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Single-Cell RNA Sequencing Reveals B Cells Are Important Regulators in Fracture Healing

ABSTRACT: The bone marrow microenvironment is composed primarily of immune and stromal cells that play important roles in fracture healing. Although immune cells have been identified in mouse bone marrow, variations in their numbers and type during the fracture healing process remain poorly defined. In this study, single-cell RNA sequencing was used to identify immune cells in fracture tissues, including neutrophils, monocytes, T cells, B cells, and plasma cells. The number of B cells decreased significantly in the early stage of fracture healing. Furthermore, B cells in mice fracture models decreased significantly during the epiphyseal phase and then gradually returned to normal during the epiphyseal transformation phase of fracture healing. The B-cell pattern was opposite to that of bone formation and resorption activities. Notably, B-cell–derived exosomes inhibited bone homeostasis in fracture healing. In humans, a decrease in the number of B cells during the epiphyseal phase stimulated fracture healing. Then, as the numbers of osteoblasts increased during the callus reconstruction stage, the number of B cells gradually recovered, which reduced additional bone regeneration. Thus, B cells are key regulators of fracture healing and inhibit excessive bone regeneration by producing multiple osteoblast inhibitors.

SUBMITTER: Zhang H

PROVIDER: S-EPMC8606664 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Skeletal muscle from meat-producing livestock such as cattle is a major source of food for humans. To improve skeletal muscle growth efficiency or quality in cattle, it is necessary to understand the genetic and physiological mechanisms that govern skeletal muscle composition, development, and growth. Satellite cells are the myogenic progenitor cells in postnatal skeletal muscle. In this study we analyzed the composition of bovine satellite cells with single-cell RNA sequencing (scRNA-seq). We isolated satellite cells from a 2-week-old male calf, cultured them in growth medium for a week, and performed scRNA-seq using the 10x Genomics platform. Deep sequencing of two scRNA-seq libraries constructed from cultured bovine satellite cells yielded 860 million reads. Cell calling analyses revealed that these reads were sequenced from 19,096 individual cells. Clustering analyses indicated that these reads represented 15 cell clusters that differed in gene expression profile. Based on the enriched expression of markers of satellite cells (PAX7 and PAX3), markers of myoblasts (MYOD1, MYF5), and markers of differentiated myoblasts or myocytes (MYOG), three clusters were determined to be satellite cells, two clusters myoblasts, and two clusters myocytes. Gene ontology and trajectory inference analyses indicated that cells in these myogenic clusters differed in proliferation rate and differentiation stage. Two of the remaining clusters were enriched with PDGFRA, a marker of fibro-adipogenic (FAP) cells, the progenitor cells for intramuscular fat, and are therefore considered to be FAP cells. Gene ontology analyses indicated active lipogenesis in one of these two clusters. The identity of the remaining six clusters could not be defined. Overall, the results of this study support the hypothesis that bovine satellite cells are composed of subpopulations that differ in transcriptional and myogenic state. The results of this study also support the hypothesis that intramuscular fat in cattle originates from fibro-adipogenic cells.

Project description:BackgroundMyocardial infarction (MI) is a cardiovascular disease that seriously threatens human health. Dysangiogenesis of endothelial cells (ECs) primarily inhibits recovery from MI, but the specific mechanism remains to be further elucidated.MethodsIn this study, the single-cell RNA-sequencing data from both MI and Sham mice were analyzed by the Seurat Package (3.2.2). The number of ECs in MI and Sham groups were compared by PCA and tSNE algorithm. FindMarkers function of Seurat was used to analyze the DEGs between the MI and Sham groups. Then, the ECs was further clustered into 8 sub-clusters for trajectory analysis. The BEAM was used to analyze the branch point 3 and cluster the results. In addition, the DEGs in the microarray data set of MI and Sham mice were cross-linked, and the cross-linked genes were used to construct PPI networks. The key genes with the highest degree were identified and analyzed for functional enrichment. Finally, this study cultured human umbilical vein endothelial cells (HUVECs), established hypoxia models, and interfered with hub gene expression in cells. The impact of hub genes on the migration and tube formation of hypoxic-induced HUVECs were verified by Wound healing assays and tubule formation experiments.ResultsThe number and proportion of ECs in the MI group were significantly lower than those in the Sham group. Meantime, 225 DEGs were found in ECs between the MI and Sham groups. Through trajectory analysis, EC4 was found to play an important role in MI. Then, by using BEAM to analyze the branch point 3, and clustering the results, a total of 495 genes were found to be highly expressed in cell Fate2 (mainly EC4). In addition, a total of 194 DEGs were identified in Micro array dataset containing both MI and Sham mice. The hub genes (Timp1 and Fn1) with the highest degree were identified. Inhibiting Timp1 and Fn1 expression promoted the migration and tube formation of HUVECs.ConclusionsOur data highlighted the non-linear dynamics of ECs in MI, and provided a foothold for analyzing cardiac homeostasis and pro-angiogenesis in MI.

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Single-Cell RNA Sequencing Reveals B Cells Are Important Regulators in Fracture Healing

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