Haematopoietic differentiation of murine embryonic stem cells

Haematopoietic stem cells (HSCs) are routinely used to treat haematological disorders, as they can engraft into the bone marrow of immuno-compromised recipients where they undergo self-renewal and multilineage differentiation to provide long-term reconstitution of the blood system. Identification of novel factors able to regulate or expand HSCs would have a significant impact in a clinical setting. Mouse embryonic stem (ES) cells can be used as a model system to investigate haematopoietic regulation, since these pluripotent cells are amenable to large-scale culture and have the capacity to differentiate into a variety of cell types in vitro, including cells of haematopoietic lineages. Mature blood cells can be generated relatively easily from ES cells; however, HSCs are generated at relatively low frequencies and there has been only limited success in the contribution of these cells to the adult haematopoietic system in vivo. Previous work demonstrated that the frequency of haematopoietic progenitors was significantly increased when ES cells were co-cultured with primary El0.5 aorta-gonad-mesonephros (AGM) tissue explants, a region which is able to give rise to HSCs in vivo. Therefore, the AGM region is a potent source of haematopoietic inductive signals both in vivo and for ES cells in vitro.This project aimed to determine which subregion(s) of the AGM were responsible for the haematopoietic enhancing effects that primary AGM explants had on differentiating ES cells. To this end, a novel co-culture system has been established to test the enhancing effects of a panel of clonal stromal cell lines derived from different subregions of the midgestational AGM. It was found that three clonal stromal cell lines derived from the dorsal aorta and surrounding mesenchyme (AM) subregion of the AGM were able to significantly enhance the frequency of ES cell derived multipotent haematopoietic progenitors, as measured by in vitro colony assays and flow cytometry. By contrast, two stromal cell lines derived from the urogenital ridges (UG) of the AGM did not enhance haematopoietic differentiation of ES cells. Interestingly, the enhancing capacity of the AM-derived stroma was comparable with that of the bone marrow derived OP9 stromal cell line, which has been widely used in the literature to promote haematopoietic differentiation of ES cells. Further investigation revealed that the enhancing capacity is not retained by extracellular matrices isolated from the AM stromal cell layers and the effects were dependent on direct ES cell-stromal cell contact. Co-culture of an ES cell line carrying a mesoderm specific Brachyury-eGFP reporter gene demonstrated that the stromal lines mediated their effects post- Brachyury (mesoderm) induction in the ES cells. In addition, co-culture of sorted ES cell populations confirmed that Brachyury*, but not Brachyury', cells gave rise to haematopoietic progenitors in AM stromal co-culture, supporting the notion that ES cell differentiation recapitulated the in vivo pattern of lineage specification. Transplantation of co-cultured ES cells into irradiated adult NOD/SCID mouse recipients led to low levels of donor cell engraftment in the spleen and bone marrow, which expanded upon serial transplantation; but full repopulation of the recipient haematopoietic system was not confirmed. Adult bone marrow cells were found to achieve repopulation more readily in the NOD/SCID animal model when transplanted intra-splenically, as compared to intra-venous injection. This suggests that transplantation of ES-derived haematopoietic cells directly into the haematopoietic niche, by intra-splenic or intrafemoral injection, could facilitate repopulation

Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs

During mouse development, definitive hematopoietic stem cells (dHSCs) emerge by late E10.5 to E11 in several hematopoietic sites. Of them, the aorta-gonad-mesonephros (AGM) region drew particular attention owing to its capacity to autonomously initiate and expand dHSCs in culture, indicating its key role in HSC development. The dorsal aorta contains characteristic hematopoietic clusters and is the initial site of dHSC emergence, where they mature through vascular endothelial (VE)-cadherin(+)CD45(–)CD41(low) (type 1 pre-HSCs) and VE-cadherin(+)CD45(+) (type 2 pre-HSCs) intermediates. Although dHSCs were also found in other embryonic niches (placenta, yolk sac, and extraembryonic vessels), attempts to detect their HSC initiating potential have been unsuccessful to date. Extraembryonic arterial vessels contain hematopoietic clusters, suggesting that they develop HSCs, but functional evidence for this has been lacking. Here we show that umbilical cord and vitelline arteries (VAs), but not veins, contain pre-HSCs capable of maturing into dHSCs in the presence of exogenous interleukin 3, although in fewer numbers than the AGM region, and that pre-HSC activity in VAs increases with proximity to the embryo proper. Our functional data strongly suggest that extraembryonic arteries can actively contribute to adult hematopoiesis

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

Analysis of <i>Runx1</i> Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an <i>In Vivo</i> Approach

Summary: Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis. : The authors found that Cre-mediated Runx1 ablation induced in vivo at pre-liver stages resulted in appearance of Runx1-null HSCs in the fetal liver. By contrast, deletion of Runx1 in cultured AGM region fully blocked HSC development. Appearance of Runx1-null HSCs in the liver is explained by presence of uncontrolled long-lasting (at least 3 days) Cre-inducing tamoxifen activity in vivo. Keywords: Runx1, hematopoietic stem cells, AGM, development, hematopoiesis, conditional knockout, tamoxife

Endothelio-hematopoietic relationship: getting closer to the beginnings

The close association between hematopoietic and endothelial cells during embryonic development led to the proposal that they may originate from a common ancestor - the hemangioblast. Due to a lack of unique specific markers for in vivo cell fate tracking studies, evidence supporting this theory derives mainly from in vitro differentiation studies. Teixeira and colleagues describe a novel enhancer that drives specific eGFP expression in blood islands of the electroporated chick embryo, thereby presenting a tool potentially suitable for analysis of hemangioblast differentiation and development of blood islands

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

Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling

Tissue progenitor cells are an attractive target for regenerative therapy. In various organs, bone marrow cell (BMC) therapy has shown promising preliminary results, but to date no definite mechanism has been demonstrated to account for the observed benefit in organ regeneration. Tissue injury and regeneration is invariably accompanied by macrophage infiltration, but their influence upon the progenitor cells is incompletely understood, and direct signaling pathways may be obscured by the multiple roles of macrophages during organ injury. We therefore examined a model without injury; a single i.v. injection of unfractionated BMCs in healthy mice. This induced ductular reactions (DRs) in healthy mice. We demonstrate that macrophages within the unfractionated BMCs are responsible for the production of DRs, engrafting in the recipient liver and localizing to the DRs. Engrafted macrophages produce the cytokine TWEAK (TNF-like weak inducer of apoptosis) in situ. We go on to show that recombinant TWEAK activates DRs and that BMC mediated DRs are TWEAK dependent. DRs are accompanied by liver growth, occur in the absence of liver tissue injury and hepatic progenitor cells can be isolated from the livers of mice with DRs. Overall these results reveal a hitherto undescribed mechanism linking macrophage infiltration to DRs in the liver and highlight a rationale for macrophage derived cell therapy in regenerative medicine

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and upregulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesi

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