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Quantifying continuous cell-cycle phase using single-cell gene expression data

ABSTRACT: The cell cycle is known to regulate cell proliferation and cell fate decisions, the underlying mechanism of which is well-studied and conserved across species and cell types. To date, the cell cycle is receiving a renewed importance due to the rapid advancement of single-cell genomics technology. Especially in the analysis of single-cell gene expression, the cell cycle plays a key role in understanding the expression variation across cell states and cell types. The current study was designed to develop effective tools for assessing and predicting the cell cycle in single-cell gene expression data analysis. We collected both Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) – for determining the cell cycle – and single-cell RNA-seq (scRNA-seq) data from each individual using STRT-seq on the Fluidigm C1 platform. The cells were collected from iPSCs derived from 6 genotyped Yoruba cell lines. The experimental design controlled for C1 processing batch effects, as well as individual and gender effects. Using these data, we developed a supervised approach for predicting the cyclical ordering of single cells in the cell cycle using single-cell gene expression data. We used the FUCCI fluorescent intensities to determine a cyclical ordering of the individual cells, and to assign cell time labels for individual cells representing each cell's position in one complete cell cycle. We estimated the cyclical trend of expression levels for each gene based on the FUCCI-derived cell times, and identified candidate sets of cyclical genes for model training. We trained our model in in 5-fold cross-validation and evaluate the trained model in held-out validation samples and external datasets. We compared the prediction results with existing approaches for estimating the cell cycle using single-cell gene expression data: including unsupervised approaches to construct the cyclical ordering of single cells and approaches to categorically assign the cell cycle to individual cells. These results provide a benchmark for assessing the cell cycle in scRNA-seq data analysis and insights into the effects of the cell cycle on gene expression variation in stem cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE121265 | GEO | 2019/02/12

REPOSITORIES: GEO

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Project description:Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying vascular development in Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous and arterial endothelial cells exhibit a propensity for different cell cycle states during development and in adulthood. That is, venous endothelial cells are predominantly FUCCI-Negative, while arterial endothelial cells are enriched for the FUCCI-Red reporter. Single cell RNA sequencing analysis of developing retinal endothelial cells reveals that venous endothelial cells are enriched for the FUCCI-Negative state and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state and TGF-b signaling. Further transcriptional analyses and live imaging of cultured endothelial cells expressing the FUCCI reporter show that reporter-negative corresponds to an early G1 state and reporter-red corresponds to late G1 state. We find the early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-b1-induced arterial gene expression. In a mouse model of endothelial cell hyperproliferation and disrupted arterial-venous specification, pharmacological inhibition of endothelial cell cycle prevents the vascular defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification.

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Quantifying continuous cell-cycle phase using single-cell gene expression data

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