Williams Charles A C, Gray Nathanael S, Findlay Greg M
The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee.
Department of Cancer Biology, Dana-Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School.
J Vis Exp. 2017 May 12(123):55515. doi: 10.3791/55515.
Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed "naïve" and "primed" pluripotency. The mechanisms that control naïve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify naïve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.
胚胎干细胞（ESCs）能够自我更新或分化为所有细胞类型，这一现象被称为多能性。已描述了不同的多能状态，即“原始态”和“启动态”多能性。控制原始态-启动态转变的机制尚不清楚。特别是，尽管用于探测激酶功能的高质量工具化合物越来越多，但我们对指定原始态和启动态多能状态的蛋白激酶仍然知之甚少。在此，我们描述了一个可扩展的平台，用于对小鼠胚胎干细胞中原始态-启动态多能转变的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以及标准试剂、材料和设备，来发现和验证对多能性有迄今未被认识到的影响的激酶抑制剂。我们讨论了该技术的潜在应用，包括筛选其他小分子集合，如日益复杂的激酶抑制剂和新兴的表观遗传工具化合物库。
