cells Scre

描述（由申请人提供）：在子宫内接触环境化学物质或药物约占所有出生缺陷的5%，并可能通过破坏生命后期激活的表观遗传发育程序（例如，青春期，衰老）对成年生活产生影响。目前公认的用于鉴定潜在致畸物的监管测试涉及使用数千只动物，成本高昂，需要两年时间才能完成，并且对人类致畸物的一致性较差（约60%）。最近欧盟禁止动物试验使得对非动物试验系统的依赖更加迫切。人类胚胎干细胞（hES）反映了一种独特的生物系统，这是毒理学中任何其他细胞类型所不能代表的。该提案的重点是开发和验证一种基于毒物基因组学的快速特征分析方法，以便无缝集成到Stemina生物标志物发现devTOX（tm）代谢组学平台。ILS和Stemina的联合专业知识将使一种创新的“系统生物学”方法能够利用人类发育起源的细胞类型——hES细胞，在与人类相关的实验中进行体外发育毒理学筛选。我们将使用生物信息学驱动的计算方法，基于独特的干细胞转录组反应（蛋白质编码mRNA和非编码rna）和细胞对毒物暴露的反应途径，确定基于毒物基因组学的特征图谱。最终，该II期SBIR将开发、验证和实施一种中等通量分析，使用基于转录组的分析，对潜在的发育毒性具有很高的预测价值。具体目标1：使用62种化合物的训练集对暴露于已知/可疑致畸物和非致畸物的hES细胞进行表达谱分析，以构建基于转录组的发育毒性预测特征；具体目标2：开发和实施生物测定标准操作程序，质量控制标准，以及仪器和glp兼容协议的验证，用于与转录组学相结合的devTOX（tm）测定；具体目标3：使用qRT-PCR方法对特定mrna、lncRNAs和miRNAs进行盲法测试，测试来自训练集的基于转录组的特征。这将允许基于高度调控的基因组和维持干细胞功能的表观基因组电路功能障碍的生物学相关特征谱的发展。

DESCRIPTION (provided by applicant): In utero exposure to environmental chemicals or drugs accounts for approximately 5% of all birth defects and can have an impact in adult life by disrupting the epigenetic developmental programs that are activated later in life (e.g., puberty, aging). The currently accepted regulatory test for identifying potential teratogens involves the use of thousands of animals, is costly, takes two years to complete, and shows poor concordance (~60%) for human teratogens. The recent European Union ban on animal testing makes the reliance on non-animal test systems more urgent. Human embryonic stem (hES) cells reflect a unique biological system that cannot be represented by any other cell type used in toxicology. This proposal is focused on developing and validating a rapid toxicogenomics-based signature profiling assay for seamless integration into the Stemina Biomarker Discovery devTOX(tm) metabolomics-based platform. The combined expertise at ILS and Stemina will enable an innovative "systems biology" approach to in vitro developmental toxicology screening in a human-relevant assay using the cell type at the origin of human development, the hES cell. We will identify a toxicogenomics-based signature profile built on the unique stem cell transcriptome response (protein coding mRNA and non-coding RNAs), and cellular response pathways to toxicant exposures, using bioinformatics-driven computational methods. Ultimately, this Phase II SBIR will result in the development, validation, and implementation of a medium throughput assay using transcriptome-based profiling with a high predictive value for potential developmental toxicants. This objective will be completed in three Specific Aims: Specific Aim 1: Conduct expression profiling of hES cells exposed to known/suspect teratogens and nonteratogens using a training set of 62 compounds to construct a predictive transcriptome-based signature of developmental toxicity; Specific Aim 2: Develop and implement bioassay standard operating procedures, quality control criteria, and validation of instrumentation and GLP-compliant protocols for the conduct of the devTOX(tm) assay integrated with transcriptomics; Specific Aim 3: Test the transcriptome-based signature derived from the training set with a blinded set of test articles using qRT-PCR assays for specific mRNAs, lncRNAs and miRNAs. This will allow development of a biologically relevant signature profile based on dysfunction of the highly regulated genome and epigenome circuitry that maintains stem cell functions.