Powered by EHA Perspective OPEN ACCESS The EHA Research Roadmap: Normal Hematopoiesis Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy Thierry Jaffredo1, Alessandra Balduini2, Anna Bigas3,4,5, Rosa Bernardi6, Dominique Bonnet7, Bruno Canque8, Pierre Charbord1, Anna Cumano9, Ruud Delwel10, Charles Durand1, Willem Fibbe11, Lesley Forrester12, Lucia de Franceschi13, Cedric Ghevaert14, Bjørn Gjertsen15,16, Berthold Gottgens17, Thomas Graf18, Olaf Heidenreich19, wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 Olivier Hermine20, Douglas Higgs21, Marina Kleanthous22, Hannes Klump23, Valerie Kouskoff24, Daniela Krause25, George Lacaud26, Cristina Lo Celso27, Joost H.A. Martens28, Simón Méndez-Ferrer29, Pablo Menendez5,30,31,32,33, Robert Oostendorp34, Sjaak Philipsen35, Bo Porse36,37,38, Marc Raaijmakers10, Catherine Robin39,40, Henk Stunnenberg19, Kim Theilgaard-Mönch36,37,38,41, Ivo Touw10, William Vainchenker42 Joan-Lluis Vives Corrons43, Laurent Yvernogeau1, Jan Jacob Schuringa44 Correspondence: Thierry Jaffredo (thierry.jaffredo@upmc.fr) or Jan Jacob Shringa (j.j.schuringa@umcg.nl). In 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 clini- cal 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. 1 Sorbonne Université, Institut de Biologie Paris Seine, Laboratoire de Biologie du 26 Cancer Research UK Manchester Institute, The University of Manchester, Développement/UMR7622, Paris, France United Kingdom 2 Department of Molecular Medicine, University of Pavia, Italy 27 Imperial College London, United Kingdom 3 Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain 28 Department of Molecular Biology, RIMLS, Radboud University, Nijmegen, The 4 Josep Carreras Leukemia Research Institute (IJC), Barcelona, Spain Netherlands 5 Centro de Investigación Biomedica en Red-Oncología (CIBERONC), Instituto de 29 University of Cambridge and NHSBT, Cambridge, United Kingdom Salud Carlos III, Madrid, Spain 30 Josep Carreras Leukemia Research Institute, Barcelona, Spain 6 IRCCS San Raffaele Scientific Institute, Milan, Italy 31 RICORS-RETAV, Instituto de Salud Carlos III, Madrid, Spain 7 The Francis Crick Institute, London, United Kingdom 32 Department of Biomedicine, School of Medicine, Universitat de Barcelona, 8 INSERM U976, Universite de Paris, Ecole Pratique des Hautes Etudes/PSL Barcelona, Spain Research University, Institut de Recherche Saint Louis, France 33 Institució Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Spain 34 Department of Internal Medicine III, Technical University of Munich, School of 9 Unité Lymphopoïèse, Département d’Immunologie, INSERM U1223, Institut Medicine, Germany Pasteur, Cellule Pasteur, Université de Paris, France 35 Department of Cell Biology, Erasmus University Medical Center Rotterdam, The 10 Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands Netherlands 36 The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of 11 Leiden University Medical Center, The Netherlands Copenhagen, Denmark 12 Centre for Regenerative Medicine, Institute for Regeneration and Repair, 37 Biotech Research and Innovation Center (BRIC), University of Copenhagen, Denmark University of Edinburgh, Scotland 38 Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of 13 Università degli Studi di Verona, Italy Health Sciences, University of Copenhagen, Denmark 14 University of Cambridge, United Kingdom 39 Hubrecht Institute-KNAW and University Medical Center Utrecht, The Netherlands 15 Department of Medicine, Hematology Section, Haukeland University Hospital, 40 Regenerative medicine center, University Medical Center Utrecht, The Netherlands Bergen, Norway 41 Department of Hematology, Rigshospitalet/National University Hospital, 16 Department of Clinical Science, Centre for Cancer Biomarkers CCBIO, University of Copenhagen, Denmark University of Bergen, Norway 42 Gustave Roussy, Villejuif, France 17 Wellcome - MRC Cambridge Stem Cell Institute and Department of 43 Red Blood Cell and Hematopoietic Disorders Research Unit, Institute for Haematology, University of Cambridge, United Kingdom Leukaemia Research Josep Carreras, Badalona, Barcelona 18 Center for Genomic Regulation, Barcelona Institute for Science and Technology 44 Department of Experimental Hematology, University Medical Center Groningen, and Universitat Pompeu Fabra, Barcelona, Spain The Netherlands 19 Prinses Máxima Centrum voor kinderoncologie, Utecht, The Netherlands Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. 20 Department of Hematology and Laboratory of Physiopathology and Treatment on behalf of the European Hematology Association. This is an open-access of Blood Disorders, Hôpital Necker, Imagine institute, University of Paris, France article distributed under the terms of the Creative Commons Attribution-Non 21 MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible United Kingdom to download and share the work provided it is properly cited. The work cannot be 22 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus changed in any way or used commercially without permission from the journal. 23 Institute for Transfusion Medicine, University Hospital Essen, Germany HemaSphere (2021) 5:12(e669). 24 University of Manchester, United Kingdom http://dx.doi.org/10.1097/HS9.0000000000000669. 