Definitive hematopoiesis is autonomously initiated by the AGM region

AbstractThe adult hematopoietic system of mammals is a dynamic hierarchy of cells with the hematopoietic stem cell at its foundation. During embryonic development, the source and expansion potential of this cell remain unclear. Two sites of hematopoietic activity, the yolk sac and aorta–gonad–mesonephros (AGM) region, function in mouse ontogeny at the pre-liver stage of hematopoiesis. However, cellular interchange between these tissues obscures the embryonic site of hematopoietic stem cell generation. Here we present the results of a novel in vitro organ culture system demonstrating that, at day 10 in gestation, hematopoietic stem cells initiate autonomously and exclusively within the AGM region. Furthermore, we provide evidence for the in vitro expansion of hematopoietic stem cells within the AGM region. These results strongly suggest that the AGM region is the source of the definitive adult hematopoietic system, which subsequently colonizes the liver

The murine placenta contains hematopoietic stem cells within the vascular labyrinth region

SummaryIn the midgestation murine embryo, several major vascular tissues contain hematopoietic stem cell (HSC) activity. These include the aorta-gonad-mesonephros region (AGM), yolk sac, and fetal liver. Recently, the placenta was demonstrated to harbor hematopoietic progenitors, but it was not examined for HSC activity. We demonstrate here that the placenta also harbors adult-repopulating HSCs. Placental HSCs begin to be detected at embryonic day (E) 11, and HSC numbers increase dramatically between E11 and E12, exceeding the numbers in the circulating embryonic blood. Furthermore, all placental HSC activity is restricted to the GFP+ fraction of cells in Ly-6A (Sca-1) GFP transgenic embryos. Cells coexpressing GFP and endothelial markers CD34 and CD31 are found in the embryonic vasculature of the placental labyrinth. Moreover, placental cell expression of other HSC markers and transcription factors suggests that HSC emergence may occur in the placenta, as has been proposed for other embryonic hematopoietic sites

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

Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region

Human HSCs appear first in the embryonic dorsal aorta, and only later in the yolk sac, liver, and placenta; a single human AGM region HSC can generate at least 300 daughter HSCs that are retransplantable into secondary recipient mice

Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.Singapore-MIT Alliance for Research and Technology ( Infectious Diseases research grant

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

Intra-Aortic Clusters Undergo Endothelial to Hematopoietic Phenotypic Transition during Early Embryogenesis

Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc