Uncovering The Emergence Of HSCs In The Human Fetal Bone Marrow By Single-cell RNA-seq Analysis

Objective:Hematopoietic stem cells(HSCs)are rare cells in the blood system,with the potential for self-renewal and multi-differentiation,which can generate all blood and immune cells and maintain the function of the body.Most studies on HSC development are based on animal models such as mice.Due to the scarcity of technologies and the limited accessibility of human embryonic tissues,our understanding of the hematopoietic development of early human embryos is still insufficient.The emergence of single-cell sequencing enables researchers to accurately decipher the developmental pattern of rare cell populations in early embryonic hematopoietic organs such as the human yolk sac,AGM and fetal liver.Previous studies have reported the precise timing of the occurrence of human functional HSCs in AGM and fetal liver.However,the development of human functional HSCs in bone marrow has not been uncovered.To this end,this study used single-cell RNA-sequencing to map the transcriptomic landscape of human fetal BM and spleen hematopoietic stem/progenitor cells(HSPCs)and their microenvironment from 10 to 14 post-conception weeks(PCWs).Combined with transplantation experiments of immunodeficient mice,we revealed the molecular characteristics of the first wave of HSCs in the human embryonic bone marrow and their interactions with microenvironment cells and provided clues and potential strategies for improving the efficiency of clinical therapy.Methods:1.We performed droplet-based scRNA-seq(10x Genomics)using hematopoietic stem/progenitor cells(HSPCS)and niche cells obtained from enzyme-digested bone marrow or spleen collected at 9 human embryonic long bone samples(10-14 PCWs)and 5 human embryonic spleen samples(12-14 PCWs).Then,quality control was performed on the sequencing data,UMI expression matrix was obtained,batch effect removal,dimensionality reduction,unsupervised cluster analysis and cell cluster annotation were conducted to construct human fetal BM and spleen HSPC and niche cell atlas.2.Primary and secondary transplantation experiments by 10-14 PCWs human fetal BM cells or 12-14 PCWs spleen cells were carried out in immunodeficient mouse models to determine the time point when functional HSC seeded the BM and spleen.3.We performed STRT-seq of HSCs cells from BM collected at 11-12 PCWs(early stage)and enriched human HSCs from the BM of recipient mice(post-trans.).Published scRNA-seq data of HSC/MPPs from the middle-stage fetal BM(17-22 PCWs),adult BM and umbilical cord blood were included to conduct data processing,cell cluster annotation,cell cycle calculation,gene set scoring,differential gene enrichment analysis,SCENIC and other biogenic analysis,to describe the characteristics of HSC/MPP at different stages.4.The published scRNA-seq data of niche cells from the middle-stage fetal BM(17-19 PCWs)(late stage)and adult BM were included to perform cell cluster annotation,pseudotime trajectory and key gene expression and other bioinformatics analysis in order to compare the differences of niche cells at different stages.5.cellphoneDB method was used to analyze the interaction between stage-corresponding HSC/MPPs and niche cells,so as to reveal the most important niche population and potential ligand-receptor interactions.and in vitro co-culture experiment was conducted to verify the supportive role of fetal CAR cells and potential niche factors in fetal HSC/MPP maintenance;6.We performed droplet-based scRNA-seq(10x Genomics)using HSPCs and niche cells obtained from enzyme-digested bone marrow collected from E16.5-18.5 mouse fetal pulp to conduct data processing,cell cluster annotation and other bioinformatics analysis to describe the differences from human and mouse hematopoietic and microenvironment development.7.cellphone DB method was used to compare the interaction between fetal liver-derived HSCs and fetal BM or fetal spleen microenvironment cells,so as to explore the reasons why fetal liver-derived HSCs preferentially colonized the BM.Results:We captured 48002 and 24721 qualified HSPCs and niche cells based on 9 human fetal long bone samples(10-14 PCWs)and 5 fetal spleen samples(12-14 PCWs)respectively.On average,we detected more than 2,000 protein-coding genes in each cell.The transcriptome landscape of human early fetal BM and spleen HSPCs and niche cells is systematically described.We detected immunophenotypic and transcriptomic-defined HSC/MPPs populations in both fetal BM at 10-14 PCWs and fetal spleen at 12-14 PCWs,but transplantation assay further validated functional HSCs capable of reconstituting long-term multi-lineage hematopoiesis in adult NOG mice do not emerge in the BM until 12 PCWs,before spleen colonization.Reconstitution in recipient mice’s peripheral blood,bone marrow or spleen exhibited lymphoid bias dominated by B cells.By co-analysis with scRNA-seq data of HSC/MPPs from early stage,post-trans.,late stage,adult BM and umbilical cord blood,we found that the early stage and post-trans.HSC/MPPs were more active,whereas late-stage HSC/MPPs were more quiescent.Early-stage HSC/MPPs showed lymphoid lineage bias.Late-stage HSC/MPPs were highly enriched for quiescence-related transcription factors(TFs)as well as several stemness maintenance-related TFs and genes,which suggested early-stage HSC/MPPs are functionally less active than late-stage cells.In conjunction with single-cell sequencing data from adult BM and cord blood HSCs/MPPs,we found that fetal BM HSC/MPPs,especially in the early stage,exhibit significant features of cell migration,adhesion,and strong interaction with the microenvironment.By reanalyzing the heterogeneity of fetal BM niche cells,we made a finer segmentation of niche cells and found that CAR cells and aECs are the main sources of important niche factors in human fetal BM.We further revealed the dramatic changes in CAR cells during development.We observed the evolution of important signals involved in the regulation of HSC function from embryo to adult stage,revealing different regulatory mechanisms of HSC function in embryonic and adult BM.Based on the analysis of the interaction between human fetal BM niche cells and HSC/MPPs,we found that CAR cells and aECs had the most and strongest interaction with HSC/MPPs,and speculated that CAR cells and aECs were the main microenvironment cells involved in HSC colonization and maintenance.We further verified that CAR cells and their secreted important molecule PTN were involved in the maintenance of fetal fetal HSPCs through in vitro co-culture experiments.At the same time,the transcriptome landscape of mouse embryonic BM HSPCs and niche cells is conducted.We also identified the CAR cells in mouse embryonic BM which had the most interaction with HSC/MPPs,further highlighting the conserved and different characteristics of hematopoietic and microenvironment development in humans and mice.Finally,through the analysis of the interaction between human fetal BM and spleen niche cells and fetal liver HSC,We observed that,compared with spleen niche cells,BM niche cells were predicted to provide critical signals to support HSC migration,maintenance,and expansion,including NOTCH signaling,CD44 and CD74 signaling,etc.which initially answered the question why HSC preferentially colonized bone marrow.Conclusion:In this study,by using 10x Genomics single-cell RNA sequencing,a transcriptomic atlas of HSPCs and niche in human early fetal BM and spleen was constructed.Combined with immunodeficient mouse transplantation experiment,it was further confirmed that functional HSC colonized BM at 12 weeks,which was earlier than that of the spleen.HSCS/MPPs are still active after entering the BM,and exhibited lymphoid bias and strong interaction with the microenvironment.However,with the development of the embryo,they gradually change to a quiescent state,and have stronger functions.CAR cells and aECs are the main sources of important niche factors in human fetal BM and the main microenvironment cells involved in the colonization and functional maintenance of HSCs.CAR cells present dramatic transcriptome changes from embryo to adult development.Embryonic CAR cells maintain the function of HSPCs by secreting PTN.The BM provides superior "soil" for HSC seeding.