The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta–gonad–mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta–gonad–mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.
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Sequencing data supporting the findings of this study have been deposited at the Gene Expression Omnibus (GEO) under accession code GSE162950. Data from published references are available at the GEO under accession code GSE135202. An interface for data browsing and links to data are also available online (http://singlecell.mcdb.ucla.edu/Human-HSC-Ontogeny). There is no restriction in data availability.
Custom code, R objects and metadata of these R objects are available at GitHub (https://github.com/mikkolalab/Human-HSC-Ontogeny).
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We thank the staff at the BSCRC FACS and sequencing cores, and TPCL, TCGB and CFAR cores (NIH AI028697-21) at UCLA; A. Pyle and H. Xi for assistance with tissue procurement and staging; F. Lay for the introduction to single-cell analysis and S. Morrison for reading the manuscript; J. Rodgers and M. Giannoni for assistance with the website generation. This work was supported by Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA Interim Research Award and Innovation awards and a Jonsson Cancer Center Foundation and UCLA David Geffen School of Medicine Regenerative Medicine Theme Award (to H.K.A.M.); NIH 1RO1DK125097-01, 1R01DK121557-01 and 5R01DK100959-07 (to H.K.A.M.), NIH R01HL148714 (to R.A.) and NIH R35HL140014 (to M.L.I.A); a Swedish Research Council International Postdoc grant IPD2 2018-06635 (to S.C.-G.); the Swiss National Science Foundation (P2ZHP3_178113 and EMBO ALTF 433-2019, to J.A.-G.); BSCRC post-doctoral fellowships (to J.A.-G. and I.F.), and T32 HL-086345-13 Developmental Hematology fellowship (to I.F. and B.N.), BSCRC Rose Hills Foundation Graduate Training Program and Ruth L. Kirschstein National Research Service Award T32HL069766 (to A.V.) and the Deutsche Forschungsgemeinschaft Cluster of Excellence iFIT (EXC 2180-390900677) (to K.S.-L.). A.G.E., E.G.S. and E.S.N. were supported by NHMRC (Australia) fellowships (GNT1117596, to A.G.E.; GNT1079004 to E.G.S.) and project grants (GNT1068866 and GNT1129861 to A.G.E. and E.G.S.; GNT1164577 to E.S.N.), by the ARC (Stem Cells Australia), and by the Stafford Fox Medical Research Foundation. Infrastructure funding was provided by NHMRC and Victorian government Infrastructure Support Programs.
The authors declare no competing interests.
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Extended data figures and tables
(a) Single-cell RNA-seq analysis of CD34+ and/or CD31+ enriched cells from CS14-15 AGM (n = 3 biologically independent samples) (Fig. 1a). tSNE plots show 20 clusters. Total haematopoietic cells (RUNX1+/CD45+, Clusters 2, 9, 12) and HSC (cluster 12) are circled in purple and black, respectively. (b) Contribution of each AGM sample to the clusters in (a). (c) Feature plots of cell type-specific genes documenting cell identities. (d) Feature plots showing co-expression of HSC surface markers in HSC cluster. (e) Dot plot with cell type-specific genes confirms cell identities in each cluster. (f) UMAP analysis showing reclustering of haematopoietic cells. (g) Contribution of each AGM to the clusters in (f). (h) Dot plot of lineage-specific genes showing the identity of haematopoietic cell types. (i) UMAP plot of haematopoietic cell types (HSC, Monocyte/macrophages-Mo/Mφ, Granulocytes-Gr, and Lymphoid cells-Ly. (j) Feature plots documenting the expression of HSC regulatory genes (left) and lack of lineage markers (right) in HSC cluster. (k) Gene ontology analysis of genes significantly enriched in HSC cluster vs other haematopoietic cells (Fisher’s exact test).
Extended Data Fig. 2 Identification of haematopoietic cells in CS14 embryo and extraembryonic tissues.
