The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. During embryonic development, HSCs derive from haemogenic endothelium (HE) in a NOTCH- and retinoic acid (RA)-dependent manner. Although a WNT-dependent (WNTd) patterning of nascent hPSC mesoderm specifies clonally multipotent intra-embryonic-like HOXA+ definitive HE, this HE is functionally unresponsive to RA. Here we show that WNTd mesoderm, before HE specification, is actually composed of two distinct KDR+ CD34neg populations. CXCR4negCYP26A1+ mesoderm gives rise to HOXA+ multilineage definitive HE in an RA-independent manner, whereas CXCR4+ ALDH1A2+ mesoderm gives rise to HOXA+ multilineage definitive HE in a stage-specific, RA-dependent manner. Furthermore, both RA-independent (RAi) and RA-dependent (RAd) HE harbour transcriptional similarity to distinct populations found in the early human embryo, including HSC-competent HE. This revised model of human haematopoietic development provides essential resolution to the regulation and origins of the multiple waves of haematopoiesis. These insights provide the basis for the generation of specific haematopoietic populations, including the de novo specification of HSCs.
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All gene expression analysis datasets are available in the Gene Expression Omnibus (GEO) under accession no. GSE139853 (scRNA-seq in Figs. 1 and 3 and Extended Data Fig. 2; CXCR4+/−mesoderm RNA-seq in Fig. 1 and Extended Data Fig. 3; RAi/RAd HE RNA-seq in Fig. 4 and Extended Data Figs. 1, 3 and 7; RAi/RAd HPC RNA-seq in Fig. 4 and Extended Data Fig. 7) or BioProjects under PRJNA352442 (WNTi mesoderm RNA-seq in Fig. 1, Extended Data Figs. 1 and 3; WNTd mesoderm RNA-seq in Extended Data Fig. 1) and PRJNA525404 (WNTi HE RNA-seq in Fig. 4 and Extended Data Figs. 1, 3 and 8). The publicly available datasets used include GEO SuperSeries GSE81102, ‘AGM CD34+’ (GSM2142333), ‘AGM PR’ (GSM2142334), ‘AGM HSPC’ (GSM2142332) for human 5 week AGM (Fig. 4 and Extended Data Fig. 7); GEO GSE135202 for human CS10-CS15 embryo (Fig. 4 and Extended Data Fig. 8); ArrayExpress E-MTAB-9388 for human CS7 embryo (Fig. 3). Genome alignments were performed with GENCODE GRCh38.p3 version 23. All source data have been deposited within the San Raffaele Open Research Data Repository and are available at https://doi.org/10.17632/pbbn55mhy9.1. Source data are provided with this paper.
All code used to analyse and visualize datasets has been shared on a repository and can be found at https://github.com/sturgeonlab/Luff-etal-2022.
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S.A.L., C.D. and J.P.C. received support from an NHLBI T32 Training Grant (HL007088-41). S.M. is supported by a Vallee Scholar Award and an Allen Distinguished Investigator Award. A.D. is supported by the Telethon Foundation (TIGET grants nos. C4 and G3b) and San Raffaele Hospital (Seed Grant). C.M.S. is supported by an American Society of Hematology Scholar Award, an American Society of Hematology Bridge Grant, a Washington University Center of Regenerative Medicine Pilot Grant, the Bill & Melinda Gates Foundation INV-002414, and NIH R01HL145290 and R01HL151777. This publication was made possible, in part, by grant no. UL1 RR024992 from the NIH National Center for Research Resources (NCRR). R.S. conducted this study as partial fulfilment of an international PhD in Molecular Medicine, Vita-Salute San Raffaele University.
The methodology described in this publication is subject to patent no. PCT/US2020/014626 (inventors: A. Ditadi and C.M.S.). The remaining authors declare no competing interests.
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Extended Data Fig. 1 Specification of HOXA+ HE from hPSCs in a WNT-dependent manner.
A, Schematic of hPSC directed differentiation towards haemogenic endothelium, as described in Sturgeon et. al2. Definitive intra-embryonic-like hematopoietic potential is specified in a WNT-dependent (‘WNTd’) manner, while extra-embryonic-like hematopoietic potential is WNT-independent (‘WNTi’). B, Representative flow cytometric analyses of the T-lymphoid potential of WNTd CD34+ cells and WNTi CD34+CD43neg and CD43+ populations. T cell potential is positively identified by the presence of a CD4+CD8+ population following 21+ days of OP9-DL4 coculture, while an absence of potential is identified by an absence of CD45+ lymphocytes3,30. C, Heatmaps visualizing the mean expression of HOXA genes across all biological replicates in mesoderm and haemogenic endothelium populations, as in (A). Grey indicates undetected gene. Scale bar: log10 FPKM. Biological replicates: WNTi/WNTd KDR+ (n=4); WNTi/WNTd HE (n=3). The expression of HOXA genes within WNTd-derived populations is suggestive of an intra-embryonic-like population6, while a lack of HOXA expression in WNTi-derived populations is suggestive of an extra-embryonic-like population7.
