The Development And Functional Heterogeneity Of Human Erythroid Precursors

Objectives:Erythroid cells are the most abundant hematopoietic cells in the human body and one of the earliest emergence blood cells.Previous studies of the development of human erythroid cells mainly focus on the adult,but little is known about the molecular characteristics of erythroid cells at the early stage of human embryonic development.Although erythroid cells are conventionally defined as the oxygen carriers,emerging studies have suggested that erythroid cells can play an important role in immunomodulatory,especially when the body is under stress conditions,such as infection or tumor.However,it is unclear whether all nucleated erythroid precursors,or just specific subsets,possess these immunomodulatory functions,and the molecular characteristics of these immunomodulatory erythroid cells remains unknown.In this study,we conducted single-cell RNA-sequencing(scRNA-seq)analysis of human erythroid precursors collected from multiple sites(Yolk Sac,YS;Fetal Liver,FL;Preterm Umbilical Cord Blood,PT-UCB;term UCB and adult Bone Marrow,BM)during ontogeny.We revealed the unique molecular characteristics of the human embryonic erythroid precursors and dissected the functional heterogeneity of erythroid precursors.We also identified and verified the existence of an immunomodulatory-erythroid subset.Methods:In this study,human erythroid precursors were collected by fluorescenceactivated cell sorting(FACS)from YS,FL,PT-UCB,term UCB and adult BM.10x Genomics and STRT-seq platforms were used to construct the library and perform single cell transcriptome sequencing,followed by further bioinformatics analysis.Firstly,we determined the heterogeneity of human erythroid precursors,the feature genes and biological pathways of each subset,and constructed the potential differentiation trajectory of the human erythroid precursors.Subsequently,we performed comparative analysis within each cluster of cells originating from different developmental stages,which uncovered the molecular changes of erythroid precursors that occur in different developmental stages.Finally,immunomodulatory-erythroid subset was analyzed from the aspects of immune-related molecular pathways,feature genes,transcriptional regulatory networks.We screened the cell surface markers and determined the existence of immunomodulatory-erythroid subset,further confirmed by in situ multicolor immunohistochemistry(IHC)staining and flow cytometry.By co-culture with classical immune cells,we determined the immunomodulatory functions of immune-prone erythroid through cytokine secretion,gene expression and protein expression assays.Interaction analysis also identified interacting molecular pairs between immune-prone erythroid and other classical immune cells,which was verified by in situ IHC staining.Results:(1)Determining the functional heterogeneous of human erythroid precursors.Four distinct heterogeneous subsets,including three classical(C1-C3)and one immune-prone subset(C4),were identified by integration analysis.Cells from each cluster were present throughout the developmental stages examined at varying frequencies.(2)lmmunomodulatory-erythroid subset has unique differentiation pathways.Pseudotime analysis and topological structure analysis showed that immunomodulatoryerythroid subset has different differentiation pathway from classical erythroid precursors.The three classical erythroid subsets were close to each other,and matured progressively from C1 to C3,the immune erythroid subset(C4)showed a divergent developmental trajectory.(3)Unique molecular characteristics of erythroid precursors derived from YS,FL and UCB.Parallel analysis of the subsets of erythroid precursors from different developmental stages showed that the cholesterol synthesis and aerobic glycolysis were enriched in YS C1 cells,the FL C1 cells showed strong signals for protein synthesis,whereas UCB C1 cells exhibited more abundant expression of globin genes.In the mature stage,YS C3 cells were characterized by expression of metallothionein genes MT2A,MT1G and MT1H,as well as the zinc transporter genes ZnT(SLC30A)and ZIP(SLC39A).YS C3 cells also showed high proliferation characteristics.