Change of Single-cell Transcription Profile and Analysis of Intercellular Communication in Myogenic Cell Differentiation

doi:10.13560/j.cnki.biotech.bull.1985.2023-1005

Abstract

Abstract:

【Objective】 Based on single-cell RNA sequencing（scRNA-seq）, the changes of gene expression profile during bovine myogenic cell differentiation were revealed, and the mechanisms of ligand-receptor interaction mediating intercellular communication were explored, which lays the foundation for the construction of dynamic regulatory network in myogenic differentiation.【Method】 The scRNA-seq raw data of bovine myogenic cells in NCBI-GEO public database were deeply analyzed, including the identification of cell cluster and the analyses of differential gene expression profile, correlation, GO enrichment, PPI, intercellular communication, and pseudotime, by using databases or softwares such as Seurat, ClusterProfiler, STRING, Cytoscape, CellChatDB, and Monocle2.【Result】 Four cell clusters with unique transcriptional characteristics, including Myoblasts, Myocytes, Fibroblasts, and FAPs, were identified based on the gene expression correlations and marker genes. By comparing the gene expression profiles and analyzing the pseudotime differentiation trajectory of myoblast subclusters, it was found that there was strong heterogeneity among subclusters, of which Myoblasts_1 was the starting point of differentiation, Myoblasts_0 and 3 were in the early stage of differentiation, while Myoblasts_2 was the most obvious subcluster with muscle characteristics and they might be the late-stage Myoblasts adjacent to forming Myocytes. There were differences in muscle-related GO terms enriched by differential genes between Myoblasts_2 and Myocytes, and there were complex protein interactions among these genes. The communication mechanisms between homotypic or heterotypic cells of Myoblasts_0-5, Myocytes, Fibroblasts, and FAPs involved various ligand-receptor interactions, such as PTN-NCL, IGF2-IGF2R, and ANGPTL2-（ITGA5 + ITGB1）.【Conclusion】 There are gradually changing of gene expression profiles and intercellular communication during the differentiation of myogenic cells, reflecting the complex dynamic heterogeneity and molecular regulatory mechanisms.

Key words: bovine myogenic cells, scRNA-seq, cell subcluster, heterogeneity, gene expression profile, intercellular communication

QIN Jian, LI Zhen-yue, HE Lang, LI Jun-ling, ZHANG Hao, DU Rong. Change of Single-cell Transcription Profile and Analysis of Intercellular Communication in Myogenic Cell Differentiation[J]. Biotechnology Bulletin, 2024, 40(6): 330-342.

Figures/Tables 7

Fig. 1 Quality control and sample integration analysis of scRNA-seq data from bovine myogenic cell samples A: A violin plot shows the number of total RNAs in the remaining high-quality cells of two samples after quality control and filtration. B: A violin plot shows the number of total genes in the remaining high-quality cells of two samples after quality control and filtration. C: A UMAP dimension reduction graph shows the singlet or doublet（single or double cells）property prediction results of two samples after integrated analysis（Blue indicates singlet and red indicates doublet）. D: UMAP dimension reduction graphs show the singlet presentations of two samples after filtering the doublet（The left shows the combined display and the right shows the separate display. Red indicates sample 1 and blue indicates sample 2）

Fig. 2 Cell type profile of bovine myogenic cell samples delineated by scRNA-seq analysis A: A UMAP dimension reduction graph shows the 12 cell clusters obtained from scRNA-seq analysis（Different colors indicate the different cell clusters）. B: A dot plot shows the expression levels of marker genes in each cell cluster（Color scale indicates the average expression level of a certain gene in the cell clusters, and dot size indicates the percentage of cells expressing a certain gene）. C: A heatmap shows the correlations among 12 cell clusters based on the differential gene expression levels（Color scale indicates correlation coefficient）. D: A UMAP dimension reduction graph shows the 12 cell clusters are annotated into 4 cell types based on the marker gene and correlation analysis（Different colors indicate the different cell types）. E: UMAP graphs combined with FeaturePlots show the expression levels of marker genes for each cell type（Color scale indicates the gene expression level）

Fig. 3 Cell subclusters and differential gene expression profile comparison of bovine Myoblasts A: Circle graphs show the proportions of four cell types from two samples of bovine myogenic cells. B: A UMAP dimension reduction graph shows the Myoblasts used to divide subclusters after the two samples are combined（Blue indicates Myoblasts）. C: A UMAP dimension reduction graph shows the six Myoblasts subclusters（Different colors indicate the different cell subclusters）. D: A dot plot shows the comparison of the representative differential gene expression profiles of the Myoblasts subclusters（Color scale indicates the average expression level of a certain gene in the cell subclusters, and dot size indicates the percentage of cells expressing a certain gene）

Fig. 4 Comparison of the major muscle-related differential genes between Myoblasts_2 and Myocytes A: A dot plot shows the expression profile of the major muscle-related differential genes between Myoblasts_2 and Myocytes（Color scale indicates the average expression level of a certain gene in the cell cluster or subcluster, and dot size indicates the percentage of cells expressing a certain gene）. B: Violin plots show the expression levels and cell distributions of some muscle-specific differential genes in Myoblasts_2 and Myocytes（Horizontal coordinate indicates the cell type and vertical coordinate indicates the gene expression level. The width of the violin indicates the cell density with the corresponding expression level, the larger the width, the more cells at the expression level）

Fig. 5 GO enrichment and PPI analysis of the Myoblasts_2 and Myocytes differential genes in muscle-related biological processes A: Muscle-related GO terms enriched by the up-regulated genes in Myoblasts_2. B: Muscle-related GO terms enriched by the up-regulated genes in Myocytes. C: PPI analysis of genes in muscle-related GO terms enriched by Myoblasts_2 and Myocytes（Yellow and green indicate the up-regulated genes respectively in Myoblasts_2 and Myocytes）

Fig. 6 Analyses of ligand-receptor for intercellular communication of various cell types in bovine myogenic cell samples A: A UMAP dimension reduction graph shows the position relationship of the six myoblasts subclusters projected on the cell clusters（Different colors indicate the different cell clusters or subclusters）. B: A circle plot shows the interaction strength between various cell types（The size of the surrounding circular nodes indicates the cell number in a certain cell type. The color indicates which cell is the sender, the cell that sends the arrow is the sender, and the cell that the arrow points to is the receiver. The thickness of the line indicates the interaction strength）. C: A dot plot shows the major ligand-receptor pairs involved in intercellular communication（Dot size indicates the P-Value, and dot color indicates the ligand-receptor communication probability）. D: A violin plot shows the expression levels and cell distributions of PTN ligand and SDC/NCL receptor genes in each cell type（Horizontal coordinate indicates the cell type and vertical coordinate indicates the gene expression level. The width of the violin indicates the cell density with the corresponding expression level, the larger the width, the more cells at the expression level）

Fig. 7 Differentiation trajectory of bovine myogenic cell subclusters based on pseudotime analysis A: Cell trajectory plot displayed by pseudotime（The color from dark to light indicates the beginning to end of pseudotime）. B: Cell trajectory plot displayed by cell type（Different colors indicate different cell types）

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