p57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion.

Hematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact ß2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

miR-130b and miR-128a are essential lineage-specific codrivers of t(4;11) MLL-AF4 acute leukemia.

t(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4-driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on 2 microRNAs (miRNAs) upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor (BCP)/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor (LMPP) population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia (ALL), maintained by a highly expanded Il7r+c-Kit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified 2 downstream targets of these miRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumor suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the preleukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.

Single-cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs.

Hematopoietic stem and progenitor cells (HSPCs) develop in distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates some of these processes; however, it has proven difficult to generate functional hematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we explored single-cell RNA sequencing (scRNAseq) in combination with single-cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors and erythroid-, megakaryocyte-, and leukocyte-committed progenitors, and we identified CD44, CD326, ICAM2/CD9, and CD18, respectively, as markers of these progenitors. Using an artificial neural network that we trained on scRNAseq derived from human fetal liver, we identified a wide range of hPSC-derived HSPCs phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded, and was completely missing in the data set that had been generated using cells selected on the basis of CD43 expression. By comparing the single-cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver, we identified transcription factors and molecular pathways that can be explored in the future to improve the in vitro production of HSCs.

Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche.

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.

Endothelial-to-haematopoietic transition: an update on the process of making blood.

The first definitive blood cells during embryogenesis are derived from endothelial cells in a highly conserved process known as endothelial-to-haematopoietic transition (EHT). This conversion involves activation of a haematopoietic transcriptional programme in a subset of endothelial cells in the major vasculature of the embryo, followed by major morphological changes that result in transitioning cells rounding up, breaking the tight junctions to neighbouring endothelial cells and adopting a haematopoietic fate. The whole process is co-ordinated by a complex interplay of key transcription factors and signalling pathways, with additional input from surrounding tissues. Diverse model systems, including mouse, chick and zebrafish embryos as well as differentiation of pluripotent cells in vitro, have contributed to the elucidation of the details of the EHT, which was greatly accelerated in recent years by sophisticated live imaging techniques and advances in transcriptional profiling, such as single-cell RNA-Seq. A detailed knowledge of these developmental events is required in order to be able to apply it to the generation of haematopoietic stem cells from pluripotent stem cells in vitro - an achievement which is of obvious clinical importance. The aim of this review is to summarise the latest findings and describe how these may have contributed towards achieving this goal.

Molecular processes involved in B cell acute lymphoblastic leukaemia.

B cell leukaemia is one of the most frequent malignancies in the paediatric population, but also affects a significant proportion of adults in developed countries. The majority of infant and paediatric cases initiate the process of leukaemogenesis during foetal development (in utero) through the formation of a chromosomal translocation or the acquisition/deletion of genetic material (hyperdiploidy or hypodiploidy, respectively). This first genetic insult is the major determinant for the prognosis and therapeutic outcome of patients. B cell leukaemia in adults displays similar molecular features as its paediatric counterpart. However, since this disease is highly represented in the infant and paediatric population, this review will focus on this demographic group and summarise the biological, clinical and epidemiological knowledge on B cell acute lymphoblastic leukaemia of four well characterised subtypes: t(4;11) MLL-AF4, t(12;21) ETV6-RUNX1, t(1;19) E2A-PBX1 and t(9;22) BCR-ABL1.

Analysis of Jak2 signaling reveals resistance of mouse embryonic hematopoietic stem cells to myeloproliferative disease mutation.

The regulation of hematopoietic stem cell (HSC) emergence during development provides important information about the basic mechanisms of blood stem cell generation, expansion, and migration. We set out to investigate the role that cytokine signaling pathways play in these early processes and show here that the 2 cytokines interleukin 3 and thrombopoietin have the ability to expand hematopoietic stem and progenitor numbers by regulating their survival and proliferation. For this, they differentially use the Janus kinase (Jak2) and phosphatidylinositol 3-kinase (Pi3k) signaling pathways, with Jak2 mainly relaying the proproliferation signaling, whereas Pi3k mediates the survival signal. Furthermore, using Jak2-deficient embryos, we demonstrate that Jak2 is crucially required for the function of the first HSCs, whereas progenitors are less dependent on Jak2. The JAK2V617F mutation, which renders JAK2 constitutively active and has been linked to myeloproliferative neoplasms, was recently shown to compromise adult HSC function, negatively affecting their repopulation and self-renewal ability, partly through the accumulation of JAK2V617F-induced DNA damage. We report here that nascent HSCs are resistant to the JAK2V617F mutation and show no decrease in repopulation or self-renewal and no increase in DNA damage, even in the presence of 2 mutant copies. More importantly, this unique property of embryonic HSCs is stably maintained through ≥1 round of successive transplantations. In summary, our dissection of cytokine signaling in embryonic HSCs has uncovered unique properties of these cells that are of clinical importance.

