Resolving The Origin Of T Lymphocytes And Thymus Organogenesis In Human At Single-cell Resolution

T lymphocytes form the major arms of adaptive immunity and play a vital role in the host's resistance to the invasion of foreign pathogens and in maintaining the host's immune homeostasis.It is generally believed that mature hematopoietic cells are mainly differentiated from hematopoietic stem cells in the bone marrow.However,unlike other hematopoietic cells,the development of T cells occur mainly in the thymus.It is well known that T cell development initiates from TSPs in the bone marrow or fetal liver.They colonize the thymus with chemokine recruitment and develop into ETPs.Subsequently,they experience CD4~-CD8~-double negative,CD4~+CD8~+double positive,CD4~+or CD8~+single positive,three major T cell differentiation stages,and develop into mature functional T cells.However,given the extreme rarity of TSP and ETP,we still know little about the developmental processes of T cells in human embryos.With the help of single-cell transcriptome sequencing,functional incubation experiments and immunohisto-chemistry,our work unveils the mysterious veil of human embryonic T cell development and thymus organogenesis and elaborates spatiotemporal heterogenicity of human embryonic lymphoid progenitors at the single-cell level.We also reveal the origin and molecular characteristics of embryonic TSP with depicting 3 successive stages including TSP-like ETP,Proliferating ETP and intermediate thymic progenitor during thymus seeding of TSPs and specialization into T cell.Beyond that,we analyze the heterogeneity and fate specialization of T cells and thymus epithelial cells in the early stage of thymus development and outline the cell-cell interaction map between hematopoietic cells and stromal cells during thymus formation at single resolution for the first time.Firstly,we interpret the heterogeneities of major cell types in the early stages of thymus development by single cell RNA sequencing of human embryonic thymus from week 8 to week 10.Interestingly,we identify and define 2 ETP populations,namely embryonic ETP and fetal ETP.They express progenitor-related genes CD34 and SPINK2,however,with different transcriptome and development stages.Fetal ETP are mainly from week 9 and week 10,while embryonic ETP are mainly from week 8 with high expression of chemotaxis and cell adhesion-related genes.These results reveal that ETP populations entering thymus around week 9 have changed and progenitors from upstream hematopoietic organs colonize the thymus primordium mainly by chemotaxis before establishment of thymic vessels.Subsequently,hematopoietic cells from aorta-gonad-mesonephros,fetal liver and blood from week 5 to week 10 were captured and subjected into single cell transcriptome sequencing.We capture CD34~+IL7R~+lymphoid progenitors at different stages and sites and reveal their transcriptome characteristics for the first time.Through joint analysis using lymphoid progenitors and ETP in the thymus,we imply high consistency of transcriptome between lymphoid progenitors in fetal liver at week 8 and ETP in thymus,suggesting that ETPs are directly derived from the CD34~+IL7R~+TSPs in fetal liver.Further analysis using the principle component analysis and pseudo-time analysis reveal that the TSPs in fetal liver need to undergo TSP-like ETP,Proliferating ETP and intermediate thymic progenitor,3 development stages,before commitment to T cells.We also depict the molecular regulation network during fate specification of TSP.Next,we verify the multi-lineage differentiation potential of the human embryonic CD34~+CD1A~-ETP population using in vitro functional experiments.Our results indicate that CD7~+ETPs account for the vast majority of CD34~+CD1A~-progenitors in human embryo.Unlike previous studies in adult,CD7~+ETPs still have multilineage differentiation potential.Subsequently,we predict and depict development trajectory of T cell fate specification in thymus,where ETP as the starting point and going through the intermediate thymic progenitor stage.Gene regulatory networks in fate selection between??T and??T are also described.Our research also captured thymus epithelial cells in the early stage of embryonic thymus development(early week 8 to week 10)for the first time.In addition to high expression of epithelial features,thymus epithelial cells still express the molecular characteristics of the third pharyngeal pouch.We also identify cortical thymus epithelial cell and medullary thymic epithelial progenitor cell populations at early stages of thymus organogenesis using unsupervised clustering and dimension reduction analysis.We further reveal significant differences in maturation speed between cortical thymus epithelial cell and medullary thymus epithelial cell and transcriptional regulation during thymus epithelial cell maturation using epithelium maturity scores and correlation analysis.Subsequently,we reveal the different roles of important signaling pathway during thymus epithelial cell fate specialization including Bmp,Hippo,Retinoic acid,Wnt and the citric acid cycle,using gene sets enrichment analysis and gene ontology analysis.Finally,we draw the microenvironment map in thymus during T cell development through cell-cell ligand-receptor interactions analysis.Our results reveal different modules of cell-cell interactions between thymocytes and stroma cells.We also show the strongest interactions between thymocytes and thymus epithelial cells compared to endothelial cells and mesenchymal cells.Next,we depict the conservation between human and mouse in chemokine signals and signaling pathways for lymphocytes development such as CXCL12-CXCR4,CCL25-CCR9,NOTCH,BMP,and IL-7,which are all involved in the migration,colonization,expansion,differentiation as reported in precious literature.In addition,we confirm the existence of IGF2-IGF1R interactions between mesenchymal cells and ETPs through bioinformatics prediction and immunofluorescence validation,suggesting that mesenchymal cells may play an important role in the proliferation and expansion of ETPs after colonization into the thymus through the insulin-like growth factor signaling pathway.In short,we demystify T lymphocytes development and thymus organogenesis using single-cell transcriptome data covering multiple hemogenic and hematopoietic sites in the early stages of human embryos,which will provide valuable data source and blueprints for our understanding of embryonic T cell development and thymus organogenesis.Undoubtedly,it will also provide important theoretical support for regeneration of T cells,reconstruction and regeneration of thymus organs in vitro.