Extended Data Fig. 2 scRNA-seq analyses of day 3 WNTi and WNTd differentiation cultures.
A, Violin plots visualizing the number of genes per cell (left), the number of unique molecular identifiers (‘UMIs’, middle), and the percent of expressed genes that are mitochondrial (right) following filtering of low quality cells from both WNTi and WNTd datasets combined (1 biological replicate each). B, UMAP visualizing before and after integration of WNTi (red) and WNTd (blue) datasets to account for batch effects between sequencing runs. C, UMAP visualizing quality control metrics, as in A, for the dataset following integration. Scale bar: values range as indicated in (A). D, Violin plots for CYP26A1 and ALDH1A2 expression within WNTi KDR+GYPA+ and WNTd KDR+GYPAneg cells, as indicated. E,F, Day 3 WNTd cultures are comprised of all germ layers. E, UMAP visualizing (i) clustering and (ii) KDR expression within WNTd differentiation cultures. Scale bar: gene expression scaled to WNTd subset. F, (i) UMAP plot with the projection of predicted germ layer type, where each label includes cells expressing the following genes: Pluripotent (SOX2, NANOS3, DND1, POU5F1, or TBXT), Ectoderm (TFAP2A, DLX5, or GATA3), Endoderm (FOXA2, APOA1, or APOA2), and Mesoderm (KDR, MEST, MESP1, TEK, or FLT1). 44 (0.64%) remaining cells were labeled based on clustering. (ii) Dot plot visualizing expression of germ layer-specific genes within each identified cell type, as in (i). Scale bar: scaled expression (iii) Violin plot visualizing the expression of KDR within all labeled populations, as in (i).
Extended Data Fig. 3 Day 3 of differentiation WNTd KDR+CXCR4neg and KDR+CXCR4+ cells are transcriptionally distinct mesodermal subsets.
A, Heatmaps visualizing the mean expression across all biological replicates (log10 FPKM) of mesodermal, endothelial, hematopoietic, and RA-related genes within hPSC-derived day 3 WNTi KDR+CD235a+ cells and WNTd KDR+CXCR4+/neg cells, day 6 WNTi HE, and day 8 WNTd HE. Grey indicates undetected gene. Hierarchical clustering based on the expression of genes shown. Scale bar: log10 FPKM. Biological replicates: WNTi KDR+ (n=4); CXCR4+/neg KDR+, WNTi HE, WNTd HE (n=3). B, Representative flow cytometric analysis for endothelial markers CD34, CD144 (VE-Cadherin/CDH5), and TIE2 (TEK) within WNTd KDR+ cells. C, Heatmaps visualizing the mean expression of HOXA genes across all biological replicates in day 3 WNTi KDR+CD235a+ cells, or WNTd KDR+CXCR4+ or KDR+CXCR4neg cells, as in A. Scale bar: log10 FPKM. Biological replicates: WNTi KDR+ (n=4); CXCR4+/neg KDR+ (n=3). D, PCA plot for batch-corrected KDR+CXCR4+ and KDR+CXCR4neg replicates, as in A. E, Expression of CDX genes within WNTi and WNTd mesodermal populations, as in A, Two-way ANOVA with Tukey’s multiple comparison test comparing all biological replicates: WNTd CXCR4+/neg (n=3), WNTi CD235a+ (n=4). SEM, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.
Extended Data Fig. 4 CXCR4neg mesoderm gives rise to HE in an RA-independent manner, while specification of HE from CXCR4+ mesoderm is RA-dependent.
A, Representative phase contrast microscopy of CD34+ cells from DMSO-treated CXCR4+/neg mesoderm (i) and retinol-treated CXCR4+ mesoderm (ii) following 1, 3, and 5 days after FACS isolation. 100X magnification, scale bar: 50um. B, Erythro-myeloid colony forming potential of HE specified from CXCR4neg mesoderm treated with DMSO or DEAB from days 3-8 (HE specification window). Two-way ANOVA with Bonferroni’s multiple comparison test comparing all biological replicates, n=13, SEM, ns=not significant, p>0.9999 for all colony types. C, Representative flow cytometric analyses of T-lymphoid RA-dependent definitive hematopoietic potential of CXCR4+ mesoderm in H1 hESC, H9 hESC, and iPSC-1 hPSC lines. CXCR4+ mesoderm was isolated and cultured as in Fig. 2a. 1 nM ATRA was applied to CXCR4+ cells immediately after FACS isolation. n≥2.