25 Goethe University Frankfurt and Georg-Speyer-Haus, Frankfurt am Main, Germany Received: 30 August 2021 / Accepted: 2 November 2021 1 Jaffredo et al Research Roadmap: Normal Hematopoiesis F undamental as well as translational research has been crit- Erythropoiesis ically important to get us where we are today in the field of hematopoiesis studies. Still, many questions remain. The major cell type in blood is the red blood cell (RBC). RBCs Among many others, these include: How do (epi)genetic transport oxygen from the lungs to other parts of the body. aberrations cause hematologic malignancies, and how can we use RBCs are formed during a process called erythropoiesis. This these insights to develop better therapeutic strategies? It is now includes the initial specification of HSCs during embryogenesis being realized that clonal heterogeneity exists in many hemato- and the decision of HSCs to contribute to the erythroid lineage.2 logic cancers and possibly even within the normal hematopoietic About a third of the world’s population has some form of Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy stem cell (HSC) compartment, but how does this affect disease anemia. Acquired anemias are often related to iron deficiency development and current treatment options remains largely or to systemic disorders such as chronic inflammation. Inherited unknown. In contrast to adult life, HSCs are rapidly expanding anemias affect erythropoiesis by diverse mechanisms, such as wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 during embryogenesis, so can we unravel those mechanisms and insufficient hemoglobin (Hb) production (thalassemias), patho- apply them to in vitro HSC expansion protocols for clinical use? logical Hb variants (sickle cell disease), defects in membrane A thorough understanding of embryonic versus adult hemato- proteins or metabolic enzymes (hemolytic anemias), impaired poiesis might also help to better understand differences between ribosome biogenesis (Diamond Blackfan anemia), and DNA childhood and adult hematologic malignancies. Reprogramming repair defects (Fanconi anemia). Activating JAK2 kinase muta- now allows the generation of patient-specific iPSCs, but the gen- tions cause excess RBC production (polycytemia vera). The eration of fully functional HSCs from these cells is still rather physiological and molecular mechanisms underlying RBC dis- challenging, can we improve this? We live in a continuously orders are still incompletely understood, hampering the devel- aging society, but how does HSC aging actually affect health and opment of new treatments. disease? Within this section, we provide an overview of the cur- Historically, research of hematopoiesis has driven novel bio- rent status of the field and an outlook on where future research logical concepts, owing to the accessibility of hematopoietic should be headed (Fig. 1). We firmly believe that combining cells for molecular and functional analyses. Early European fundamental and translational research will result in not only contributions include the Nobel Prize-winning discovery of a better understanding of the hematopoietic system but also the the structure of Hb3 and understanding the epidemiology of development of better therapeutic approaches for hematologic inherited anemias, leading to implementation of prenatal diag- malignancies, many of which are still difficult to treat. nostic programs.4 Additional European contributions include Figure 1. Overview of the current field and an outlook on where future research should be headed. 2 (2021) 5:12 www.hemaspherejournal.com determining the origin of HSCs, the transcriptional circuitry of several myeloid diseases, leading to the identification of underlying erythropoiesis, the role of iron metabolism, and novel myeloid regulators, and the development of knowledge DNA sequences driving Hb expression which are now applied bank tools allowing for prognostication and guidance of in gene therapy. A database dedicated to erythroid disorders has therapeutic stratification of patients with myeloid diseases.8,9 been established.5 Translational research has resulted in opti- (3) Transcriptomics studies comparing identical normal and mized stem cell transplantation protocols, magnetic resonance malignant myeloid progenitors have mapped gene expression imaging for monitoring of iron overload, improved iron che- signatures of disease states in patients, which reflect aberrant lation therapies, and targeted inhibition of signaling pathways signaling pathway activities that contribute to dissect disease Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy under (pre)leukemic conditions. biology, and are both prognostic relevant and druggable by Previous research has laid the foundation from which under- novel therapeutic modalities.10–12 standing erythropoiesis will be extended. For instance, single The advent of novel comprehensive omics technologies such wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 cell analysis allows hierarchical relationships between cells to as single-cell RNA-seq, single-cell ATAC-seq, ChIP-seq analyses be determined and the impact of cell–cell interactions and sig- of transcriptional and epigenetic regulators, and more recently naling cascades to be unraveled. The protocols developed will be single-cell proteomics allows to map the phenotypic cellular quickly translated to clinical applications, as demonstrated by states of the myeloid differentiation landscape in health and dis- CRISPR-mediated gene editing.6 The goal is to apply this deep ease, at a hitherto unprecedent resolution. understanding of erythropoiesis to improve diagnosis, progno- We propose a coordinated European effort that will develop sis, and treatment of patients with RBC conditions. and apply standards for comprehensive single cell omics analy- The fundamental understanding of erythropoiesis will have ses and establish a Pan-European “reference” single cell library direct impact on medical care of patients with RBC conditions. of myeloid cells in health and disease. Ultimately, this single-cell First, improved diagnosis will lead clinicians