Identification of molecular pathways regulating quiescence in pluripotent cells

Division Of Basic Sciences - Nci

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Abstract

During FY2021 we also have made great progress in this project. Here, we are interested in understanding the molecular mechanisms inducing quiescence in pluripotent cells. For this, we generated two different reporter cell lines: 1) A pluripotent cell line expressing a fluorescently labelled p27-mutant form unable to interact with CDKs and only visible in quiescent cells (p27Mut-mCherry). 2) A pluripotent cell line expressing a recently described live cell sensor to measure CDK2 activity. This sensor included amino acids 994-1087 of the human DNA helicase B fused to the fluorescent protein mVenus (DHBmVenus). In addition, it contains four CDK consensus phosphorylation sites, a nuclear localization signal, and a nuclear export signal making this fusion protein able to shuttle between the nuclei and the cytoplasm. Cells in G0/G1 with low CDK2 activity will accumulate DHBmVenus in the nuclei while cells traversing through cell cycle will progressively export the chimeric reporter out to the cytoplasm. Using p27-mutant Cherry embryonic stem cell lines (ESCs), we assayed a chemical library of almost 1000 compounds, and selected 15 as potent inducers of quiescence based on p27-Cherry levels. After a secondary validation screen, we have further proceeded with 5 compounds. Currently, we are exploring the molecular mechanisms by which these compounds induce quiescence in ESCs. Using the same fluorescently labelled p27-mutant protein we have identified the existence of endogenous quiescent cells residing within mESCs cultures. We are currently identifying the proliferation dynamics by live cell imaging and transcriptional profile by single cell RNAseq of these cells. Finally, we generated similar fluorescently labelled reporter cell lines in human embryonic stem cell lines (hESC) to screen the same library of chemical compounds. Moreover, we set up a system to induce a gastrulation-like system in vitro using these reporter hESC lines to visualize changes in CDK activity as cells commit and differentiate. In addition, we targeted mESCs with one single copy of the CDK2 live-sensor described above in the ROSA26 loci. We have generated a mouse model containing this reporter to determine the spatiotemporal CDK2 activity landscape during early development. Using this CDK2 activity map we will evaluate the causal relation between cell cycle regulation and cell specification.