An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

MyD88-dependent TLR signaling oppositely regulates hematopoietic progenitor and stem cell formation in the embryo.

Hemogenic endothelial (HE) cells in the dorsal aorta undergo an endothelial-to-hematopoietic transition (EHT) to form multipotent progenitors, lympho-myeloid biased progenitors (LMPs), pre-hematopoietic stem cells (pre-HSCs) and adult-repopulating HSCs. These briefly accumulate in intra-arterial hematopoietic clusters (IAHCs) before being released into the circulation. It is generally assumed that the number of IAHC cells correlates with the number of HSCs. Here, we show that changes in the number of IAHC cells, LMPs and HSCs can be uncoupled. Mutations impairing MyD88-dependent toll-like receptor (TLR) signaling decreased the number of IAHC cells and LMPs, but increased the number of HSCs in the aorta-gonad-mesonephros region of mouse embryos. TLR4-deficient embryos generated normal numbers of HE cells, but IAHC cell proliferation decreased. Loss of MyD88-dependent TLR signaling in innate immune myeloid cells had no effect on IAHC cell numbers. Instead, TLR4 deletion in endothelial cells (ECs) recapitulated the phenotype observed with germline deletion, demonstrating that MyD88-dependent TLR signaling in ECs and/or in IAHCs regulates the numbers of LMPs and HSCs.

Concealed expansion of immature precursors underpins acute burst of adult HSC activity in foetal liver.

One day prior to mass emergence of haematopoietic stem cells (HSCs) in the foetal liver at E12.5, the embryo contains only a few definitive HSCs. It is thought that the burst of HSC activity in the foetal liver is underpinned by rapid maturation of immature embryonic precursors of definitive HSCs, termed pre-HSCs. However, because pre-HSCs are not detectable by direct transplantations into adult irradiated recipients, the size and growth of this population, which represents the embryonic rudiment of the adult haematopoietic system, remains uncertain. Using a novel quantitative assay, we demonstrate that from E9.5 the pre-HSC pool undergoes dramatic growth in the aorta-gonad-mesonephros region and by E11.5 reaches the size that matches the number of definitive HSCs in the E12.5 foetal liver. Thus, this study provides for the first time a quantitative basis for our understanding of how the large population of definitive HSCs emerges in the foetal liver.

Runx1 is required for progression of CD41+ embryonic precursors into HSCs but not prior to this.

Haematopoiesis in adult animals is maintained by haematopoietic stem cells (HSCs), which self-renew and can give rise to all blood cell lineages. The AGM region is an important intra-embryonic site of HSC development and a wealth of evidence indicates that HSCs emerge from the endothelium of the embryonic dorsal aorta and extra-embryonic large arteries. This, however, is a stepwise process that occurs through sequential upregulation of CD41 and CD45 followed by emergence of fully functional definitive HSCs. Although largely dispensable at later stages, the Runx1 transcription factor is crucially important during developmental maturation of HSCs; however, exact points of crucial involvement of Runx1 in this multi-step developmental maturation process remain unclear. Here, we have investigated requirements for Runx1 using a conditional reversible knockout strategy. We report that Runx1 deficiency does not preclude formation of VE-cad+CD45-CD41+ cells, which are phenotypically equivalent to precursors of definitive HSCs (pre-HSC Type I) but blocks transition to the subsequent CD45+ stage (pre-HSC Type II). These data emphasise that developmental progression of HSCs during a very short period of time is regulated by precise stage-specific molecular mechanisms.

HSC-independent definitive hematopoiesis persists into adult life.

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Vast Self-Renewal Potential of Human AGM Region HSCs Dramatically Declines in the Umbilical Cord Blood.

Human hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region during Carnegie stages (CS) 14-17. Although we previously reported that these HSCs can generate no less than 300 daughter HSCs, their actual number has never been established. Here, we show that a single human AGM region HSC can generate 600-1,600 functional daughter HSCs. The presence of HSCs in the CS 17 liver in one case gave us a unique opportunity to describe a reduction of HSC self-renewal potential after liver colonization. From a clinical perspective, the efficacy of long-term hematopoietic regeneration depends on HSC self-renewal capacity. We quantitatively show that this capacity dramatically declines in the umbilical cord blood compared with HSCs in the AGM region. A full appreciation of the vast regenerative potential of the first human embryo-derived HSCs sets a new bar for generation of clinically useful HSCs from pluripotent stem cells.