Activity of a heptad of transcription factors is associated with stem cell programs and clinical outcome in acute myeloid leukemia.

Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

Concise review: From greenhouse to garden: the changing soil of the hematopoietic stem cell microenvironment during development.

The hematopoietic system has been intensely studied for many decades. For this reason, it has become the best understood stem cell-derived system that serves as a paradigm for stem cell biology and has found numerous applications in the clinics. While a lot of progress has recently been made in describing the bone marrow components that maintain and control blood stem cell function in the adult, very little is currently known about the regulatory microenvironment in which the first adult-repopulating hematopoietic stem cells are formed during development. Knowledge of these processes is crucial for understanding the basic regulation of hematopoietic stem cell production and behavior and to allow their in vitro expansion and generation from embryonic stem cells or iPS cells for clinical and research purposes. This review summarizes the recent advances that have been made in defining the cellular components, as well as the soluble and physical factors, that are part of the niche involved in regulating hematopoietic stem cell generation in the embryo. The findings are compared with what is known about the adult bone marrow niche to find common pathways for stem cell regulation, but also to highlight processes uniquely required for de novo hematopoietic stem cell generation, as these are the conditions that will need to be recreated for the successful production of blood stem cells in culture.

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.

Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells.

The first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.5, thus pointing to the possible existence of both positive and negative regulators of emerging hematopoietic stem cells. Our recent expression analysis of the aorta-gonad-mesonephros region showed that the Delta-like homologue 1 (Dlk1) gene is up-regulated in the region of the aorta-gonad-mesonephros where hematopoietic stem cells are preferentially located. To analyze its function, we studied Dlk1 expression in wild-type and hematopoietic stem cell-deficient embryos and determined hematopoietic stem and progenitor cell activity in Dlk1 knockout and overexpressing mice. Its role in hematopoietic support was studied in co-culture experiments using stromal cell lines that express varying levels of Dlk1. We show here that Dlk1 is expressed in the smooth muscle layer of the dorsal aorta and the ventral sub-aortic mesenchyme, where its expression is dependent on the hematopoietic transcription factor Runx1. We further demonstrate that Dlk1 has a negative impact on hematopoietic stem and progenitor cell activity in the aorta-gonad-mesonephros region in vivo, which is recapitulated in co-cultures of hematopoietic stem cells on stromal cells that express varying levels of Dlk1. This negative effect of Dlk1 on hematopoietic stem and progenitor cell activity requires the membrane-bound form of the protein and cannot be recapitulated by soluble Dlk1. Together, these data suggest that Dlk1 expression by cells of the aorta-gonad-mesonephros hematopoietic microenvironment limits hematopoietic stem cell expansion and is, to our knowledge, the first description of such a negative regulator in this tissue.

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia.

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F-positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2(V617F) mice develop reduced numbers of lineage(-)Sca-1(+)c-Kit(+) cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2(V617F) mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Endothelial-specific Gata3 expression is required for hematopoietic stem cell generation.

To generate sufficient numbers of transplantable hematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-hematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments, and that is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for GATA3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in hemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing GATA3 at the core of the EHT regulatory network.

An unexpected role for IL-3 in the embryonic development of hematopoietic stem cells.

Cytokines are important in adult hematopoiesis, yet their function in embryonic hematopoiesis has been largely unexplored. During development, hematopoietic stem cells (HSCs) are found in the aorta-gonad-mesonephros (AGM) region, yolk sac (YS), and placenta and require the Runx1 transcription factor for their normal generation. Since IL-3 is a Runx1 target and this cytokine acts on adult hematopoietic cells, we examined whether IL-3 affects HSCs in the mouse embryo. Using Runx1 haploinsufficient mice, we show that IL-3 amplifies HSCs from E11 AGM, YS, and placenta. Moreover, we show that IL-3 mutant embryos are deficient in HSCs and that IL-3 reveals the presence of HSCs in the AGM and YS prior to the stage at which HSCs are normally detected. Thus, our studies support an unexpected role for IL-3 during development and strongly suggest that IL-3 functions as a proliferation and/or survival factor for the earliest HSCs in the embryo.

Identification of novel regulators of hematopoietic stem cell development through refinement of stem cell localization and expression profiling.