Extended Data Fig. 5 RA-dependent HE, similar to RAi HE, is a CD34+CD43negCD73negCXCR4neg population and undergoes the endothelial-to-hematopoietic transition (EHT) in a NOTCH-dependent manner.
A, Schematic for the differentiation of WNTd cultures towards either RAi and RAd CD34+ cells and their respective hematopoietic progenitor cells (HPCs) in the presence or absence of NOTCH inhibitor L-685458. B, Representative flow cytometric analyses and FACS isolation strategies within DEAB-treated (RAi) or retinol (‘ROH’)-treated (RAd) cultures. Isolated populations were then assessed for hematopoietic potential. C(i), Representative flow cytometric analyses of T-lymphoid potential of populations, as in (B). (ii) Quantification of the erythro-myeloid CFC potential from the populations in (B), averaged across all biological replicates. Two-way ANOVA with Tukey’s multiple comparison test comparing all biological replicates: RAd HE plus NOTCH inhibitor (n=3), remaining samples (n=4), SEM, statistics shown for BFU-E (RAi HE vs. RAi HE+gSI (p=0.0059), RAi HE vs. RAi CXCR4+ (p=0.008), RAi HE vs. RAi CD73+ (p=0.0044), RAi HE vs. RAd CXCR4+ (p=0.0128), RAi HE vs. RAd CD73+ (p=0.0033), p<0.0001 for RAi HE vs. RAd HE, RAi HE+gSI vs. RAd HE, RAi CXCR4+ vs. RAd HE, RAi CD73+ vs. RAd HE, RAd HE vs. RAd HE+gSI, RAd HE vs. RAd CXCR4+, RAd HE vs. RAd CD73+), all comparisons not shown are not significant. All colony counts and statistical analyses are included in Source Extended Data Fig. 5. (iii) Representative flow cytometric analysis of CD34 and CD45 at 9 days after HE FACS isolation and percentage of CD34+CD45+ cells from each culture, averaged across all biological replicates (n=4). Two-tailed paired t-test, p=0.0458, SEM. D, Average expression across all biological replicates of embryonic (HBE), fetal (HBG), and adult (HBB) globin genes and BCL11A in BFU-E derived from WNTi CD34+ cells (purple), WNTd RAi HE (green), and RAd HE (blue). Ordinary one-way ANOVA comparing all biological replicates: HBE (n=3; WNTi vs. RAi, p=0.4636, WNTi vs. RAd, p=0.8811; RAi vs. RAd, p=0.3841), HBG (n=3; WNTi vs. RAi, p=0.234; WNTi vs. RAd, p=0.0018; RAi vs. RAd, p=0.007), HBB (n=3, WNTi vs. RAi, p=0.3253; WNTi vs. RAd, p=0.0662; RAi vs. RAd, p=0.286), BCL11A (n=6, p=0.002552), SEM, ns=not significant.
Extended Data Fig. 6 Xenograft analyses of hPSC-derived HE populations.
A, Transient xenograft persistence of RAd hematopoietic progenitors following injection in neonatal mice. Percent chimerism observed of hCD45 cells present in either the peripheral blood and bone marrow following injection with hPSC-derived CD34+ cells. B, Lineage distribution of hCD45+ cells present in the peripheral blood (PB) 8 weeks post-transplant. C-E, Detailed analysis of human engraftment in the bone marrow or peripheral blood of mice transplanted with RAd HE cells. C, Two independent CD45 antibodies (‘CD45-1’ and ‘CD45-2’) were used to label human cells. Representative flow cytometric 4-week analysis of the bone marrow of a non-injected control mouse (top), a recipient of 105 CD34+ cord blood cells (middle) and a recipient of 5x104 RAd CD34+ cells. D-E, Alternative strategy for human chimerism analysis based on mCD45 and hCD45 exclusive staining. D, Representative flow cytometric analysis for single stains of both mCD45 and hCD45, from the bone marrow of an non-injected control mouse or a recipient of 2x105 RAd CD34+ cells. E, Representative flow cytometric strategy for the detection of human chimerism in the peripheral blood, from a recipient of 105 RAd CD34+ cells.
Extended Data Fig. 7 Whole-transcriptome analysis of hPSC and human embryonic CD34+ populations.