to better-informed “reference” library will improve our understanding of normal decisions on disease management. Second, novel curative treat- myeloid differentiation (and hematopoiesis in general) and ments will be developed, such as those involving gene correc- innate immune defense at the system level but also how genetic tion/addition and drugs targeting specific signaling pathways. aberrations of myeloid regulators cause perturbations of normal Third, in vitro generation of RBCs will bring guaranteed dis- cellular states can lead to myeloid disease, and how these states ease-free transfusion units to the clinic. Finally, fundamental can be targeted by novel therapeutic approaches and modalities. insights in erythropoiesis will continue to facilitate discoveries A coordinated European effort involving basic and clinical in other fields of medicine, leading to better clinical care. researchers including bioinformaticians is required to achieve the following key aims of the proposed research program: Myelopoiesis 1. Establishment of a European expert group that will discuss, establish, and continuously update standard operating The body´s immediate defense against invading microor- procedures (SOPs) for single-cell omics analyses of bone ganisms is orchestrated by myeloid effector cells of the innate marrow and blood cells in general and myeloid cells in par- immune system including granulocytes, monocytes/macro- ticular. These SOPs should include (a) general processing of phages, dendritic cells, and rare mast cells. These cells are gener- normal bone marrow and blood cells, (b) single-cells omics ally short-lived, and require life-long replenishment by HSCs and protocols and technology platforms, and (c) the develop- their progeny, which gradually transit along their differentiation ment of bioinformatics methodologies for integrated single trajectories and ultimately become highly specialized myeloid cells omics and clinical data analysis. effector cells. This complex process, referred to as myelopoiesis, 2. Implementation of designated European core facilities/hos- is tightly regulated by growth factors, epigenetic and transcrip- pitals that will implement standardized collection and bio- tional regulators, which coordinate early lineage-specification, banking of human bone marrow and blood samples from cell cycle exit, and terminal differentiation. Consequently, healthy subjects and patients for single cell omics analyses. genetic aberrations of key regulators of myeloid differentiation 3. Implementation of specialized European core facilities/ programs have been causally linked to the majority of myeloid research teams for standardized single-cell omics analysis diseases such as acute myeloid leukemias (AMLs), myeloid dys- of bone marrow and blood cells according to the single-cell plastic syndromes and proliferative neoplasms (MDSs/MPNs), omics SOPs defined by the expert group. and severe congenital as well as cyclic neutropenia. Hence, in 4. Implementation of a European core computational consor- the context of disease, functional characterization of myeloid tium for concerted standardized processing and analysis of regulators during steady-state hematopoiesis and infections at single cell omics data to generate a “reference” single-cell both the molecular and system levels is pivotal to understand omics library of normal and genetically perturbated cells (1) the biology of myeloid effector cells and their role in innate from healthy subjects and patients with myeloid disease. immune defense and (2) how genetic aberrations affect normal Importantly, the computational consortium should develop myelopoiesis and cause myeloid disease. open-access web-based platforms allowing researchers and For more than 2 decades, European scientists have contrib- clinicians worldwide to download and match single-cell uted substantially to the understanding of myelopoeisis as high- omics data with their own data including those from lighted in the 2016 EHA roadmap for European Hematology patients with myeloid diseases enrolled in clinical trials. Research.1 Since then, the European research community has continued this endeavor with a particular focus on myeloid reg- The proposed research program conducted by a multi- ulators and networks in health and disease, and the of char- disciplinary concerted European effort will generate a com- acterization of novel myeloid differentiation trajectories in the prehensive single-cell omics reference library of myeloid hematopoietic hierarchy. The latter is exemplified by a selection differentiation in health and disease. The resultant single cells of recent key European achievements: (1) Paul et al7 conducted a library represents a most powerful tool for the research com- seminal single-cell transcriptomics study highlighting that com- munity, as it can be used in part as a reference of how expres- mitment toward specific myeloid lineages is a very early event sion and activity of genes, proteins, or signaling pathways during hematopoiesis, which is not linked to transitional stages change during normal myeloid differentiation and are per- of differentiation via progenitors with multi- and bi-lineage turbed by genetic aberrations in myeloid diseases. Significantly, potential, as described in the “classic” models of hematopoiesis. this will have substantial impact on complementary European (2) European research consortia have pioneered the mapping clinical research programs and trials aiming at identifying (1) of mutational landscapes and aberrant regulatory networks novel diagnostic markers for improved prognostication and 3 Jaffredo et al Research Roadmap: Normal Hematopoiesis (2) therapeutic targets for effective personalized combination 3. Mutual regulation of MKs and bone marrow environ- therapies of patients with AML, MDS, MPN, and other rare ment. There is evidence that MKs are able to express and myeloid diseases. release different molecules that may regulate bone marrow homeostasis in both, steady-state and pathological con- ditions. Furthermore, MKs are implicated in the immune Megakaryopoiesis and inflammatory responses. On this basis, it is important Megakaryopoiesis is the differentiation process that leads to to identify the molecules actively expressed by MKs, the platelet production. This is a unique cell biology system because release of which is implicated in the bone marrow and Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy platelets arise from the fragmentation of their marrow precur- immune regulation. sors, namely the megakaryocytes (MKs), in the blood shear. The precise understanding of the molecular mechanism of Platelet production is both regulated by the MK size through MK differentiation as well as of platelet production may have wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 polyploidization and number. The regulation of megakaryopoi- important clinical consequences in: understanding numerous esis is dependent on a cytokine/hormone called thrombopoie- disorders affecting MK lineage; developing new technologies for tin (TPO), which signals through the thrombopoietin receptor large platelet production in vitro; and the identification of new MPL. Megakaryopoiesis is affected by numerous acquired and molecules capable to modify platelet production in vivo. hereditary disorders that involve transcription factors, TPO/ MPL signaling and the actin and tubulin cytoskeletons. The classical treatments of thrombocytopenia include TPO mimetics Lymphopoiesis and platelet transfusion from donors but novel approaches are required. Lymphoid lineage development is by far the most com- European researchers have played a central role in the under- plex differentiation process within the hematopoietic system. standing of the regulation of megakaryopoiesis both at the cel- Following gradual acquisition of the lymphoid identity, lym- lular and transcription level and have developed cutting edge phoid progenitors undergo subsequent diversification into T, B, cell/molecular biology technologies. and natural killer/innate lymphoid cells (NK/ILC) lineage-spec- Key advances have been achieved among the last 5 years by ified precursors. While NK/ILC rapidly acquires cytotoxic or the European researchers. cytokine producing effector functions, differentiation of the T and B precursors continues with somatic V(D)J recombination 1. Two different pathways of MK commitment have been of T cell receptor (TCR) or Ig loci and subsequent elimination of identified, one directly from a MK-biased HSC playing a auto-reactive clones, resulting in mature T and B cells that leave major role in MK stress, and the second by a hierarchi- the thymus and the bone marrow, respectively. In this section, cal pathway through an MK-Erythroid progenitor.13,14 The we will focus on the initial steps of lymphoid differentiation pre- presence of megakaryopoiesis in the lung that may lead ceding TCR/Ig rearrangement, a field in which European groups to a platelet production by atypical hypoploid megakary- have a strong research record.22–25 ocytes expressing an hybrid phenotype between classical Future studies in the field should lead to a better integra- MKs and dendritic cells was also identified.15,16 tion of the synchronic and diachronic time scales of lymphoid 2. Proplatelet formation is not the unique mechanism to pro- development. duce platelets as they may be produced by MK rupture in At the synchronic differentiation level, recent technological stress conditions and membrane budding in both basal and advances in vivo fate-mapping genetic models and multiomics stress conditions.17 approaches at single-cell resolution have largely confirmed ear- 3. Characterization of 2 physic parameters involved in plate- lier studies based on analyses of phenotypic/function relation- let shedding; shear stress18 and turbulence19 that is associ- ships. However, despite recent progresses, there are still major ated with the secretion of at least 3 mediators increasing gaps in our knowledge of the molecular machinery driving lym- platelet production. An approach combining immortalized phoid development trajectories. It remains difficult to accom- MK cell lines derived from iPSC and these new parameters modate microenvironment-driven instructive differentiation has allowed the in vitro production for transfusion pur- schemes with the Waddington-based cell-autonomous models pose.19 In parallel, the development of 3D scaffolds has of lineage-commitment. A limitation of current models of lym- allowed an increase in production of functional platelets phoid development is that they do not take into account the link from iPSC and hematopoietic progenitors.20,21 between cell proliferation, that is, between symmetric or asym- The major topics that require intense research resources and metric cell division, and lineage diversification at the origin of efforts include: increasing heterogeneity across cell generations. These issues are all the more important that they are beginning to be addressed 1. Mechanisms of MK commitment and differentiation from in myeloid development studies.26,27 HSC. The new techniques on single cells allowing to study Another conceptual consideration for development of syn- transcriptome, chromatin accessibility and occupancy, as thetic models of lymphoid differentiation is to accommo- well as DNA methylation