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Mapping human haematopoietic stem cells from haemogenic endothelium to birth

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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.
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.
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Evaluation of HSC in the AGM and liver during first and second trimester (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.
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534 | Nature | Vol 604 | 21 April 2022
Article
Mapping human haematopoietic stem cells
from haemogenic endothelium to birth
Vincenzo Calvanese1,2,3,16  ✉, Sandra Capellera-Garcia1,2,16 , Feiyang Ma1,2,4,16 , Iman Fares1,2,
Simone Liebscher5, Elizabeth S. Ng6, Sophia Ekstrand1,2, Júlia Aguadé-Gorgorió1,2,
Anastasia Vavilina1,2, Diane Lefaudeux7,8, Brian Nadel1, Jacky Y. Li6, Yanling Wang1,
Lydia K. Lee9, Reza Ardehali2,1 0, M. Luisa Iruela-Arispe11, Matteo Pellegrini1,2, Ed G. Stanley6,12,13 ,
Andrew G. Elefanty6,12,13, Katja Schenke-Layland5,10,14,15 & Hanna K. A. Mikkola1,2 ✉
The ontogeny of human haematopoietic stem cells (HSCs) is poorly dened owing to
the inability to identify HSCs as they emerge and mature at dierent haematopoietic
sites1. Here we created a single-cell transcriptome map of human haematopoietic
tissues from the rst 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 dierent 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 reecting 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 immunouorescence, 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
denitive haematopoietic stem and progenitor cells from human pluripotent stem
cells, and serves as a guide to improve their maturation to functional HSCs.
Developmental haematopoiesis consists of multiple waves of blood
cell production that culminate in the generation of self-renewing HSCs.
The steps to generate human HSCs rather than HSC-independent pro-
genitors are poorly defined, compromising the efforts to differentiate
HSCs from human pluripotent stem (PS) cells for transplantation and
disease modelling. Human HSCs emerge in the aorta–gonad–meso-
nephros (AGM) region between Carnegie stages 13and17 (CS13–17;
4–6 weeks) through intra-aortic haematopoietic clusters (IAHCs)
2
.
Although IAHCs contain numerous haemogenic cells, transplantable
HSCs are rare (one per AGM)
3
, suggesting functional immaturity
4
. Nas-
cent HSCs colonize the liver, where they mature and acquire robust
bone marrow engraftment ability
4
. In contrast to mouse HSCs, there
are no methods to recapitulate human HSC maturation in culture or
pinpoint their maturation stage5,6.
HSC emergence is preceded by several HSC-independent pro-
genitor waves
7,8
. The yolk sac first generates primitive erythrocytes
and macrophages, followed by erythro-myeloid progenitors (EMPs)
that initiate fetal liver haematopoiesis, at least in mice
8,9
. In humans,
yolk-sac-derived myeloid progenitors seed the liver by CS12 (ref.
10
).
Recent lineage-tracing studies have challenged the dogmas by show-
ing that progenitors generate long-lived progeny, including microglia
and tissue-resident macrophages
11,12
. Moreover, lymphoid potential,
which was previously considered to be an exclusive trait of HSCs, has
been reported in HSC-independent progenitors
7,10,13,14
. Innate-like B1 B
cells were linked to developmentally restricted haematopoietic stem
and progenitor cells (HSPCs) that can acquire self-renewal ability after
transplantation15. Single-cell technologies uncovered an early (CS10–11)
HSC-independent intraembryonic human haematopoietic wave
16
, and
https://doi.org/10.1038/s41586-022-04571-x
Received: 31 December 2020
Accepted: 22 February 2022
Published online: 13 April 2022
Check for updates
1Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA. 2Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell
Research, University of California Los Angeles, Los Angeles, CA, USA. 3Laboratory for Molecular Cell Biology, University College London, London, UK. 4Chongqing International Institute for
Immunology, Chongqing, China. 5Institute of Biomedical Engineering, Department for Medical Technologies and Regenerative Medicine, Eberhard Karls University, Tübingen, Germany.
6Murdoch Children’s Research Institute, The Royal Children’s Hospit al, Parkville, Victoria, Australia. 7Signaling Systems Laboratory, Department of Microbiology Immunology and Molecular
Genetics (MIMG), University of California Los Angeles, Los Angeles, CA, USA. 8Institute for Quantitative and Computational Biosciences (QCB), University of California Los Angeles, Los
Angeles, CA, USA. 9Department of Obstetrics and Gynecology, University of California Los Angeles, Los Angeles, CA, USA. 10Department of Medicine/Cardiology, CVRL, University of California
Los Angeles, Los Angeles, CA, USA. 11Cell and Developmental Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA. 12Department of Paediatrics, Faculty of
Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria, Australia. 13Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria,
Australia. 14Cluster of Excellence iFIT (EXC 2180) ‘Image-Guided and Functionally Instructed Tumor Therapies’, Eberhard Karls University Tübingen, Tübingen, Germany. 15NMI Natural and
Medical Sciences Institute, University Tübingen, Reutlingen, Germany. 16These authors contributed equally: Vincenzo Calvanese, Sandra Capellera-Garcia, Feiyang Ma.
e-mail: v.calvanese@ucl.ac.uk; hmikkola@mcdb.ucla.edu
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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