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Updated: Nov 11, 2025
A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
Dissection of two routes to naïve pluripotency using different kinase inhibitors
Ana Martinez-Val1,2, Cian J Lynch3, Isabel Calvo3
1Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
Abstract:
Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.
Insights
Two small molecule inhibitors, 2i and Cdk8/19i, maintain embryonic stem cells (ESCs) in a naïve state. Both treatments reveal overlapping molecular events, supporting naive cell mitochondrial capacity via post-transcriptional de-repression.
Area of Science:
- Stem cell biology
- Molecular and Cellular Biology
Background:
- Embryonic stem cells (ESCs) can be maintained in a pluripotent naïve state using specific inhibitors.
- The 2i inhibitor cocktail (Mek1/2 and Gsk3 inhibitors) and Cdk8/19 inhibition (Cdk8/19i) are known to stabilize this naïve state.
Purpose of the Study:
- To comprehensively analyze the molecular events (phosphoproteome, proteome, metabolome) induced by 2i and Cdk8/19i in ESCs.
- To compare the overlapping and distinct mechanisms employed by 2i and Cdk8/19i in maintaining ESC naïve pluripotency.
Main Methods:
- Mass spectrometry-based phosphoproteomics, proteomics, and metabolomics were employed.
- Comparative analysis of molecular profiles under 2i and Cdk8/19i treatments.
Main Results:
- Both 2i and Cdk8/19i treatments exhibit significant molecular commonalities, suggesting overlapping pathways.
- Post-transcriptional de-repression by both inhibitors appears to enhance mitochondrial capacity in naïve ESCs.
- Distinct proteome reprogramming mechanisms were observed: Cdk8/19i directly activates transcription, while 2i stabilizes via de-phosphorylation.
Conclusions:
- 2i and Cdk8/19i share common downstream effects in maintaining the naïve ESC state, particularly in supporting mitochondrial function.
- The study highlights divergent upstream mechanisms by which these inhibitors achieve proteome reprogramming and naïve pluripotency maintenance.
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