Abstract
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.
Highlights
Embryonic stem cells (ESCs) have the capacity to self-renew or differentiate into any cell type in the adult body, a phenomenon known as pluripotency[1]
It is essential to triage inhibitors based on both Nanog:Dnmt3b ratio and overall signal, to ensure that kinase inhibitors which adversely affect mouse ESCs (mESCs) survival are not selected for further analysis
The library is prepared at a concentration of 0.1 mM for a 1:100 dilution and final screening concentration of 1 μM in mESCs. 48 h later, mESCs were lysed and extracts prepared for quantitative dot blot analysis of Nanog and Dnmt3b expression (Figure 2, top)
Summary
Embryonic stem cells (ESCs) have the capacity to self-renew or differentiate into any cell type in the adult body, a phenomenon known as pluripotency[1]. Naïve and primed pluripotent states are marked by distinct gene regulatory networks. In mouse ESCs (mESCs), primed pluripotency is characterized by reduced expression of naïve markers and a specific gene expression signature which includes the de novo DNA methyltransferase Dnmt3b7. When cultured in leukemia inhibitory factor (LIF) and fetal bovine serum (FBS), mESCs undergo dynamic transition between naïve and primed pluripotent states[9,10]. LIFJak-Stat[3] signaling functions to promote a naïve gene regulatory network[11], whilst autocrine signaling via the fibroblast growth factor 4 (Fgf4) Erk1/2 pathway drives transition to the primed state[12]. We describe a quantitative and scalable platform by which to perform targeted small molecule screens for kinase regulators of the naïveprimed pluripotent transition. We discuss potential extended applications for this technology, including for screening of other small molecule collections such as emerging inhibitor libraries targeting epigenetic regulators
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