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

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

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

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

Indian Hedgehog release from TNF activated renal epithelia drives local and remote organ fibrosis

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell–resolution transcriptomic analysis, we identified an “inflammatory” proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)–induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis

Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Background: Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. Methods: We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively. Results: Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo. Conclusions: Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency

Additional file 3: Table S1. of Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Primer sequences. (DOCX 14 kb