单细胞基因组学比较人诱导多能干细胞来源巨噬细胞与外周血来源巨噬细胞的异质性  

Comparison of human-induced pluripotent stem cell-derived macrophages with peripheral blood-derived macrophages using single-cell genomics

作　　者：张羽桐 侯国俊 沈南[1] ZHANG Yutong;HOU Guojun;SHEN Nan(Department of Rheumatology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai Institute of Rheumatology,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院风湿科,上海风湿病学研究所,上海200127

出　　处：《上海交通大学学报(医学版)》2024年第12期1477-1489,共13页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(31930037,82302024,32141004);上海市高水平地方高校创新团队(SSMU-ZDCX20180100)。

摘　　要：目的·探究人诱导多能干细胞(induced pluripotent stem cell,iPSC)来源的巨噬细胞(iPSC-derived macrophage,IPSDM)与人外周血来源的巨噬细胞(peripheral blood-derived macrophage,PBDM)的单细胞基因组学异质性。方法·利用无饲养层无血清方案体外诱导iPSC分化为IPSDM,通过流式细胞术及反转录实时荧光定量聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)分别检测IPSDM表面白细胞分化抗原14(cluster of differentiation antigen 14,CD14)与单核-巨噬细胞标志基因的表达情况。随后对IPSDM进行单细胞测序,同时从基因表达综合数据库中下载单细胞测序数据集GSE126085作为PBDM参考数据集。通过R软件seurat包处理分析IPSDM与PBDM测序数据,利用singleR包注释PBDM。以PBDM的高变基因构建参考数据集,利用scmap包将IPSDM的高变基因投影于PBDM,根据高变基因的相似性推断IPSDM的细胞身份。随后参考细胞类型注释工具及相关研究完成对IPSDM的注释,并比较IPSDM和PBDM巨噬细胞标志基因的表达分布。最后,利用seurat包寻找IPSDM与PBDM的差异表达基因(differentially expressed gene,DEG),通过基因本体(Gene Ontology,GO)分析与京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析探究DEG潜在的生物学功能。结果·体外诱导分化29 d后得到悬浮的IPSDM,经流式细胞术及RT-qPCR检测证实约23.1%的IPSDM表达CD14,且与U-937细胞系相比,IPSDM表达更高水平的单核-巨噬细胞标志基因。PBDM数据集中的细胞全部被注释为巨噬细胞,随后构建scmap参考数据集,将IPSDM的高变基因映射于PBDM参考数据集后发现59.8%的IPSDM可以依据PBDM的高变基因索引被注释为巨噬细胞,而余下40.2%的IPSDM细胞无法匹配PBDM的高变基因索引。进一步人工注释IPSDM发现其由97.15%巨噬细胞、2.71%造血前体细胞样细胞和0.14%树突状细胞组成。对IPSDM和PBDM的巨�Objective·To explore the heterogeneity in single-cell genomics between human-induced pluripotent stem cell(iPSC)-derived macrophages(IPSDM)and human peripheral blood-derived macrophages(PBDM).Methods·iPSCs were differentiated into IPSDMs in vitro using a feeder-free and serum-free protocol.The expression of cluster of differentiation antigen 14(CD14)and monocyte-macrophage marker genes in IPSDMs was analyzed using flow cytometry and real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR),respectively.Single-cell sequencing was then performed on IPSDMs.Simultaneously,the single-cell sequencing dataset GSE126085 was downloaded from the Gene Expression Omnibus database as a reference dataset for PBDMs.Sequencing data for both IPSDMs and PBDMs were processed and analyzed using the seurat package in R software,with PBDMs annotated using the singleR package.A reference dataset was constructed with highly variable genes from PBDMs,and the highly variable genes of IPSDMs were projected onto the PBDM dataset using the scmap package to infer IPSDMs cell identities based on variable gene similarity.IPSDMs were annotated using cell-type annotation tools and referenced against relevant studies.The expression distribution of macrophage marker genes was compared between IPSDMs and PBDMs.Differentially expressed genes(DEGs)between IPSDMs and PBDMs were identified using the seurat package,and their potential biological functions were explored through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.Results·Suspended IPSDMs were obtained after 29 d of in vitro differentiation.Flow cytometry and RT-qPCR confirmed that approximately 23.1%of IPSDMs expressed CD14,and IPSDMs exhibited higher expression of monocyte-macrophage marker genes compared to the U-937 cell line.All cells in the PBDM dataset were annotated as macrophages.After constructing a scmap reference dataset using PBDMs,59.8%of IPSDMs were annotated as macrophages through mapping their highly variabl

关 键 词：诱导多能干细胞 细胞分化 巨噬细胞 单细胞测序 

分 类 号：R392.12[医药卫生—免疫学]