A novel analytical assay for predictive embryotoxicity using human embryonic stem

描述(申请人提供)：制药公司定期对新物质进行生物毒性筛选，但未能预测化合物对人类的毒性是制药行业的一大财务成本。体内多代研究是昂贵、费力和耗时的。或者，体外细胞模型可以用更少的时间和成本完成。体外细胞模型试验在药物筛选中的预测潜力很大程度上受细胞类型和试验终点的影响。原代细胞的增殖能力有限，永生化的细胞系并不代表体内的细胞系。在这里，我们建议开发一种新的、简单的、低成本的基于微珠的流式细胞仪分析方法，该方法具有潜在的高预测性，使用人类胚胎干细胞进行体外胚胎毒性评估。为了评估化学物质对胚胎的毒性作用，需要开发检测早期化学诱导的应激状态的分析方法。当处于细胞毒性或遗传毒性培养条件下时，未分化的多能ES细胞通过激活凋亡过程或通过诱导分化过程来响应。这些存活过程保护hESC免受携带带有潜在致癌突变的受损DNA的细胞的繁殖。在次优条件下，小鼠ES细胞保持其多能性和基因组稳定性的时间长于hESC。这表明HES细胞比MES细胞更适合于对环境诱导的应激条件更敏感的体外毒性系统进行自发分化和凋亡。在培养诱导应激条件下，在控制未分化的hESC自发凋亡和分化过程中起核心作用的蛋白是P53蛋白。P53在早期和晚期凋亡的hESC中均有较强的表达。在这项建议的第一阶段，我们将开发一种基于微珠的分析流式细胞仪免疫分析方法，以捕获和定量化学诱导的未分化hESCs培养基中P53的积聚，以响应已知的胚胎毒性物质。已知的具有高、中和无胚胎毒性作用的胚胎毒素化合物的相对胚胎毒性(REP)值将使用P53表达来计算，并与已发表的REP值进行比较以进行验证。在第二阶段，我们将应用这项技术来筛选更广泛的化学物质用于胚胎毒性，使用未分化的hESCs和特定的hESC来源的分化细胞。最终，这项技术将被改造成高通量的筛选平台。 公共卫生相关性：这项提案将开发一种新的基于流式细胞术的分析方法，利用未分化的人类胚胎干细胞来预测胚胎毒性化合物。

DESCRIPTION (provided by applicant): New substances are routinely screened by pharmaceutical companies for their biological toxicity but failure to predict the toxicity of compounds to humans is a major financial cost for the pharmaceutical industry. In vivo multigeneration studies are expensive, laborious, and time consuming. Alternatively, in vitro cellular models can be completed in substantially less time and cost. The predictive potential of in vitro cellular model assays in drug screening is largely influenced by the cell types and the endpoint in the assays. Primary cells are limited in their proliferation capacity and the immortalized cell lines do not represent the in vivo counterparts. Here we propose to develop a novel, simple, low cost bead-based flow cytometric analytical assay with potentially high predictive in vitro embryotoxicity assessment using human embryonic stem cells. In order to assess the embryotoxic effects of chemicals, assays need to be developed to detect the early chemically-induced stress conditions. When placed in cytotoxic or genotoxic culture conditions, undifferentiated pluripotent ES cells respond by either activating apoptosis processes, or by inducing differentiation process. These survival processes protect hESC against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. Murine ES cells maintain their pluripotency and genomic stability longer than hESC under suboptimum conditions. This indicates that hES cells are a better choice than mES cells for a more sensitive in vitro toxicity system to environmental-induced stress conditions for spontaneous differentiation and apoptosis. The protein with a central role in controlling undifferentiated hESC to spontaneous apoptosis and differentiation under culture-induced stress condition is the p53 protein. P53 was shown to express strongly in both early and late apoptotic hESC. In Phase I of this proposal, we will develop an analytical bead-based flow cytometry immunoassay to capture and quantify the chemically-induced p53 accumulation in undifferentiated hESCs culture medium in response to known embryotoxic substances. The Relative Embryotixic Potency (REP) values of known embryotxic compounds with high-, medium-, and no-embryotoxicity effects will be calculated using p53 expression and compared with the published REP values for validation. In Phase II we will apply this technology to screen a broader spectrum of chemicals for embryotoxicty using undifferentiated hESCs and specific hESC-derived differentiated cells. Ultimately, the technique will be adapted to a high- throughput screening platform. PUBLIC HEALTH RELEVANCE: This proposal will develop a novel flow cytometric based assay to predict embryotoxic compounds using undifferentiated human embryonic stem cells.