associated with cell biology may date phenotype/function and transcriptional approaches with allow to understand the mechanisms of MK/platelet bias recently developed chromatin conformation analyses that allow and commitment. It may also permit to precisely under- for dissection of enhancer/promoter interactions at the base- stand the stress megakaryopoiesis. Furthermore, it will be pair resolution.28 Human lymphopoiesis is far less well charac- possible to precisely determine the ontogeny of lung MKs terized than in the mouse. Earlier studies led to identification of and the MK heterogeneity. two CD7+ or CD10+ lymphoid-restricted subsets which are now 2. Regulation of platelet formation. There is evidence that at referred multilymphoid progenitors, but the developmental sta- least three different processes (proplatelet formation, MK tus of these populations remains uncertain. Their relationships budding, and MK rupture) are involved in platelet forma- with recently described CD127−/+ early lymphoid progenitors tion. How these different mechanisms are regulated, and also need to be clarified.22 In coming years, lymphoid devel- how they contribute to the basal and stress platelet produc- opment studies in humans should focus on the establishment tion still remains to be determined. These approaches may of clonal scale maps of developmental trajectories. A special also provide candidate molecules implicated in the final attention should also be given to human–mouse cross-species regulation of MK differentiation and which may permit to approaches that will permit to distinguish species-specific from increase platelet production. evolutionary conserved regulatory mechanisms. 4 (2021) 5:12 www.hemaspherejournal.com At the diachronic development level, future models of lym- homing of the transplanted (expanded or not) HSCs and better phoid development will need to integrate ontogeny-related defined stem-cell niche properties and location is still warranted. changes in lymphocyte production patterns in order to deter- mine whether ontogeny-related changes in lymphocyte produc- tion patterns are based on independent progenitors and/or if are HSC transformation, cell of origin, and accompanied by concomitant changes in lymphoid architecture. preleukemia Progress in the field will be essential to understand the differ- ences in the age incidence of B versus T cell leukemias helping to It has become clear that the identity of the hematopoietic stem Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy devise therapeutic strategies to reconstitute lymphoid cell sub- or progenitor cell in which a leukemic event first arises plays a sets in disease and aging. crucial role in the biology of the disease. Preleukemic lesions have been identified in healthy aged individuals. Understanding what influences the expansion of these preleukemic clones, over- wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 Hematopoietic stem cells time, and why certain individuals are more likely to develop a full-blown leukemia will allow us to decrease the risk of leu- HSCs were the first adult stem cells to find their way into the kemia development. Additionally, understanding how leukemic clinic. Therefore, HSCs are frequently regarded as a role model (stem) cells outcompete the normal HSC overtime and altered in adult stem cell biology. Notwithstanding their very success- the BM microenvironment might help us device new treatment ful and beneficial clinical applicability, many aspects of basic strategies.34 HSC biology remain unresolved, precluding additional rational approaches to even further expand them, or their more mature cellular derivatives’ usage in the clinic. Here, we briefly summa- Developmental hematopoiesis rize the most recent advances in the field of stem cell research in the last five years and proposed new roadmaps research topics Throughout adult life, the hematopoietic system is a highly that will need to be addressed to further expand and maximize dynamic hierarchy of cells founded by robust self-renewing and the impact of the use of HSCs in the clinic. multipotent HSCs that produce billions of mature blood cells daily. The use of HSCs for cell and gene therapies is continuously growing, although with the limitation of obtaining a source of HSC hierarchy and contribution of HSC HLA-compatible cells. During the last 5 years, significant prog- ress has been made by the European researchers regarding a The contribution of stem and multipotent progenitor cells better identification of the different hematopoietic cell popula- to steady-state blood maintenance has been recently chal- tions and the microenvironments or niches where HSCs emerge lenged. Similarly, a number of recent studies obtained from (ie, in the embryonic aorta) and expand (ie, in the fetal liver) to single-cell analysis have also questioned the routes by which form the pool of adult HSCs, as well as the molecular orches- lineage differentiation occurs.29 These evolving views of hema- tration of HSC emergence and expansion processes. This precise topoiesis have broad implications for our understanding of fundamental knowledge is salient since it will make the foun- adult stem cell function. Nevertheless, most of these data have dation of future translational research. However, despite these been obtained in mice and few in human.30 Furthermore, most advances, the generation of HSCs from pluripotent stem cells or of the new data relied heavily on scRNAseq analysis, which using direct cell reprogramming remains elusive. This is likely have limitations on identifying closely related cell types. Thus, because the intrinsic and extrinsic cues regulating HSC spec- further