(a) Single-cell RNA-seq analysis of different tissues from CS14 (4.5 weeks) conceptus. tSNE clustering indicating the main cell types, and feature plots documenting the expression of selected HSC molecular signature genes RUNX1, HLF and SPINK2+ are shown. Total haematopoietic cells (RUNX1+/CD45+) and HSCs are circled in purple and black, respectively. (b) Presence of HLF+ HSPC in HSC-containing clusters in each CS14 tissue. (c) Nascent HSC scorecard genes evaluated in HLF+ HSPC in each tissue. (d) UMAP analysis of haematopoietic cells from the merge of all indicated tissues from the 4.5 weeks/CS14 embryo, using MAGIC for imputation of gene expression. (e) Feature plots showing MAGIC-imputed expression of the six HSC signature genes RUNX1, HLF, HOXA9, MLLT3, MECOM, HLF and SPINK2. (f) Visualization of HSCs defined by the “HSC signature” module score on MAGIC-imputed expression. (g) Nascent HSC scorecard on HSC signature module -defined HSCs identified in CS14 tissues. (h) Nascent HSC scorecard on all SPINK2+ cells from haematopoietic clusters from CS14 tissues. (i) GO categories and example genes enriched SPINK2+ AGM HSC (top) or in SPINK2+ Liver haematopoietic progenitors (bottom) (Fisher’s exact test). (j) Dot plot of genes enriched in SPINK2+ Liver haematopoietic progenitors in SPINK2+ cells from CS14 tissues. (k) UMAP analysis of CS14 liver haematopoietic clusters, showing 10 clusters and the main cell types. (l) Feature plots of HSPC genes in haematopoietic cells in CS14 liver. SPINK2+ progenitor cells are circled. (m) Feature plots of lineage-specific genes in CS14 liver.
(a) Single-cell RNA-seq analysis of individual embryonic and fetal tissues: AGM at 5 weeks (CS15a), AGM and liver at 5 weeks (CS15b), AGM and liver at 6 weeks (CS17) embryo, livers at 8, 11 and 15 weeks. For each tissue, tSNE clustering indicating the main cell types and feature plots showing the expression of selected HSC molecular signature genes RUNX1, HLF and SPINK2 are shown. Total haematopoietic cells (RUNX1+/PTPRC+) and HSCs are circled in purple and black, respectively (n = 8 biologically independent samples). (b) Expression of nascent HSC scorecard genes in HLF+ HSCs from HSC-containing clusters in the different tissues. (c) HLF+ HSCs from tissues containing > 10 HSCs, and cord blood were selected, and analysed in Monocle. (d) GO categories and example genes up- or downregulated during HSC maturation in pseudotime analysis are shown. (Parametric Correlation test) (e) Dot plots of HOXA and HOXB cluster genes during HSC maturation. (f) UMAP analysis of haematopoietic cells from the merge of all indicated tissues in (a) and CS14 AGM and liver, using MAGIC imputed gene expression. (g) Feature plots showing MAGIC-imputed expression of the six HSC signature genes. (h) Visualization of HSCs defined based on the “HSC signature” module score on MAGIC-imputed expression. (i) Quantification of HSC module-defined HSCs from each CS14 to 15wk tissue analysed. (j) Module-defined HSCs at different ages shown in UMAP analysis. (k) Feature plots visualizing immaturity and maturity module scores defined by the indicated genes, which were calculated on MAGIC-imputed expression. (l) Quantification of immaturity and maturity modules in the MAGIC-imputed, module selected HSCs in the indicated tissues. (m) Representative flow cytometry plots of the expression of HSC maturation markers HLA-DR and CD133(PROM1) in fetal liver HSPC (CD43+CD45midCD34+CD38low/-CD90+GPI-80+) are shown. (n) Schematic depicting molecular programs and HSC surface markers that change during human HSC developmental maturation.
(a) UMAP plot showing contribution of each AGM sample (CS14-15, 4.5-5 weeks) (n = 3 biologically independent samples) to haemato-vascular clusters. (b) Dot plot showing HOXA expression in AGM haematovascular cells. (c) Pseudotime analysis of 833 cells from clusters 0–5 (endothelium and HSC). (d,e) Pseudotime trajectory plots showing the progression of pseudotime variable (d) and contribution of each AGM sample to trajectory (e). (f) Feature plots displaying the expression of markers of the different stages of EHT in pseudotime trajectory plot. (g) Heat map displaying unsupervised clustering of 11514 genes whose expression significantly changes over pseudotime, divided into 10 gene groups. (h) Summary table of enriched GO categories from each gene group (Fisher’s exact test). (i-k) Dot plots showing endothelial and haematopoietic lineage genes (i), cell cycle and metabolism-related genes (j) and signalling pathway associated genes (k) identified from pseudotime analysis, in clusters 0–8 and HE. HE is highlighted in blue.