A, Comparison of whole transcriptomes of hPSC-derived HE and HPCs (3 biological replicates each). (i) PCA plot demonstrating the similarity between batch-corrected biological replicates of WNTi, RAi, and RAd HE, along with CD34+CD45+ HPCs derived from RAi or RAd HE. (ii) Heatmap and hierarchical clustering showing the Euclidean distance between all biological replicates of WNTd RAi and RAd HE, as in (i). (iii) Selection of significant normalized enrichment scores (NES) from pre-ranked GSEA between RAi HE (green) and RAd HE (blue), as in Supplementary Table 5,c. RAi was enriched in embryonic development (NES=−1.80, FDR=0.23), endothelium development (NES=−1.85, FDR=0.23), cellular adhesion (NES=−1.78, FDR=0.24), the epithelial-to-mesenchymal transition (NES=−1.95, FDR 0.14), and response to mechanical stimuli (NES=−1.71, FDR=0.23). In contrast, RAd HE was enriched for RA signaling (NES=1.99, FDR=0.22) and several histone modification pathways (NES=2.12, FDR=0.14). B, (i) Mean expression (log10 FPKM) of hemato-endothelial genes for all biological replicates within hPSC-derived WNTi HE, RAi HE, RAd HE, RAi HPC, and RAd HPC, as in (A), with CD34+CD90+CD43neg cells (‘AGM CD34+’), CD34+CD90+CD43+ hematopoietic stem/progenitors (‘AGM HSPC’), and CD34+CD90negCD43+ hematopoietic progenitors (‘AGM PR’) isolated from the aorta-gonad mesonephros (AGM) region of 5th-week of gestation human embryos6. Grey indicates undetected gene. Scale bar: log10 FPKM. Biological replicates: WNTi HE, RAi HE, RAd HE, RAi HPC, RAd HPC (n=4); AGM CD34+, AGM HSPC, AGM PR (n=1, GEO SuperSeries GSE81102). (ii) Selection of significant normalized enrichment scores (NES) from preranked GSEA comparing RAi HE to AGM CD34+ cells (green) and RAd HE to AGM CD34+ cells (blue), as in Supplementary Table 5,e,f. RAi and RAd HE was enriched for hematopoietic stem and progenitor differentiation (NES≥1.63, FDR≤0.015), while AGM CD34+ cells were enriched for aorta and vascular development (NES≥1.82, FDR≤0.01), and BMP and VEGF signaling pathways (NES≥1.89, FDR≤0.004).
Extended Data Fig. 8 Establishment of human embryonic dataset for comparative analysis with hPSC-derived haemogenic and arterial endothelial populations.
A, UMAPs visualizing (i) Carnegie Stages and (ii) cell type labels from the complete dataset of Zeng, et al.19. Biological replicates: CS10, CS11, CS12, CS14, CS15 (n=1); CS13 (n=2, 1 each for CS13X (10X genomics) and CS13D (Modified STRT-Seq). B, UMAP visualizing the cells categorized as arterial endothelial cells (‘AEC’, defined as CDH5+CXCR4+GJA5+DLL4+HEY2+SPNnegPTPRCneg) and haemogenic endothelial cells (‘HEC’, defined as CDH5+RUNX1+HOXA+ITGA2BnegSPNnegPTPRCneg), as in Fig. 3b. C, UMAP visualizing the numbered groups for embryonic cells, as in Fig. 3b, within the categorized AEC and HEC. Di, Heatmap visualizing the expression of select broadly and arterial endothelial genes and RUNX1 in human CS10-14 AEC and HEC. Clusters of AEC and HEC are segregated by their relative similarity to hPSC-derived RAi or RAd HE, as in Fig. 3b. Scale bar: gene expression scaled to subset. (ii) Violin plot for scaled expression of select arterial endothelial genes across 6 AEC/HEC clusters, as in (i).
Extended Data Fig. 9 Gating strategy and controls for flow cytometric analyses.
A, Universal gating strategy for all hPSC-derived flow cytometric analyses. B, Single stain controls for markers assessed at the mesodermal stage (day 3 of differentiation). C, Single stain controls for markers assessed at the HE stage (day 8 of differentiation). D, Gating strategy and single stain controls for T cell assay (day 21 of OP9-DL4 co-culture). E, Gating strategy for assessment of xenograft persistence established using human cord blood CD34+ cells.
Source Data Fig. 1
Source data for Fig. 1e(ii),f,g(ii).
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Source data for Figs. 2b,c,d(iii).
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Source data for Extended Data Fig. 3.
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Source data for Extended Data Fig. 5c(ii),(iii),d.
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Source data for Extended Data Fig. 6a,b.
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Luff, S.A., Creamer, J.P., Valsoni, S. et al. Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells. Nat Cell Biol 24, 616–624 (2022). https://doi.org/10.1038/s41556-022-00898-9