studies are needed to define HSC heterogeneity and their cell fate decision both at steady state and under stress. ification, expansion, and homing as it occurs physiologically Another aspect that will need to be addressed is to if/how the during embryogenesis and in successive ontogenic microenvi- BM niche influences of the activity of HSC subsets and their ronments are not fully understood. This knowledge is, however, differentiation. indispensable if one intends to understand/treat hematologic diseases and childhood leukemias. Developmental hematopoiesis is characterized by the follow- HSC aging and rejuvenation ing rules: The effects of aging on HSCs have started to be defined.31 1. HSC generation is currently limited to the embryo and Changes in the composition of the HSC pool as well as loss of occurs through the transdifferentiation of endothelial cells cell polarity can be due to intrinsic properties, extrinsic micro- endowed with an hemogenic potential. environmental perturbations and accumulation of mutations. 2. Extrinsic cues and cell-intrinsic cues, yet to be fully defined, Reversion or prevention of stem cell aging should help delay govern the hematopoietic generative program. age-related hematopoietic deficiencies. In this subsection, we will summarize the advances made over the past 5 years and propose new issues that need to be Generation of editing HSC for therapy addressed to create and amplify HSCs for therapeutic purposes. The discovery of the (CRISPR)-Cas9 nuclease (Cas9/sgRNA) systems in 2012, has revolutionised gene therapies for HSCs. The Hemogenic endothelium commitment number of genome-editing strategies in HSCs that could offer How endothelial cells acquire a hemogenic potential is a key therapeutic potential for blood diseases and immune system have question. Interestingly, both the yolk sac and the embryo carry dramatically increased. The HSC-based genome-editing field is hemogenic endothelium but only the one from the embryo is primed to enter clinical trials in Europe in the coming years.32 capable of generating HSCs.35 An in depth knowledge of the intrinsic transcriptional program(s) operating to specify hemo- HSC expansion, transplantation, and homing genic endothelium able to give rise to HSCs is required, prefer- ably at the single cell level. This is required since hematopoietic A number of small molecules-based approaches and/or production in vitro from the hemogenic endothelium is limited refined cultured conditions have suggested that ex vivo expan- to progenitors. Recently, hemogenic endothelium generating sion of HSCs could be achievable.33 Still, efforts to increase the HSCs has also been described in the forming bone marrow.36 5 Jaffredo et al Research Roadmap: Normal Hematopoiesis The comparison between hemogenic endothelial cells speci- pregnancy and are accounting for 30% of childhood and ado- fied in different anatomical sites and at different time points lescent cancers.44 Understanding the initial mutation events trig- of development will certainly shed light on this cell commit- gering leukemia and modelling the initiation and progression of ment, which is still elusive. For a comprehensive view, several the disease will be of prime importance for the future. Access to species including human should be studied and compared since human samples and manipulation of human pluripotent stem the cellular and to many extents the molecular control of hema- cells will certainly be a key issue. Bringing together people from topoietic stem cell production in the aorta is strongly conserved developmental hematopoiesis and from childhood leukemia will between species. This allows to functionally test specific genes help in the success. Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy or signaling pathways. Taken together, progress into these major axes should signifi- cantly enhance our understanding of the hematopoietic hierar- chy, how the hematopoietic microenvironment is shaped, which Gene regulatory networks operating to drive wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 mechanisms co-operate in regulating the commitment of hemo- hemogenic endothelium to HSCs genic endothelial cells, how HSCs emerge and amplify, how this relates to changes in HSC heterogeneity during ontogeny, and Based on previous studies mostly conducted in bulk pop- how leukemia is initiated at early developmental stages. One ulations, some genes, mostly transcription factors, have been of the strongest challenges will be to collect and integrate the identified and used to transduce cells to produce HSCs.37 This multiple dimensions of omics applied to the hematopoietic cells production however remains extremely low and cannot be used and to combine them with similar if not identical approaches for therapeutic applications. Despite the proof of concept, the applied to the niches. These dimensions span from genomics, efficiency and robustness in HSC generation of these studies is population or single-cell transcriptomics, spatial transcriptom- extremely low. Little is known about the gene cascades regulat- ics, single-cell epigenomics, and metabolomics. This unprece- ing the fine-tuned progression steps leading to HSC production. dented effort should gather biologists, computational biologists Transcriptomic data either in bulk and/or at the single cell level and, if required, clinicians to produce a comprehensive view have recently started to be generated from different types of sam- of HSC production and hematopoietic development in health ples but the number of cells collected is small in the embryo and and disease. In depth knowledge of these should quickly pave reliable systems of ex vivo differentiation of HSCs are still lack- the way toward ex vivo generation of HSCs for therapeutic ing, for example, pluripotent stem cell differentiation (see chapter purposes. “Reprogramming/induced pluripotent stem cells/embryonic stem cells”), gastruloids, aorta reconstruction/on a chip. In particular, gastruloids should give exciting insights into how extrinsic envi- Mesenchymal stromal cells ronmental signals instruct hemogenic endothelium and trigger the endothelial to hematopoietic transition to produce HSCs. Hemopoiesis is critically regulated by nonhematopoietic cells that support the production of blood and immune cells according to the organismal demands. These stromal cells Dynamics of the hematopoietic waves in the compose the so-called hematopoietic microenvironment. It is different embryonic sites becoming increasingly clear that different stromal populations regulate distinct subsets of hematopoietic cells, and vice versa. Despite active research on the main hematopoietic sites in The complexity of these networks is further increased by the the embryo, that is, yolk sac, dorsal aorta, placenta, and fetal recognition of heterogeneity among bone marrow stem cells liver,38–41 the precise hematopoietic cell types and the hemato- and their progenies. Recent technological developments (most poietic hierarchies present in these different locations are not notably single-cell studies) have allowed to describe candidate fully identified. More importantly, most of these data have been subpopulations. However, functional studies will be critical in obtained using the mouse embryo model as the mammalian the near future for fulfilling the enormous potential of stromal embryo reference, while very few studies focused on the human cells in immune modulation, tissue regeneration, and cancer embryo due to the lack of tissues. In a dynamic point of view, treatment. innovative barcoding systems are needed to identify the differ- ent stem and progenitor cell compartments and their routes of differentiation. Combined approaches generating bulk and Understanding the specific functions of MSC single cell RNA-Seq approaches are also necessary to precisely subsets identify these cell types and establish their close relationships. HSCs and their microenvironment represent probably the best-characterized hierarchical stem cell system in vertebrates, Embryonic niches paving the path to understanding how other organs function. In addition to establishing the intrinsic programs driving Recent European research has revealed the cellular and spa- hemogenic endothelium specification, HSCs, and progeni- tial organization of different bone marrow MSC subsets.45 tor cell differentiation, the microenvironments should also Computational methods have been developed to infer candi- be strongly scrutinized. Little is known about the first niches date gene networks in HSC-supporting MSCs46 and potential harboring/specifying hemogenic endothelium in the yolk sac ligand-receptor interactions.47 Future studies will be essen- and the embryo. Recently, 2 articles using spatial transcrip- tial to reveal the specific functions and potential of MSC tomics revealed the molecular complexity of the aortic hema- subpopulations. topoietic microenvironment.42,43 Of note, the fetal liver—and the placenta in the case of the mouse embryo—are 2 organs Understanding the role of MSCs in hematological orchestrating HSC amplification that have hardly been studied regarding this point. malignancies Recent European research has demonstrated a key role for MSCs in the initiation, progression, and drug resistance of a The embryonic origins of leukemia variety of hematological neoplasms through paracrine or Even though many leukemic events occur after birth, sev- cell-contact-dependent mechanisms.48 Extracellular vesicles eral hematological diseases were shown to take place during from lymphoma B cells render MSCs protumorigenic.49 Notch2 6 (2021) 5:12 www.hemaspherejournal.com signaling on MSCs triggers Wnt-dependent survival of chronic subject in the past decade (eg, Epigenome NoE, HEROIC, lymphocytic leukemia.50 Particularly, Wnt5A from MSCs is EPITRON, Epigenesys, BLUEPRINT, Human Cell Atlas). These required for the engraftment of normal and leukemic HSCs.51 projects revealed many new insights into the interplay of TFs MSCs remodel the extracellular matrix to support the progres- with chromatin to establish epigenetic patterns that define cell sion of B-cell acute lymphoblastic leukemia (ALL),52 support the type functionality, but also led to the realization that much can growth of myeloproliferative neoplasms53 or differentiate into still be learned about how blood cell types develop and how we myofibroblasts in myelofibrosis.54 Finally, mitochondrial trans- could modulate their activities to prevent or counteract disease. fer from MSCs protects ALL and AML cells from oxidative This will require a concerted effort to better understand regu- Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy stress induced by chemotherapy.55,56 Therefore, elucidating and latory networks in both normal hematopoiesis and disease and targeting niche-dependent mechanisms of tumorigenesis and will be accomplished only through close collaboration between resistance will be key to eradicate cancer. experimentalists, computational biologists, statisticians, and wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 05/29/2023 clinicians. Mutations in transcriptional and epigenetic regulators are MSCs in hematopoietic regeneration some of the most common mutations in hematologic malignan- cies. Given that these proteins function as regulatory network Over the past 5 years, European research