(4)Immunomodulatoryerythroid cells coupled with dual erythroid and immune regulatory networks.The analysis of transcription factor activity showed that MYC,GATA1 and KLF1 were downregulated along the erythroid maturation,whereas NFE2 and TAL1 were sequentially activated to promote terminal erythroid maturation in three classical erythroid subsets.Immunomodulatory-erythroid cells not only shared the core erythroid lineage regulons of GATA1,MYB and MYC,but also highly enriched in immune-related regulons such as GATA2,MEF2C,SPI1,FOS,and JUN.The target genes of these immune-related regulons were enriched in categories such as chemokine signaling pathway and antigen processing and presentation.Genes associated with activation of immune response and cytokinemediated signaling pathway,as well as VSIR,TGFB1,TNFSF10 were specifically highly expressed in immunomodulatory-erythroid cells.Through screening for surface markers,CD63 was highest and most specifically expressed in immunomodulatory-erythroid cells.CD63 positive erythroid subsets were exited in the erythroid cells from FL and UCB,accounting for about 2.58%and 6.36%of the GYPA+CD71+ erythroid precursors.Expression of CD63 on GYPA+erythroid precursors was also detected by IHC staining and they also expressed the immune signature proteins,such as HLA-E and TGF-β,which were detected in GYPA+CD63+cells by IHC staining.(5)Immunomodulatory-erythroid cells exert immunomodulatory functions.CD63+and CD63-erythroid precursors were sorted by FACS and co-cultured with peripheral blood mononuclear cells(PBMCs).Compared with GYPA+CD71+CD63--PBMC culture,the expressions of CCL4,CXCL8,IFITM3 and other immune-related genes were highly expressed in the GYPA+CD71+CD63+-PBMC culture.The levels of IL-8,IL-6 and TNF were also significantly increased in CD63 positive group.In addition,enhanced TNF expression in CD11b+ cells were observed after co-culture with GYPA+CD71+ CD63+cells.(6)Comparative analysis of immunomodulatory-erythroid cells at the distinct development stages and the interaction analysis between immunomodulatoryerythroid cells and immune cells.Correlation analysis was conducted based on expression of the signature genes in corresponding immunomodulatory-erythroid cells.It was found that a greater resemblance of immunomodulatory-erythroid cells among the YS,FL and neonatal UCB stages compared with the features of the corresponding population in adult BM.Enrichment analysis based on the higher expression of stage specific genes revealed that YS stage-specific genes were associated with hypoxia and embryonic organ development,those in FL were associated with active ribosomal and mitochondrial activities,whereas those in UCB displayed enhanced immune properties through enrichment of the leukocyte proliferation and the antigen receptor-mediated signaling pathway,those in adult BM associated with adaptive immunomodulatory functions were evident in the immunomodulatory-erythroid counterparts,such as the regulation of lymphocyte activation and T cell activation.Interaction analysis showed that more significantly enriched pairs and stronger signal intensities were detected among immunomodulatory-erythroid and immune cells than between classical-erythroid and immune cells across all developmental stages,which were also verified by in situ IHC staining,we found the physical juxtaposition of HLA-E+GYPA+CD63+immunomodulatory-erythroid cells and KLRC1+CD56+NK cells,MIF+GYPA+ CD63+immunomodulatory-erythroid cells and CD74+CD20+B cells in FL tissue sections.Conclusions:In this study,integrated,single-cell transcriptomic studies of erythroid precursors from the human YS,FL,preterm UCB,term UCB and adult BM,constructed a cellular and molecular panorama of human erythroid precursors from embryo to adult,this systematic and comprehensive molecular characterization and heterogeneity analyses provides a valuable data resource and important insights into erythropoiesis.In addition,the comparative analysis of in vitro and in vivo erythroid cells also found that insufficient protein synthesis and multiple aberrant metabolic and biological processes were detected in hESC-derived cells during erythroid maturation.These findings could improve the in vitro erythropoietic system and enhance the efficiency of the erythroid cells production from in vitro system.In conclusion,this study not only revealed the unique molecular characteristics and the functional heterogeneity of human erythroid precursors,but also successfully identified and verified an immunomodulatory-erythroid subset throughout human ontogenesis,bring a new field of functional research on nucleated erythroid cells.