Identification of molecular pathways regulating quiescence in pluripotent cells

多能细胞中调节静止的分子途径的鉴定

• 批准号: 10486991
• 负责人: Sergio Ruiz
• 金额: $ 34.23万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486991
• 关键词: Amino Acids; Biological Assay; CDK2 gene; Cell Cycle; Cell Cycle Regulation; Cell Line; Cell Nucleus; Cells; Chimeric Proteins; Consensus; Cytoplasm; Development; Diapause; ES Cell Line; Embryo; Food; Gene Expression Profile; Goals; HELB gene; In Vitro; Label; Maintenance; Mammals; Maps; Measures; Molecular; Mus; Nuclear Export; Nuclear Localization Signal; Pathway interactions; Phosphorylation Site; Physiological; Process; Proteins; Reporter; Signal Transduction; System; Validation; base; cancer stem cell; gastrulation; human DNA; human embryonic stem cell line; interest; live cell imaging; mouse model; mutant; sensor; single-cell RNA sequencing; small molecule libraries; spatiotemporal; tumor

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.

在2021财年期间，我们在该项目中也取得了长足的进步。在这里，我们有兴趣了解多能细胞中诱导静止的分子机制。为此，我们生成了两个不同的报告基细胞系：1）表达荧光标记的P27突变形式的多能细胞系无法与CDK相互作用，并且仅在静态细胞中可见（p27mut-mcherry）。 2）表达最近描述的活细胞传感器以测量CDK2活性的多能细胞系。该传感器包括与荧光蛋白MVenus（DHBMVenus）融合的人DNA解旋酶B的氨基酸994-1087。此外，它包含四个CDK共有磷酸化位点，一个核定位信号以及核输出信号，使该融合蛋白能够在细胞核和细胞质之间穿梭。具有低CDK2活性的G0/G1中的细胞将在细胞核中积累DHBMVENUS，而穿越细胞周期的细胞将逐渐将嵌合报告子导出到细胞质中。使用P27突变的樱桃胚胎干细胞系（ESC），我们根据P27-Cherry水平分析了一个近1000种化合物的化学库，并选择15种作为有效的静态诱导剂。在二级验证屏幕之后，我们进一步进行了5种化合物。当前，我们正在探索这些化合物在ESC中诱导静止的分子机制。使用相同的荧光标记的p27突变蛋白，我们已经确定了存在于MESCS培养物中的内源性静态细胞的存在。我们目前正在通过实时细胞成像和这些细胞的单细胞RNASEQ进行转录曲线来识别增殖动力学。最后，我们在人类胚胎干细胞系（HESC）中生成了类似的荧光标记的报告细胞系，以筛选相同的化合物库。此外，我们建立了一个系统，使用这些报告基因hESC线在体外诱导胃样系统，以可视化CDK活性的变化，因为细胞的作用和分化。此外，我们将MESC靶向上面在Rosa26基因座中描述的CDK2 Live-Sensor的单个副本。我们已经生成了一个包含此报告基因的小鼠模型，以确定早期发育过程中时空CDK2活性景观。使用此CDK2活性图，我们将评估细胞周期调节和细胞规范之间的因果关系。