Catherine Robin

In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium

Haematopoietic stem cells (HSCs), responsible for blood production in the adult mouse, are first detected in the dorsal aorta starting at embryonic day 10.5 (E10.5). Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize. The origin of HSCs has long been controversial and several candidates of the direct HSC precursors have been proposed (for review see ref. 7), including a specialized endothelial cell population with a haemogenic potential. Such cells have been described both in vitro in the embryonic stem cell (ESC) culture system and retrospectively in vivo by endothelial lineage tracing and conditional deletion experiments. Whether the transition from haemogenic endothelium to HSC actually occurs in the mouse embryonic aorta is still unclear and requires direct and real-time in vivo observation. To address this issue we used time-lapse confocal imaging and a new dissection procedure to visualize the deeply located aorta. Here we show the dynamic de novo emergence of phenotypically defined HSCs (Sca1+, c-kit+, CD41+) directly from ventral aortic haemogenic endothelial cells.

Runx1 Expression Marks Long-Term Repopulating Hematopoietic Stem Cells in the Midgestation Mouse Embryo

Hematopoietic stem cells (HSCs) are first found in the aorta-gonad-mesonephros region and vitelline and umbilical arteries of the midgestation mouse embryo. Runx1 (AML1), the DNA binding subunit of a core binding factor, is required for the emergence and/or subsequent function of HSCs. We show that all HSCs in the embryo express Runx1. Furthermore, HSCs in Runx1(+/-) embryos are heterogeneous and include CD45(+) cells, endothelial cells, and mesenchymal cells. Comparison with wild-type embryos showed that the distribution of HSCs among these various cell populations is sensitive to Runx1 dosage. These data provide the first morphological description of embryonic HSCs and contribute new insight into their cellular origin.

Hematopoietic Stem Cells Localize to the Endothelial Cell Layer in the Midgestation Mouse Aorta

The emergence of the first adult hematopoietic stem cells (HSCs) during mammalian ontogeny has been under intense investigation. It is as yet unresolved whether these first HSCs are derived from intraembryonic hemangioblasts, hemogenic endothelial cells, or other progenitors. Thus, to examine the spatial generation of functional HSCs within the mouse embryo, we used the well-known HSC marker, Sca-1, and a transgenic approach with an Ly-6A (Sca-1) GFP marker gene. Our results show that this transgene marker is expressed in all functional HSCs in the midgestation aorta. Immunohistology of aorta-gonads-mesonephros (AGM) regions show that GFP(+) cells are specifically localized to the endothelial layer lining the wall of the dorsal aorta but not to the mesenchyme, strongly suggesting that HSC activity arises within a few cells within the endothelium of the major vasculature.

Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta–gonad–mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, β-NGF (a neurotrophic factor), MIP-1γ (a C–C chemokine family member) and Bmp4 (a TGF-β family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

The Ly‐6A (Sca‐1) GFP Transgene is Expressed in all Adult Mouse Hematopoietic Stem Cells

The Sca‐1 cell surface glycoprotein is used routinely as a marker of adult hematopoietic stem cells (HSCs), allowing a >100‐fold enrichment of these rare cells from the bone marrow of the adult mouse. The Sca‐1 protein is encoded by the Ly‐6A/E gene, a small 4‐exon gene that is tightly controlled in its expression in HSCs and several hematopoietic cell types. For the ability to sort and localize HSCs directly from the mouse, we initiated a transgenic approach in which we created Ly‐6A (Sca‐1) green fluorescent protein (GFP) transgenic mice. We show here that a 14‐kb Ly‐6A expression cassette directs the transcription of the GFP marker gene in all functional repopulating HSCs in the adult bone marrow. A >100‐fold enrichment of HSCs occurred by sorting for the GFP‐expressing cells. Furthermore, as shown by fluorescence‐activated cell sorting and histologic analysis of several hematopoietic tissues, the GFP transgene expression pattern generally corresponded to that of Sca‐1. Thus, the Ly‐6A GFP transgene facilitates the enrichment of HSCs and presents the likelihood of identifying HSCs in situ.

Long-Term Maintenance of Hematopoietic Stem Cells Does Not Require Contact with Embryo-Derived Stromal Cells in Cocultures

We recently established that two midgestation‐derived stromal clones—UG26‐1B6, urogenital ridge–derived, and EL08‐1D2, embryonic liver–derived—support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC‐stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly‐6C− side population (K6‐SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08‐1D2 and UG26‐1B6 with four HSC‐nonsupportive clones. We found that EL08‐1D2 and UG26‐1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor‐7, insulin‐like growth factor‐binding proteins 3 and 4, pleiotrophin, pentaxin‐related, and thrombospondin 2, whereas 11 genes, including GPX‐3 and HSP27, were expressed at a lower level. In summary, we show for the first time long‐term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.

GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta–gonad–mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.

CD41 is developmentally regulated and differentially expressed on mouse hematopoietic stem cells

CD41 expression is associated with the earliest stages of mouse hematopoiesis. It is notably expressed on some cells of the intra-aortic hematopoietic clusters, an area where the first adult-repopulating hematopoietic stem cells (HSCs) are generated. Although it is generally accepted that CD41 expression marks the onset of primitive/definitive hematopoiesis, there are few published data concerning its expression on HSCs. It is as yet uncertain whether HSCs express CD41 throughout development, and if so, to what level. We performed a complete in vivo transplantation analysis with yolk sac, aorta, placenta, and fetal liver cells, sorted based on CD41 expression level. Our data show that the earliest emerging HSCs in the aorta express CD41 in a time-dependent manner. In contrast, placenta and liver HSCs are CD41⁻. Thus, differential and temporal expression of CD41 by HSCs in the distinct hematopoietic territories suggests a developmental/dynamic regulation of this marker throughout development.

Progressive maturation toward hematopoietic stem cells in the mouse embryo aorta

Clusters of cells attached to the endothelium of the main embryonic arteries were first observed a century ago. Present in most vertebrate species, such clusters, or intraaortic hematopoietic clusters (IAHCs), derive from specialized hemogenic endothelial cells and contain the first few hematopoietic stem cells (HSCs) generated during embryonic development. However, some discrepancies remained concerning the spatio-temporal appearance and the numbers of IAHCs and HSCs. Therefore, the exact cell composition and function of IAHCs remain unclear to date. We show here that IAHCs contain pre-HSCs (or HSC precursors) that can mature into HSCs in vivo (as shown by the successful long-term multilineage reconstitution of primary neonates and secondary adult recipients). Such IAHC pre-HSCs could contribute to the HSC pool increase observed at midgestation. The novel insights in pre-HSC to HSC transition represent an important step toward generating transplantable HSCs in vitro that are needed for autologous HSC transplantation therapies.