Multi-layered Spatial Transcriptomics Identify Secretory Factors Promoting Human Hematopoietic Stem Cell Development.

Hematopoietic stem cells (HSCs) first emerge in the embryonic aorta-gonad-mesonephros (AGM) region. Studies of model organisms defined intersecting signaling pathways that converge to promote HSC emergence predominantly in the ventral domain of the dorsal aorta. Much less is known about mechanisms driving HSC development in humans. Here, to identify secreted signals underlying human HSC development, we combined spatial transcriptomics analysis of dorsoventral polarized signaling in the aorta with gene expression profiling of sorted cell populations and single cells. Our analysis revealed a subset of aortic endothelial cells with a downregulated arterial signature and a predicted lineage relationship with the emerging HSC/progenitor population. Analysis of the ventrally polarized molecular landscape identified endothelin 1 as an important secreted regulator of human HSC development. The obtained gene expression datasets will inform future studies on mechanisms of HSC development in vivo and on generation of clinically relevant HSCs in vitro.

A Modified Semisolid Clonal Culture for Identification of B-1 and B-2 Progenitor Colony Forming Ability of Mouse Embryonic Hemogenic Endothelial Cells.

The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported to be biased to innate-like B-1 cell lineage that can develop from hemogenic endothelial cells (HECs) independently of HSCs. On the other hand, conventional adaptive immune B cells (B-2) cells are considered to be exclusively derived from HSCs. Therefore, segregating B-1 and B-2 progenitor potential is important to understand the developmental process of HSCs that are also produced from HECs through intermediate precursors referred to as pre-HSCs. Both HECs and pre-HSCs show endothelial surface phenotype and require stromal support to detect their hematopoietic activity. The method utilizing stromal cell culture followed by modified semisolid clonal culture enables us to detect the number of colony forming units for B-1/B-2 progenitors originally derived from HECs/pre-HSCs, which will reflect the potential of B-1 biased or multi-lineage repopulating HSCs.

Embryonic Intra-Aortic Clusters Undergo Myeloid Differentiation Mediated by Mesonephros-Derived CSF1 in Mouse.

The aorta-gonad-mesonephros (AGM) region contains intra-aortic clusters (IACs) thought to have acquired hematopoietic stem cell (HSC) potential in vertebrate embryos. To assess extrinsic regulation of IACs in the AGM region, we employed mouse embryos harboring a Sall1-GFP reporter gene, which allows identification of mesonephros cells based on GFP expression. Analysis of AGM region tissue sections confirmed mesonephros GFP expression. Mesonephric cells sorted at E10.5 expressed mRNA encoding Csf1, a hematopoietic cytokine, and corresponding protein, based on real-time PCR and immunocytochemistry, respectively. Further analysis indicated that some IACs express the CSF1 receptor, CSF1R. Expression of Cebpa and Irf8 mRNAs was higher in CSF1R-positive IACs, whereas that of Cebpε and Gfi1 mRNAs was lower relative to CSF1R-negative IACs, suggesting that CSF1/CSF1R signaling functions in IAC myeloid differentiation by modulating expression of these transcription factors. Colony formation assays using CSF1R-positive IACs revealed increased numbers of myeloid colonies in the presence of CSF1. Analysis using an intra-cellular signaling array indicated the greatest fold increase of Cleaved Caspase-3 in AGM cells in the presence of CSF1. Immunohistochemistry revealed that Cleaved Caspase-3 is primarily expressed in IACs in the AGM region, and incubation of IACs with CSF1 up-regulated Cleaved Caspase-3. Overall, our findings suggest that CSF1 secreted from mesonephros accelerates IAC myeloid differentiation in the AGM region, possibly via Caspase-3 cleavage.