The EHA Research Roadmap: Normal Hematopoiesis.

International audienceIn 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1–2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy

How the avian model has pioneered the field of hematopoietic development

International audienceThe chicken embryo has a long history as a key model in developmental biology. Because of its distinctive developmental characteristics, it has contributed to major breakthroughs in the field of hematopoiesis. Among these, the discovery of B lymphocytes and the three rounds of thymus colonization; the embryonic origin of hematopoietic stem cells and the traffic between different hematopoietic organs; and the existence of two distinct endothelial cell lineages one angioblastic, restricted to endothelial cell production, and another, hemangioblastic, able to produce both endothelial and hematopoietic cells, should be cited. The avian model has also contributed to substantiate the endothelial-to-hematopoietic transition associated with aortic hematopoiesis and the existence of the allantois as a hematopoietic organ. Because the immune system develops relatively late in ayes, the avian embryo is used to probe the tissue-forming potential of mouse tissues through mouse-into-chicken chimeras, providing insights into early mouse development by circumventing the lethality associated with some genetic strains. Finally, the avian embryo can be used to investigate the differentiation potential of human ES cells in the context of a whole organism. The combinations of classic approaches with the development of powerful genetic tools make the avian embryo a great and versatile model

Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence of HSCs has never been formally proven in chicken embryos. Here, we have established a complete cartography and quantification of hematopoietic cells in the aorta during development. We demonstrate the existence of bona fide HSCs, originating from the chicken embryo aorta (and not the yolk sac, allantois or head), through an in vivo transplantation assay. Embryos transplanted in ovo with GFP embryonic tissues on the chorio-allantoic membrane provided multilineage reconstitution in adulthood. Historically, most breakthrough discoveries in the field of developmental hematopoiesis were first made in birds and later extended to mammals. Our study sheds new light on the avian model as a valuable system to study HSC production and regulation in vivo

Etude ontogénique des lignages endothélial, musculaire lisse et musculaire squelettique de souris

Notre étude porte sur la contribution du mésoderme para-axial à la mise en place des cellules endothéliales et musculaires au cours de l embryogenèse murine. La technique de greffe chimère embryonnaire Souris-Poulet constitue un outil de choix pour évaluer le comportement in vivo du mésoderme présomitique. Nous montrons que les premières cellules qui se différencient à partir du mésoderme présomitique sont toujours de type endothélial. Elles participent au remodelage de l aorte dorsale ainsi qu à la musculature lisse des vaisseaux du tronc qui se mettent en place parallèlement à la myogenèse épaxiale. Les greffes hétérospécifiques combinées à la technique de rétro-greffe du bourgeon de membre sur la membrane chorio-allantoïdienne nous ont permis d établir que la migration des progéniteurs endothéliaux en direction du bourgeon de membre commence en même temps que son développement c est-à-dire au stade 15 somites. Ce n est qu à partir du stade 21 somites que les progéniteurs myogéniques colonisent le membre. L ensemble de nos observations associées à l exploitation des mutants murins Pax3-GFP et Flk1-LacZ démontrent que chez l embryon de souris, les lignages, endothélial et musculaire squelettique ou lisse, se mettent en place indépendamment. De façon intéressante, nos résultats suggèrent que si la musculature lisse du tronc dérive du mésoderme présomitique, celle du bourgeon de membre serait issue du mésoderme somatopleural et ne dépendrait pas de l expression de Flk1.The aim of our study was to evaluate the contribution of the para-axial mesoderm to endothelial and muscular cell development during mouse embryogenesis. The Mouse-Chick chimera constitutes an excellent tool to evaluate, in vivo, presomitic mesoderm behavior. We showed that the first cells that differentiate from the presomitic mesoderm are of an endothelial cell type. They participate in dorsal aorta remodeling and the establishment of the vessel wall of the body at the same time as epaxial myogenesis. Heterospecific grafts combined with limb bud retro-grafting on chorio-allantoic membrane allowed us to determine that endothelial progenitors migration toward the limb bud occurred simultaneously to its development (i.e. 15 somite stage). In contrast, myogenic progenitors only start to migrate from 21 somite stage. All our observations, concomitant with the study of Pax3-GFP and Flk1-LacZ mouse mutants demonstrate that, in the mouse embryo, endothelial and skeletal or smooth muscular lineages take place independently. Interestingly, our results suggest that if the smooth musculature of the trunk originates from the presomitic mesoderm, that of the limb bud is probably derived from the somatopleural mesoderm and would not depend on Flk1 expression.NANTES-BU Sciences (441092104) / SudocSudocFranceF

An in vitro model of hemogenic endothelium commitment and hematopoietic production

