A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

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.

An ERK5–KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells

“…Finally, we employed Erk5/Mapk7 −/− mESCs [ 22 ] to confirm the role of ERK5 in the regulation of Zscan4 /2C genes. Erk5 −/− mESCs expressing MEK5DD express a basal level of Zscan4, Zfp352 and Tdpoz3 mRNAs ( Figure 2C ).…”

“…ERK5 is a unique kinase encoding both a kinase domain and a transcriptional activation domain. It was identified as a transcriptional activator of pluripotency genes in mESCs, including the Klf2 transcription factor [ 22 ]. However, other functions of ERK5 in regulating gene expression and proteome dynamics in mESCs have not been systematically explored.…”

An ERK5-KLF2 signalling module regulates early embryonic gene expression dynamics and stem cell rejuvenation

“…Finally, we employed Erk5/Mapk7 -/- mESCs (Williams et al, 2017) to confirm the role of ERK5 in regulation of Zscan4 /2C genes. Erk5 -/- mESCs expressing MEK5DD express a basal level of Zscan4, Zfp352 and Tdpoz3 mRNAs (Figure 2C).…”

“…ERK5 is a unique kinase encoding both a kinase domain and a transcriptional activation domain. It was identified as a transcriptional activator of key pluripotency genes in mESCs, including the Klf2 transcription factor (Williams et al, 2017). However, other functions of ERK5 in regulating gene expression and proteome dynamics in mESCs have not been systematically explored.…”