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J Vis Exp. 2017 May 12;(123). doi: 10.3791/55515.

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

Author information

1
The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee.
2
Department of Cancer Biology, Dana-Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School.
3
The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee; g.m.findlay@dundee.ac.uk.

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.

PMID:
28570543
PMCID:
PMC5607952
DOI:
10.3791/55515
[Indexed for MEDLINE]
Free PMC Article

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