Analysis of the types and functions of CD34+cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing
Objective To analyze the types and functions of CD34+cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing.Methods This study was an experimental study.The CD34+cell lineage tracing mouse was produced,and the visualization of CD34+cells under the fluorescent condition was realized.Six male CD34+cell lineage tracing mice aged 7-8 weeks(designated as diabetic group)were intraperitoneally injected with streptozotocin to establish a diabetic model,and full-thickness skin defect wounds were prepared on their backs when they reached 13 weeks old.Another 6 male CD34+cell lineage tracing mice aged 13 weeks(designated as control group)were also subjected to full-thickness skin defect wounds on their backs.On post-injury day(PID)4,wound tissue was collected from 3 mice in control group and 2 mice in diabetic group,and digested to prepare single-cell suspensions.CD34+cells were screened using fluorescence-activated cell sorting,followed by single-cell RNA sequencing.The Seurat 4.0.2 program in the R programming language was utilized for dimensionality reduction,visualization,and cell clustering analysis of CD34+cell types,and to screen and annotate the marker genes for each CD34+cell subpopulation.Kyoto encyclopedia of genes and genomes(KEGG)and gene ontology(GO)enrichment analysis was performed to analyze the differentially expressed genes(DEGs)of CD34+fibroblasts(Fbs),smooth muscle cells(SMCs),keratinocytes(KCs),and chondrocyte-like cells(CLCs)in the wound tissue of two groups of mice for exploring cellular functions.Results On PID 4,CD34+cells in the wound tissue of both groups of mice were consisted of 7 cell types,specifically endothelial cells,Fbs,KCs,macrophages,T cells,SMCs,and CLCs.Among these,Fbs were further classified into 5 subpopulations.Compared with those in control group,the proportions of CD34+endothelial cells,Fbs subpopulation 1,Fbs subpopulation 4,KCs,and CLCs in the wound tissue of mice were increased in diabetic group,while the proportions of CD34+Fbs subpopulation 2,Fbs subpopulation 3,and SMCs were decreased.The marker genes for annotating CD34+CLCs,endothelial cells,Fbs subpopulation 1,Fbs subpopulation 2,Fbs subpopulation 3,Fbs subpopulation 4,Fbs subpopulation 5,KCs,macrophages,SMCs,and T cells were respectively metastasis-associated lung adenocarcinoma transcript 1,fatty acid binding protein 4,Gremlin 1,complement component 4B,H19 imprinted maternally expressed transcript,Dickkopf Wnt signaling pathway inhibitor 2,fibromodulin,keratin 5,CD74 molecule,regulator of G protein signaling 5,and inducible T-cell co-stimulator molecule.KEGG and GO enrichment analysis revealed that,compared with those in control group,DEGs with significant differential expression(SDE)in CD34+Fbs from the wound tissue of mice in diabetic group on PID 4 were significantly enriched in terms related to inflammatory response,extracellular matrix(ECM)organization,regulation of cell proliferation,and aging(with P values all＜0.05),DEGs with SDE in CD34+SMCs were significantly enriched in terms related to cell migration,apoptotic process,positive regulation of transcription,and phagosome(with P values all＜0.05),DEGs with SDE in CD34+KCs were significantly enriched in terms related to mitochondrial function,transcription,and neurodegenerative diseases(with P values all＜0.05),and DEGs with SDE in CD34+CLCs were significantly enriched in terms related to rhythm regulation,ECM,and viral infection(with P values all＜0.05).Conclusions CD34+cells display high heterogeneity in the healing process of full-thickness skin defect wounds in both normal mice and diabetic mice.The significantly enriched functions of DEGs with SDE in CD34+cell subpopulations in the wound tissue of the two mouse groups are closely related to the wound healing process.
Wound healingSkinCD34Single-cell RNA sequencingCell subpopulationsMarker genesCellular heterogeneityCell function
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