has shown that components, it will be important to gain an understanding of MSCs enhance HSC mobilization for subsequent apheresis the malignant state as a perturbation of wider regulatory net- from peripheral blood and transplantation (HSCT).57 Targeting works. Research on the concerted actions as well as posttran- MSCs could help mitigate the damage to normal hematopoiesis scriptional regulation of TFs and on how these regulate the local by aging, AML, infection or iron overload in β-thalassemia.58–61 and global transcriptomic and epigenetic environment should Additionally, MSC coinfusion could increase the efficiency of be intensified. HSCT62 and minimize the risks of graft failure in gene therapy Although main TFs involved in disease development have applications associated with low conditioning regimens and been identified, many components that drive the functional infusion of limited numbers of gene-edited HSCs.63 fine-tuning of blood cell types and individual cells within are still not known. These are likely controlled by the niche in which MSCs in tissue regeneration, immune modulation, the cells reside, the availability of metabolites-both endogenous (amino acids, sugars, and vitamins) and exogenous (drugs, food and systemic disease additives, and toxins)-and other environmental factors. In addi- MSCs have a profound impact on different immune cells, tion, it has become clear that not only the adaptive immune sys- having therapeutic benefits in sepsis, autoimmune disorders tem confers memory potential, but that also cells of the innate or graft-versus-host disease, and may influence the outcome of immune system can be trained and are functionally dependent immunotherapies. Additionally, research on MSCs could influ- on past and present behavior, offering another entry point to ence the outcome and treatment of steadily increasing systemic utilize cells of the hematopoietic system in disease prevention conditions, such as diabetes, obesity, and aging. and control. Increasing research in the hematopoietic microenvironment Apart from creating additional comprehensive data sets on and particularly MSCs will, scientifically, feed into other stem single-cell genomics, epigenomics, and metabolomics, integra- cell systems and, clinically, provide new ways to modulate and tive analysis of data is mandatory. Computational, conditional treat hematopoietic diseases, immune responses, and regenera- dependency models need to be established, for example by cre- tive processes. As a result of this productive research, Europe ating Bayesian networks, to formulate the relationship between continues to be the world region with the second highest num- the individual cell’s niche, environmental factors, TF binding, ber of registered clinical trials using MSCs. epigenetic alterations, and the presence of various hematologic disorders. Transcriptional/epigenetic networks Reprogramming/induced pluripotent stem Maintaining a balanced output of hematopoietic cell lin- cells/embryonic stem cells eages is critically dependent on cell fate choices at the stem and progenitor level within the hematopoietic hierarchy. Pluripotent stem cells (PSCs) including embryonic stem cells These choices are executed at the single-cell level through the (ESCs) and induced PSCs (iPSCs), represent a limitless source interplay of extracellular signaling pathways with the intra- of cells for investigations ranging from developmental pro- cellular decision-making machinery. The latter is driven by cesses to drug discovery. One of the most significant promises networks of transcriptional and epigenetic regulators that of the stem cell field is the in vitro derivation of cell populations interact in a combinatorial fashion and can form larger pro- usable in the clinic for therapeutic purposes. A comprehensive tein complexes with different functions dependent on compo- understanding of the molecular and cellular processes leading to sition and cellular context. These establish cell type-specific blood cell emergence and maintenance is of paramount impor- transcriptional and epigenetic programs and mediate devel- tance to fulfil this promise. opmental transitions when cells differentiate down a particu- Over the last 5 years, European researchers have made semi- lar hematopoietic lineage. nal contributions to several interconnected areas in this research European researchers have contributed significantly to the field. Studies into embryonic hematopoiesis using murine and identification and characterization of individual transcription human PSCs have further defined, sometimes even redefined, factors (TFs), TF networks and epigenomic changes involved our understanding of hemogenic potential, cellular intermedi- in normal and aberrant hematopoieisis. In addition, they have ates, signalling pathways, and molecular players implicated in extended many of these analyses toward mutated TFs in pri- blood cell emergence. High throughput sequencing has led to mary hematopoietic (single) cells from healthy and diseased a wealth of data at both, population and single-cell levels.37,67 individuals, thereby revealing multiple unanticipated functions These data revealed the importance of cell cycle regulation of these proteins.25,41,64–66 as a driver of differentiation, demonstrated the generation of European researchers have been acting at the forefront of hematopoietic stem cells-like cells from differentiated human epigenetic research, a prime research interest of the European PSCs, and highlighted gene regulatory network dynamics driv- Union, which has supported a multitude of projects on this ing blood cell emergence. 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