Single-Cell Atlas Of Multisource Hematopoietic Stem/Progenitor Cells And Bone Marrow T Cells In Severe Aplastic Anemia

Background:Human hematopoietic stem cells(HSC)begin to appear from embryogenesis and participate in the formation of the human hematopoietic system through progressive differentiation and development.A full understanding of the hematopoietic developmental process is essential for the treatment of hematologic diseases.Human hematopoietic development is a multitemporal and continuous process.The earliest primitive hematopoietic system appears in the yolk sac(YS)during the 2nd-6th weeks of embryonic development.The fetal liver(FL)becomes the main site of hematopoiesis during the 7th week.The hematopoietic activity of bone marrow(BM)becomes active after the 4th month of embryonic development,and BM becomes the main site of hematopoiesis after birth in humans.The megakaryocyte progenitor cells(MkP)in hematopoietic stem/progenitor cells(HSPC)are capable of differentiating and developing into polyploid mature Mk.Previous studies have dissected the heterogeneity of Mk and identified a subpopulation of Mk with immune characteristics,and it is worthwhile to further investigate whether MkP have the similar heterogeneity and non-classical immune functions as mature Mk.In recent years,single cell RNA sequencing(scRNA-seq)technology has been widely used to study the hematopoietic system,however,transcriptome studies of HSPC at different developmental stages in humans lack a unified analytical platform to directly compare the changes in hematopoietic development at different stages.Objective:1.To integrate and construct single-cell transcriptome profiles of human multisource HSPC using a unified analytical platform.2.To probe the transcriptome characteristics of megakaryocyte progenitors/megakaryocytes(MkP/Mk).Methods:1.BM from 4 healthy donors was collected and published single-cell transcriptome datasets from 2 YS and 2 FL samples were downloaded.2.Flow sorting of CD34+cells from BM.3.Performing 10X Genomics 3’ single cell transcriptome library construction and sequencing.4.Cell Ranger was used for raw data preprocessing.Seurat was used for quality control and filtering,sample merging,clustering,dimensionality reduction and differentially expressed genes(DEGs)estimation.ClusterProfiler was used for functional enrichment analysis.pySCENIC was used for transcription factor regulatory network analysis.Monocle 3 and PAGA were used for developmental trajectory construction.Results:1.Single-cell sequencing data collection and quality control results:single-cell datasets from 4 healthy adult BM-derived CD34+HSPC and 2 published YS and 2 FL-derived hematopoietic tissues were integrated,and 56381 cells of high quality were retained for subsequent analysis after filtering.2.Clustering of multisource hematopoietic tissue cells and their characterization:24 cell populations were defined based on the characteristic genes of each subpopulation of HSPC.The directions of hematopoietic differentiation in the lymphoid,myeloid and megakaryocytic/erythroid lineages were marked.Hematopoietic developmental trajectories were constructed using Monocle 3.The characteristic transcription factors of each cell population reflect the specific functions in each population.The gene expression patterns of HSC in different source groups were explored,with genes and pathways about immunity in BM,signature genes closely associated with early erythropoiesis in YS,and biological processes related to cell division and energy metabolism in FL.3.Single-cell transcriptome characteristics of MkP/Mk:there was functional heterogeneity within MkP/Mk,with different gene expression profiles of MkP/Mk from different sources.Pathways associated with regulation of hematopoietic differentiation and lymphocyte-mediated immunity were enriched in BM.Classical coagulation-related functions were enriched in YS,and pathways such as DNA replication were enriched in FL.4.Subpopulation reclassification and heterogeneity analysis of MkP/Mk:some MkP and Mk subpopulations presented both immune and coagulation characteristics and were associated in developmental trajectories.One subpopulation of MkP with dual characteristics showed different functional preferences and sustained immune potential in different source groups.Conclusion:1.The HSPC single-cell transcriptome of human BM,YS and FL was drawn.A total of 24 cell clusters were identified and the hematopoietic development trajectories were constructed.The characteristic genes and transcription factors of each cell cluster were consistent with their definition and functions.2.The transcriptional expression profiles of HSC from different sources differed,with BM,YS and FL enriched to genes and biological processes related to immunity,early erythropoiesis,cell division and energy metabolism,respectively.3.There is functional heterogeneity in MkP/Mk from different sources,with BM,YS,and FL enriched to genes and biological processes related to immunity,coagulation,and DNA replication,respectively.。