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: 10702687
• 负责人: Sergio Ruiz
• 金额: $ 30.52万
• 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/10702687
• 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.

2021财年期间，我们在该项目上也取得了很大进展。在这里，我们有兴趣了解诱导多能细胞静止的分子机制。为此，我们生成了两种不同的报告细胞系：1）表达荧光标记的 p27 突变体形式的多能细胞系，无法与 CDK 相互作用，并且仅在静止细胞中可见（p27Mut-mCherry）。 2) 表达最近描述的活细胞传感器的多能细胞系，用于测量 CDK2 活性。该传感器包含与荧光蛋白 mVenus (DHBmVenus) 融合的人类 DNA 解旋酶 B 的氨基酸 994-1087。此外，它还包含四个 CDK 共有磷酸化位点、一个核定位信号和一个核输出信号，使该融合蛋白能够在细胞核和细胞质之间穿梭。 CDK2 活性低的 G0/G1 细胞将在细胞核中积累 DHBmVenus，而穿过细胞周期的细胞将逐渐将嵌合报告基因输出到细胞质中。使用 p27 突变型 Cherry 胚胎干细胞系 (ESC)，我们分析了近 1000 种化合物的化学库，并根据 p27-Cherry 水平选择了 15 种作为有效的静止诱导剂。经过二次验证筛选后，我们进一步研究了 5 种化合物。目前，我们正在探索这些化合物诱导ESCs静止的分子机制。使用相同的荧光标记 p27 突变蛋白，我们已经鉴定出 mESC 培养物中存在内源性静止细胞。我们目前正在通过活细胞成像和这些细胞的单细胞 RNAseq 的转录谱来识别增殖动态。最后，我们在人胚胎干细胞系（hESC）中生成了类似的荧光标记报告细胞系，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 hESC 系在体外诱导原肠胚形成样系统，以可视化细胞定型和分化时 CDK 活性的变化。此外，我们在 ROSA26 位点中使用上述 CDK2 活体传感器的单个副本来靶向 mESC。我们已经生成了包含该报告基因的小鼠模型，以确定早期发育过程中的时空 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格之间的因果关系。