HTS Screen-Neural Differentiation in Human ES Cells(RMI)

描述(申请人提供)：这项研究的目标是开发一种高通量(HTS)分析来鉴定诱导或抑制人类胚胎干细胞(HES)神经分化的化合物。该建议的基础是：i)需要确定推动HES细胞谱系特异性分化的试剂；ii)我们使用BAC转基因产生谱系特异性报告ES系的能力；iii)改善hES细胞标准化生长的方案；iv)用于HTS实施的最先进的机器人和筛查工具的可用性。这项研究应该是使用HTS分析来研究人类早期发育的一个原则验证应用，为控制神经分化的途径提供新的见解，并提供基于HES细胞的HTS分析的原型应用，包括未来基于我们内部生成的siRNA文库的分析。本研究的具体目的是：1.建立转BAG基因的人ES细胞HTS检测报告系(Wa-09)；2.HTS检测方法的发展(Wa-09)；3.候选化合物的生物学验证和二次筛选，(Wa-09、Wa-01、ES-03、ES-04)。SA-01、SA-02、MI-01)。在确定的体外条件下，鉴定能够指导HES细胞神经分化的新化合物也将对翻译干细胞的努力做出重要贡献。尤其重要的是神经分化条件的发展，避免使用类胚体形成或基质小鼠饲养器共培养方案。明确的基于化合物的神经分化最终可能为未来的临床应用提供最具重复性和安全性的分化策略。

DESCRIPTION (provided by applicant): The goal of this study is to develop a high throughput (HTS) assay to identify chemical compounds that induce or repress neural differentiation of human embryonic stem (hES) cells. The proposal is based on i) the need to identify agents that drive lineage specific differentiation in hES cells, ii) our ability to generate lineage specific reporter ES lines using BAC transgenesis, iii) improved protocols for the standardized growth of hEScells, iv) the availability of state-of-the-art robotic and screening tools for HTS implementation. This study should be a proof-of-principle application for the use of HTS assays to study early human development, provide novel insights into the pathways that control neural differentiation, and offer a prototype application for hES cell based HTS assays the will include future assays based on the use of our in-house generated siRNA library. The Specific Aims of the study are: 1. Generation of BAG transgenic human ES cell reporter lines for HTS assay (line WA-09) 2. Assay development to HTS format (line WA-09) 3. Biological validations and secondary screens of candidate compounds, (lines WA-09, WA-01, ES-03, ES-04. SA-01, SA-02, MI-01). The identification of novel compounds that direct neural differentiation of hES cells under defined in vitro conditions will also be an important contribution to translational stem cell efforts. Of particular importance will be the development of neural differentiation conditions that obviate the use of embryoid body formation or stromal mouse feeder co-culture protocols. Defined compound based neural differentiation may ultimately provide the most reproducible and safe differentiation strategy for future clinical applications.