Adult-type hematopoietic stem and progenitor cells are formed during ontogeny from a specialized subset of endothelium, termed the hemogenic endothelium, via an endothelial-to-hematopoietic transition (EHT) that occurs in the embryonic aorta and the associated arteries. Despite efforts to generate models, little is known about the mechanisms that drive endothelial cells to the hemogenic fate and about the subsequent molecular control of the EHT. Here, we have designed a stromal line-free controlled culture system utilizing the embryonic pre-somitic mesoderm to obtain large numbers of endothelial cells that subsequently commit into hemogenic endothelium before undergoing EHT. Monitoring the culture for up to 12 days using key molecular markers reveals stepwise commitment into the blood-forming system that is reminiscent of the cellular and molecular changes occurring during hematopoietic development at the level of the aorta. Long-term single-cell imaging allows tracking of the EHT of newly formed blood cells from the layer of hemogenic endothelial cells. By modifying the culture conditions, it is also possible to modulate the endothelial cell commitment or the EHT or to produce smooth muscle cells at the expense of endothelial cells, demonstrating the versatility of the cell culture system. This method will improve our understanding of the precise cellular changes associated with hemogenic endothelium commitment and EHT and, by unfolding these earliest steps of the hematopoietic program, will pave the way for future ex vivo production of blood cells

CLASP2 safeguards hematopoietic stem cell properties during mouse and fish development

Hematopoietic stem cells (HSCs) express a large variety of cell surface receptors that are associated with acquisition of self-renewal and multipotent properties. Correct expression of these receptors depends on a delicate balance between cell surface trafficking, recycling, and degradation and is controlled by the microtubule network and Golgi apparatus, whose roles have hardly been explored during embryonic/fetal hematopoiesis. Here we show that, in the absence of CLASP2, a microtubule-associated protein, the overall production of HSCs is reduced, and the produced HSCs fail to self-renew and maintain their stemness throughout mouse and zebrafish development. This phenotype can be attributed to decreased cell surface expression of the hematopoietic receptor c-Kit, which originates from increased lysosomal degradation in combination with a reduction in trafficking to the plasma membrane. A dysfunctional Golgi apparatus in CLASP2-deficient HSCs seems to be the underlying cause of the c-Kit expression and signaling imbalance

Endoglin expression level discriminates long-term hematopoietic from short-term clonogenic progenitor cells in the aorta

International audienceCD105 is an auxiliary receptor for the transforming growth factor beta superfamily, highly expressed on proliferating endothelial cells and adult hematopoietic stem cells. Because CD105 mRNA expression was reported in the developing aortic region, we further characterized its expression profile in the aorta and examined the hematopoietic potential of CD105(+) cells. Aortic endothelial cells, intra-aortic hematopoietic cell clusters and the purified cell fraction enriched in progenitor/hematopoietic stem cell activity expressed CD105. Aortic hematopoietic short-term clonogenic progenitors were highly enriched in the CD105(intermediate) population whereas more immature long-term progenitors/hematopoietic stem cells are contained within the CD105(high) population. This places CD105 on the short list of molecules discriminating short-term versus long-term progenitors in the aorta. Furthermore, decreasing transforming growth factor beta signaling increases the number of clonogenic progenitors. This suggests that CD105 expression level defines a hierarchy among aortic hematopoietic cells allowing purification of clonogenic versus more immature hematopoietic progenitors, and that the transforming growth factor beta pathway plays a critical role in this process

Single-cell transcriptomics reveal the dynamic of haematopoietic stem cell production in the aorta

Haematopoietic stem cells (HSCs) are generated from haemogenic endothelial (HE) cells via the formation of intra-aortic haematopoietic clusters (IAHCs) in vertebrate embryos. The molecular events controlling endothelial specification, endothelial-to-haematopoietic transition (EHT) and IAHC formation, as it occurs in vivo inside the aorta, are still poorly understood. To gain insight in these processes, we performed single-cell RNA-sequencing of non-HE cells, HE cells, cells undergoing EHT, IAHC cells, and whole IAHCs isolated from mouse embryo aortas. Our analysis identified the genes and transcription factor networks activated during the endothelial-to-haematopoietic switch and IAHC cell maturation toward an HSC fate. Our study provides an unprecedented complete resource to study in depth HSC generation in vivo. It will pave the way for improving HSC production in vitro to address the growing need for tailor-made HSCs to treat patients with blood-related disorders

Single-cell transcriptomics reveal the dynamic of haematopoietic stem cell production in the aorta

Haematopoietic stem cells (HSCs) are generated from haemogenic endothelial (HE) cells via the formation of intra-aortic haematopoietic clusters (IAHCs) in vertebrate embryos. The molecular events controlling endothelial specification, endothelial-to-haematopoietic transition (EHT) and IAHC formation, as it occurs in vivo inside the aorta, are still poorly understood. To gain insight in these processes, we performed single-cell RNA-sequencing of non-HE cells, HE cells, cells undergoing EHT, IAHC cells, and whole IAHCs isolated from mouse embryo aortas. Our analysis identified the genes and transcription factor networks activated during the endothelial-to-haematopoietic switch and IAHC cell maturation toward an HSC fate. Our study provides an unprecedented complete resource to study in depth HSC generation in vivo. It will pave the way for improving HSC production in vitro to address the growing need for tailor-made HSCs to treat patients with blood-related disorders