(a) CS15d/5 weeks human embryo processed for Visium Spatial transcriptomics and H&E stainings of seven transverse sections that were sequenced are shown, with key anatomical landmarks highlighted (top). Seurat cluster analysis is shown on the embryo sections (middle) and as UMAP plots (bottom). Bars = 1mm. (b) tSNE plots of scRNA-seq data from the AGM region (CS14-15) documenting the main cell types and the expression of cell type-specific genes. (c) Spatial expression of landmark genes for neural tube (NEUROD1), myotome (MYOD), and haematopoietic cell types (GYPA for erythroid cells, RUNX1 for HE, HSPC and other haematopoietic cells, and HLF for HSC). Note HLF expression also in liver epithelium in ED Fig. 2. The default colour scale from Loupe browser was applied, which represents the log2 expression from 0 to the maximum value in the spots. Each dot is 55 μm and shows combined expression of 1–10 cells.
(a) tSNE plot documenting the main cell types in CS14-15 (4.5-5 weeks) AGM tissues (top, n = 3 biologically independent samples). Feature plots displaying the expression of arterial (GJA5), pre-HE (IL33, ALDH1A1), HE (ALDH1A1, KCNK17), HSC (KCNK17 and SPINK2) and liver SPINK2 progenitor (SPINK2, IL7R) markers in CS14-15 AGM samples (bottom). (b) First row, H&E staining of seven transverse sections, featuring dorsal aorta. Red arrows indicate intra-aortic haematopoietic cluster (IAHC) and green arrows red blood cells. Spatial sequencing plots showing the expression of arterial (GJA5), pre-HE (IL33, ALDH1A1), HE (ALDH1A1, KCNK17), HSC (KCNK17 and SPINK2) and liver SPINK2 progenitor (SPINK2, IL7R) markers. The default colour scale from Loupe browser was applied, which represents the log2 expression from 0 to the maximum value in the spots. Each dot is 55 μm and shows combined expression of 1–10 cells. White bars = 250μm, black bars = 1mm. (c) Immunofluorescence staining of CS15c (5 weeks) aorta for IL33, ALDH1A1, CD31/PECAM and DAPI (Section #251), CXCR4, KCNK17, CD31/PECAM and DAPI (section #254) and SPINK2, PTPRC/CD45, CD31/PECAM and DAPI (Section) #239. White bars = 200μm, black bar = 20μm. Individual antibody staining was performed minimum three times in independent embryos with comparable staining pattern.
(a) UMAP showing the contribution of each embryo/AGM (CS10-CS17) or YS (CS11) to CDH5+/RUNX1+ haemato-vascular cells (n = 8 biologically independent samples). (b) Feature plots displaying EC, pre-HE, HE and HSC landmark genes. (c) UMAP plots highlighting HSPC (HLF+SPINK2+), HE (CDH5+RUNX1+and/orKCNK17+PTPRC-SPN-SPINK2-), pre-HE (CDH5+RUNX1-PTPRC-SPN-SPINK2-IL33+and/orALDH1A1+) and EC (remaining cells in HE-containing clusters) from early (CS10-11, blue) and HSC-forming (CS13–17, red) waves. (d) “Nascent HSC scorecard” genes in CS10 embryo HPC, CS11 YS HPC and CS13–17 AGM HSCs. (e) “HSPC waves scorecard” dot plot showing genes co-regulated in EC, pre-HE, HE and HSPC from distinct waves. Genes shown are identified through differential expression analysis and GO term enrichment between early HPC (CS10 embryo and CS11YS) vs HSCs. (f) “Endo waves scorecard” dot plot showing selected genes co-regulated in EC, pre-HE, HE and HSPC populations from the early and HSC-forming waves. Genes shown are identified through differential expression analysis and GO term enrichment between early HE (embryo CS10 and CS11, YS CS11) vs. HSC-forming HE (CS13–15). (g) UMAP feature plots displaying the expression of stage-specific markers.
(a) UMAP analysis of haematopoietic and erythroid cells in embryonic and extraembryonic tissues from CS10 to 15 weeks concepti (CS14/15 AGM, CS14 placenta, yolk sac, vitelline vessels, umbilical cord, head, heart, CS14 to 15 weeks livers) (n = 19 biologically independent samples), using MAGIC imputed gene expression. (b) Feature plots displaying MAGIC imputed expression of HSC signature genes. (c) Visualization of HSCs defined based on the “HSC signature” module score on MAGIC imputed expression. (d) Quantification of module-selected HSCs in CS10 to 15 weeks concepti. (e) Mature HSC and Immature HSC module scores in module-defined HSCs. (f) UMAP analysis showing relative similarity of HLF+ HSPCs from each tissue and stage (CS14/15 AGM, CS14 placenta, yolk sac, vitelline vessels and umbilical cord, CS17 to 15 weeks livers, 40 weeks cord blood) (n = 12 biologically independent samples). (g) “HSPC waves scorecard” documenting the expression of wave-specific genes in HLF+ HSPCs. (h) “HSC maturation scorecard” documenting the maturation stage. (i) Feature plots show the expression of HSC signature genes, nascent HSC genes and maturation genes in HLF+ HSPC in the indicated tissues over time. (j) UMAP analysis including haematopoietic (RUNX1+PTPRC+) and erythroid (RUNX1-GYPA+) clusters from the indicated tissues and stages (CS14–17 AGM, CS14 other haematopoietic tissues, CS17 to 15 weeks livers, n = 19 biologically independent samples. Colours show the contribution of each tissue to haematopoietic cell types (top). Selected tissues highlighted in black on the UMAP plot display HSC and progenitor populations and their differentiation trajectories in each tissue (bottom). (k) Feature plots show the expression of HSPC signature genes and lineage markers at different ages. HLF+ HSC are circled in black.
(a) Schematic depicting protocols A or B for hPS cell differentiation using swirler EB method and different cytokine combinations that generate adherent and suspension fractions at day 14. Created with BioRender.com. (b) UMAP plot showing strongest ACTINN matches for PS-cell-derived CDH5+ and/or RUNX1+ cells. (c) Feature plots depicting ACTINN probability scores for different cell identities in PS-cell-derived CDH5+/RUNX1+ cells. (d) Dot plot showing “Nascent HSC scorecard” genes in PS-cell-derived SPINK2+HLF+ HSPC. (e) Upper left, UMAP plot showing the adherent and suspension fractions of PS-cell-derived CDH5+/RUNX1+ haematovascular cells. Feature plots displaying the expression of HSC genes (upper) and endothelial genes (lower) in PS-cell-derived CDH5+/RUNX1+ cells. Lower right, UMAP highlighting the selection of HE (CDH5+RUNX1+ and/or KCNK17+PTPRC-SPN-SPINK2-) in PS-cell-derived CDH5+/RUNX1+ cells. (f,g) Dot plots showing “EHT scorecard” genes (f) and “Endo waves scorecard” genes (g) in PS-cell-derived HE and HSPC from differentiation protocols A and B, compared to their in vivo counterparts.
(a) Summary table displaying patterns of gene expression for selected markers that distinguish different cell types and stages during human HSPC ontogeny. Colour gradient from gray to dark red represents increase in gene expression levels and/or the frequency of positive cells within a population. (b) A schematic displaying the key cell types, stages and markers involved in human HSPC specification, emergence, and maturation. Scorecards used to evaluate key stages of HSC development are shown below. Created with BioRender.com.
The legends for Supplementary Tables 1–8.
Inventory of tissues.
Genes enriched in nascent HSC in human AGM.
Differential gene expression between SPINK2 cells from human CS14 AGM and liver.
Gene expression changes during functional maturation of human HSCs.
Differential gene expression analysis between cells at distinct stages of EHT.
Gene expression programs that evolve during EHT.
Cluster analysis of spatial transcriptomics data.
Differential gene expression analysis between early and HSC-forming haemogenic waves.
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Calvanese, V., Capellera-Garcia, S., Ma, F. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534–540 (2022). https://doi.org/10.1038/s41586-022-04571-x
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