4.Both MkP and Mk contain the subpopulations with immune and coagulation characteristics,and MkP with dual characteristics have different functional preferences and sustained immune potential in different hematopoietic tissues.Background:Aplastic anemia(AA)is a bone marrow hematopoietic failure syndrome caused by multiple etiologies.Severe aplastic anemia(SAA)has a high early mortality rate and is a serious threat to lives.Exploring the pathogenesis of AA is important for establishing better treatment plans,improving survival rates,and reducing the risk of relapse.In most cases,AA is considered to be an immune-mediated disease.Bone marrow is the main site of T cell immune response and HSPC destruction in AA patients,and the analysis of bone marrow-derived T cells is the focus of AA research,which plays a key role in revealing the mechanism of immune damage in AA.In addition,changes in the T cell receptor(TCR)repertoire constitute an important part of the pathogenesis of AA,and identification of disease-associated TCRs can help to discover potential biomarkers and provide new insights into the therapeutic targets of AA.scRNA-seq is increasingly being used in the study of immune-related diseases.scRNA-seq studies of bone marrow T cells from SAA patients are still lacking and have further value for transcriptome characterization of T cell subpopulations,changes in TCR repertoire,and antigen specificity prediction.Objective:1.To analyze the single-cell transcriptome and V(D)J sequencing dataset of bone marrow T cells from SAA patients to dissect the transcriptome characteristics of T cell subpopulations and the composition and differences of TCR clones in SAA.2.To probe the potential features of SAA by correlation analysis of TCR repertoire and gene expression data.Methods:1.T cells were flow-sorted from bone marrow of 5 SAA patients and 4 healthy controls(HC)for 10X Genomics 5’ single cell transcriptome and V(D)J library preperation and sequencing.2.Bone marrow was collected from 6 SAA patients and 6 HC for analyses of the proportion of CD4+tissue-resident memory T cells(CD4+Trm)by flow cytometry.3.Cell Ranger was used for raw data preprocessing.Seurat was used for quality control and filtering,sample merging,clustering,dimensionality reduction and differentially expressed genes(DEGs)estimation.ClusterProfiler was used for functional enrichment analysis.scvelo was used for RNA velocity analysis.Monocle 2 was used for T cell developmental trajectory construction.scRepertoire,scirpy and CoNGA were used for TCR repertoire related analysis.Results:1.Single-cell sequencing data collection and quality control results:single-cell datasets of bone marrow T cells from 4 HC and 5 SAA patients were integrated,and 78391 cells of high quality were retained for subsequent analysis after filtering.2.Clustering of bone marrow T cells and their characterization:14 T cell subpopulations were defined based on the characteristic genes of each subpopulation of T cells.T cell developmental trajectories were constructed using RNA velocity analysis and Monocle 2.3.Single-cell transcriptome characteristics of bone marrow CD8+cytotoxic T cells(CD8+CTL)in SAA:CD8+CTL in SAA expressed a strong immune hyperfunction,reflected by enhanced expression of cytotoxicity-related genes and pathways of antiviral responses.4.Single-cell transcriptome characteristics of bone marrow mucosa-associated constant T cells(MAIT)in SAA:MAIT of SAA were associated with lymphocyte regulation,antiviral infection,increased proliferation and impaired apoptosis.5.Altered ratio and single-cell transcriptome characteristics of bone marrow CD4+ Trm in SAA:altered ratio and abnormal function of bone marrow CD4+Trm were found in SAA,showing abnormal activation of antigen processing and T cell signaling,and defective protein folding function.6.Altered ratio and single-cell transcriptome characteristics of bone marrow CD8+Trm in SAA:showing activation of T cell activation,differentiation,and enhanced intercellular adhesion.7.Immune repertoire analysis of bone marrow T cells:abnormal TCR clonal alterations were found in SAA,but heterogeneity existed in different individuals and subpopulations.8.Correlation analysis of TCR repertoire and gene expression data:CD8+CTL and MAIT activated by viral induction in SAA were involved in the immune response.Conclusion:1.The single-cell transcriptome and V(D)J profiles of bone marrow T cells were drawn.14 T cell subpopulations were identified and the T cell developmental trajectories were constructed.2.The transcriptional expression profiles of bone marrow T cells suggested that CD8+CTL and MAIT presented enhanced antiviral responses,CD4+Trm showed altered ratio and abnormal functions,and CD8+Trm were in an activated proliferative state.3.Aberrant TCR clonal alterations were present in SAA,but heterogeneity existed in different individuals and subpopulations.The CD8+CTL and MAIT activated by viral induction in SAA were involved in the immune response.