The first adult-repopulating hematopoietic stem cells (HSCs) are detected starting at day 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. Despite the importance of the AGM in initiating HSC production, very little is currently known about the regulators that control HSC emergence in this region. We have therefore further defined the location of HSCs in the AGM and incorporated this information into a spatial and temporal comparative gene expression analysis of the AGM. The comparisons included gene expression profiling (1) in the newly identified HSC-containing region compared with the region devoid of HSCs, (2) before and after HSC emergence in the AGM microenvironment, and (3) on populations enriched for HSCs and their putative precursors. Two genes found to be up-regulated at the time and place where HSCs are first detected, the cyclin-dependent kinase inhibitor p57Kip2/Cdkn1c and the insulin-like growth factor 2, were chosen for further analysis. We demonstrate here that they play a novel role in AGM hematopoiesis. Interestingly, many genes involved in the development of the tissues surrounding the dorsal aorta are also up-regulated during HSC emergence, suggesting that the regulation of HSC generation occurs in coordination with the development of other organs.

HOXA9/IRX1 expression pattern defines two subgroups of infant MLL-AF4-driven acute lymphoblastic leukemia.

Infant t(4;11) acute lymphoblastic leukemia is the most common leukemia in infant patients and has a highly aggressive nature. The patients have a dismal prognosis, which has not improved in more than a decade, suggesting that a better understanding of this disease is required. In the study described here, we analyzed two previously published RNA-sequencing data sets and gained further insights into the global transcriptomes of two known subgroups of this disease, which are characterized by the presence or absence of a homeobox gene expression signature. Specifically, we identified a remarkable mutually exclusive expression of the HOXA9/HOXA10 and IRX1 genes and termed the two subgroups iALL-HOXA9 and iALL-IRX1. This expression pattern is critical as it suggests that there is a fundamental difference between the two subgroups. Investigation of the transcriptomes of the two subgroups reveals a more aggressive nature for the iALL-IRX1 group, which is further supported by the fact that patients within this group have a worse prognosis and are also diagnosed at a younger age. This could be reflective of a developmentally earlier cell of origin for iALL-IRX1. Our analysis further uncovered critical differences between the two groups that may have an impact on treatment strategies. In summary, after a detailed investigation into the transcriptional profiles of iALL-HOXA9 and iALL-IRX1 patients, we highlight the importance of acknowledging that these two subgroups are different and that this is of clinical importance.

Infant leukaemia - faithful models, cell of origin and the niche.

For patients and their families, the diagnosis of infant leukaemia is devastating. This disease has not seen the improvements in outcomes experienced with other paediatric leukaemias and it is becoming ever more apparent that infant leukaemia is a distinct biological entity. Insights into some of the distinguishing features of infant leukaemia, such as a single mutation - the MLL-gene rearrangement, the biology of disease aggressiveness and lineage plasticity, and the high incidence of central nervous system involvement, are likely to be gained from understanding the interactions between leukaemic cells and their environment or niche. The origins of infant leukaemia lie in the embryonic haematopoietic system, which is characterised by shifting locations and dynamic changes in the microenvironment. Understanding this foetal or embryonic context is integral to understanding infant leukaemia development. Owing to its rarity and prenatal origins, developing accurate modelling systems for further investigation of infant leukaemia is essential. In this Review, we discuss how available in vitro, ex vivo and in vivo infant leukaemia models contribute to our current understanding of the leukaemia niche in embryonic development, established disease and specialised non-haematopoietic niches. The mechanistic insights provided by accurate models will help identify viable novel therapeutic options.

Defining the fetal origin of MLL-AF4 infant leukemia highlights specific fatty acid requirements.

Infant MLL-AF4-driven acute lymphoblastic leukemia (ALL) is a devastating disease with dismal prognosis. A lack of understanding of the unique biology of this disease, particularly its prenatal origin, has hindered improvement of survival. We perform multiple RNA sequencing experiments on fetal, neonatal, and adult hematopoietic stem and progenitor cells from human and mouse. This allows definition of a conserved fetal transcriptional signature characterized by a prominent proliferative and oncogenic nature that persists in infant ALL blasts. From this signature, we identify a number of genes in functional validation studies that are critical for survival of MLL-AF4+ ALL cells. Of particular interest are PLK1 because of the readily available inhibitor and ELOVL1, which highlights altered fatty acid metabolism as a feature of infant ALL. We identify which aspects of the disease are residues of its fetal origin and potential disease vulnerabilities.

GATA-2 plays two functionally distinct roles during the ontogeny of hematopoietic stem cells.

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs), whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac, fetal liver, and adult bone marrow. However, HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also, cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus, GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow.