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Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT) 1-4 . This process generates hundreds of blood progenitors, of which a fraction go on to become definitive HSCs. It is generally thought that most adult blood is derived from those HSCs, but to what extent other progenitors contribute to adult haematopoiesis is not known. Here we use in situ barcoding and classical fate mapping to assess the developmental and clonal origins of adult blood in mice. Our analysis uncovers an early wave of progenitor specification-independent of traditional HSCs-that begins soon after EHT. These embryonic multipotent progenitors (eMPPs) predominantly drive haematopoiesis in the young adult, have a decreasing yet lifelong contribution over time and are the predominant source of lymphoid output. Putative eMPPs are specified within intra-arterial haematopoietic clusters and represent one fate of the earliest haematopoietic progenitors. Altogether, our results reveal functional heterogeneity during the definitive wave that leads to distinct sources of adult blood., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Hematopoiesis changes over life to meet the demands of maturation and aging. Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis. Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood. By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7's control of hematopoietic maturation. We find that juvenile Cbx2 -/- hematopoietic tissues show impairment of B-lymphopoiesis, a precocious adult-like myeloid bias, and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors. These findings define a mechanism of regulation of HSPC output via chromatin modification as a function of age with potential impact on age-biased pediatric and adult blood disorders., Competing Interests: Declaration of interests G.Q.D. is a founder and shareholder of 28/7 Therapeutics., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41 + HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production., Competing Interests: Declaration of Interests V.L. is currently at the Karolinska Institutet, L.N.T. is at Sanofi, and A.G.C. is at the Peter MacCallum Cancer Centre. G.Q.D. holds equity interest in True North Therapeutics and 28/7 Therapeutics; D.E.I. holds equity in Emulate Inc. and chairs its scientific advisory board., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL / KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9 , leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia., (© 2019 Rowe et al.)

All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.

Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)green fluorescent protein (GFP)(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-)GFP(+/+) HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs., (© 2015 by The American Society of Hematology.)

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial-cadherin(+) hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis., (© 2015 by The American Society of Hematology.)

Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF-β signaling. Msi2-null HSCs are insensitive to TGF-β-mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias.

Hematopoiesis is a paradigm for stem cell biology in that it centers on differentiation of a self-renewing pluripotent precursor into multiple committed cell types with specific functions. The use of induced pluripotent stem cells (iPSCs) as a disease modeling tool has revealed numerous insights into the underlying pathophysiology of hematological diseases - those disorders arising from defective hematopoiesis. Likewise, studying hematopoiesis and the defects that can arise offer clues to understanding general stem cell survival and differentiation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Notch signaling regulates several cellular processes including cell fate decisions and proliferation in both invertebrates and mice. However, comparatively less is known about the role of Notch during early human development. Here, we examined the function of Notch signaling during hematopoietic lineage specification from human pluripotent stem cells of both embryonic and adult fibroblast origin. Using immobilized Notch ligands and small interfering RNA to Notch receptors we have demonstrated that Notch1, but not Notch2, activation induced hairy and enhancer of split 1 (HES1) expression and generation of committed hematopoietic progenitors. Using gain- and loss-of-function approaches, this was shown to be attributed to Notch-signaling regulation through HES1, which dictated cell fate decisions from bipotent precursors either to the endothelial or hematopoietic lineages at the clonal level. Our study reveals a previously unappreciated role for the Notch pathway during early human hematopoiesis, whereby Notch signaling via HES1 represents a toggle switch of hematopoietic vs endothelial fate specification.

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryonic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage.

Members of the caudal gene family (in mice and humans: Cdx1, Cdx2, and Cdx4) have been studied during early development as regulators of axial elongation and anteroposterior patterning. In the adult, Cdx1 and Cdx2, but not Cdx4, have been intensively explored for their function in intestinal tissue homeostasis and the pathogenesis of gastrointestinal cancers. Involvement in embryonic hematopoiesis was first demonstrated in zebrafish, where cdx genes render posterior lateral plate mesoderm competent to respond to genes specifying hematopoietic fate, and compound mutations in cdx genes thus result in a bloodless phenotype. Parallel studies performed in zebrafish embryos and murine embryonic stem cells (ESCs) delineate conserved pathways between fish and mammals, corroborating a BMP/Wnt-Cdx-Hox axis during blood development that can be employed to augment derivation of blood progenitors from pluripotent stem cells in vitro. The molecular regulation of Cdx genes appears complex, as more recent data suggest involvement of non-Hox-related mechanisms and the existence of auto- and cross-regulatory loops governed by morphogens. Here, we will review the role of Cdx genes during hematopoietic development by comparing effects in zebrafish and mice and discuss their participation in malignant blood diseases., (© 2012 New York Academy of Sciences.)

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs.

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear., Design and Methods: We inactivated Cdx4 in mice through either a germline or conditional knockout approach and analyzed requirement for Cdx4 in both normal adult hematopoiesis and leukemogenesis initiated by the MLL-AF9 fusion oncogene., Results: Here, we report that loss of Cdx4 had a minimal effect on adult hematopoiesis. Indeed, although an increase in white blood cell counts was observed, no significant differences in the distribution of mature blood cells, progenitors or stem cells were observed in Cdx4-deficient animals. In addition, long-term repopulating activity in competitive transplantation assays was not significantly altered. In vitro, B-cell progenitor clonogenic potential was reduced in Cdx4-deficient animals but no significant alteration of mature B cells was detected in vivo. Finally, induction of acute myeloid leukemia in mice by MLL-AF9 was significantly delayed in the absence of Cdx4 in a retroviral transduction/bone marrow transplant model., Conclusions: These observations indicate that Cdx4 is dispensable for the establishment and maintenance of normal hematopoiesis in adult mammals. These results, therefore, outline substantial differences in the Cdx-Hox axis between mammals and zebrafish and support the hypothesis that Cdx factors are functionally redundant during mammalian hematopoietic development under homeostatic conditions. In addition, our results suggest that Cdx4 participates in MLL-AF9-mediated leukemogenesis supporting a role for Cdx factors in the pathogenesis of myeloid leukemia.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia-associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML).

Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1(+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4(-/-) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.

A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generating pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) pluripotent stem cell lines. Current research is focusing on improving reprogramming protocols (e.g., circumventing the use of retroviral technology and oncoproteins), and on methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. Here, we explore hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data show robust differentiation of iPS cells to mesoderm and to blood lineages, as shown by generation of CD34(+)CD45(+) cells, hematopoietic colony activity, and gene expression data, and suggest conservation of blood patterning pathways between mouse and human hematopoietic development.

Osteoblasts and endothelium constitute functional niches that support haematopoietic stem cells in mammalian bone marrow. Adult bone marrow also contains adipocytes, the number of which correlates inversely with the haematopoietic activity of the marrow. Fatty infiltration of haematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia. To explore whether adipocytes influence haematopoiesis or simply fill marrow space, we compared the haematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. Here we show, by flow cytometry, colony-forming activity and competitive repopulation assay, that haematopoietic stem cells and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 'fatless' mice, which are genetically incapable of forming adipocytes, and in mice treated with the peroxisome proliferator-activated receptor-gamma inhibitor bisphenol A diglycidyl ether, which inhibits adipogenesis, marrow engraftment after irradiation is accelerated relative to wild-type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone-marrow microenvironment, and indicate that antagonizing marrow adipogenesis may enhance haematopoietic recovery in clinical bone-marrow transplantation.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

The transcription factor SCL is critically required for establishing hematopoiesis and for proper endothelial development, but not for maintenance of hematopoietic stem cells or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely, whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic precursors. To answer this question, we have engineered murine embryonic stem cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of SCL expression at defined time points during in vitro development. Continual SCL expression during differentiation results in markedly increased hematopoiesis. By using pulses of gene expression over a 6-day differentiation time course, we map and characterize windows of SCL responsiveness. Notably, a pulse of SCL expression during early mesodermal patterning (48 to 72 hours of differentiation) promoted Flk1+ PDGFRalphaneg presumptive extraembryonic/lateral plate mesoderm at the expense of PDGFRalpha+ Flk1neg presumptive paraxial mesoderm. Consistent with this, the early pulse of SCL expression also expanded hematopoietic colony-forming cell numbers, while concomitantly repressing expression of paraxial and cardiac markers, and inhibited development of beating cardiomyocytes. By mixing the inducible embryonic stem cells with fluorescently labeled wild-type cells in chimeric embryoid bodies, we show that these effects of SCL are cell autonomous. These data support a master-regulatory role for SCL in mesodermal patterning, in addition to its established later roles in hematopoietic differentiation.

Cdx genes (Cdx1, Cdx2, and Cdx4) encode a family of caudal-related transcription factors that mediate anterior-posterior patterning during embryogenesis through Hox gene regulation. Homologues in the zebrafish have been shown to play key roles in blood formation. To define the role of Cdx genes during embryonic hematopoiesis in a mammalian system, we examined the hematopoietic potential of Cdx-deficient mouse embryonic stem cells (ESCs) in vitro and in vivo. Individual Cdx-deficient ESCs exhibited impaired embryonic hematopoietic progenitor formation and altered Hox gene expression, most notably for Cdx2 deficiency. A more severe hematopoietic defect was observed with compound Cdx deficiency than loss of function of any single Cdx gene. Reduced hematopoietic progenitor formation of ESCs deficient in multiple Cdx genes could be rescued by ectopic expression of Cdx4, concomitant with partially restored Hox gene expression. These results reveal an essential and partially redundant role for multiple Cdx genes during embryonic hematopoiesis in the mouse.

Embryonic stem (ES) cells serve as a potentially unlimited source of cells and tissues to treat a number of genetic and malignant diseases. The differentiation of these cells into specific cell types is an area of very active investigation. One method of manipulating ES cell differentiation is through the alteration of gene expression. There are a multitude of different methods for expressing a target gene in ES cells, but most are limited in their ability to provide spatial, temporal, and quantitative control of gene expression. These properties are important because many developmentally interesting genes are regulated in at least one of these ways. This chapter will address these limitations through the use of an ES cell line with a doxycycline-inducible transgene system. A characterization of this inducible transgene system will be discussed, as well as the use of this system to develop ES-derived long-term engrafting hematopoietic stem cells. This demonstration is one of many possible uses for this powerful and versatile system.

Though a topic of medical interest for centuries, our understanding of vertebrate hematopoietic or "blood-forming" tissue development has improved greatly only in recent years and given a series of scientific and technical milestones. Key among these observations was the description of procedures that allowed the transplantation of blood-forming activity. Beyond this, other advances include the creation of a variety of knock-out animals (mice and more recently zebrafish), microdissection of embryonic and fetal blood-forming tissues, hematopoietic stem (HSC) and progenitor cell (HPC) colony-forming assays, the discovery of cytokines with defined hematopoietic activities, gene transfer technologies, and the description of lineage-specific surface antigens for the identification and purification of pluripotent and differentiated blood cells. The availability of both murine and human embryonic stem cells (ESC) and the delineation of in vitro systems to direct their differentiation have now been added to this analytical arsenal. Such tools have allowed researchers to interrogate the complex developmental processes behind both primitive (yolk sac or extraembryonic) and definitive (intraembryonic) hematopoietic tissue formation. Using ES cells, we hope to not only gain additional basic insights into hematopoietic development but also to develop platforms for therapeutic use in patients suffering from hematological disease. In this review, we will focus on points of convergence and divergence between murine and human hematopoiesis in vivo and in vitro, and use these observations to evaluate the literature regarding attempts to create hematopoietic tissue from embryonic stem cells, the pitfalls encountered therein, and what challenges remain.

Cellular therapies derived from embryonic stem (ES) cells have acquired new interest and urgency with the demonstration that embryonic stem cells can be established from human blastocyst-stage embryos. Our ability to derive therapeutic cells from differentiating ES cell cultures will ultimately depend on our understanding of the embryonic developmental processes that direct the differentiation of pluripotent cells into transplantable lineage-specific stem cells, and on our ability to recapitulate these processes in vitro. In this review, we evaluate the work that has been done to date on the hematopoietic differentiation of ES cells, and discuss this in the context of what is known about the embryonic origin of the hematopoietic stem cell.

Differentiation of embryonic stem (ES) cells in vitro yields abundant hematopoietic progenitors, but achieving stable hematopoietic engraftment of irradiated mice has proven difficult, begging the question of whether ES cells give rise to hematopoietic stem cells in vitro, and limiting the application of ES cells as experimental and therapeutic models. We have employed a number of hematopoietic regulatory genes to probe the nature and developmental potential of ES-derived blood precursors. The chronic myeloid leukemia-associated BCR/ABL oncoprotein transforms a novel class of ES-derived embryonic hematopoietic stem cell that represents a common progenitor of primitive erythropoiesis and definitive lymphoid-myeloid blood development. Expression of the homeobox gene HoxB4 generated normal, non-leukemic hematopoietic progenitors that enabled long-term, multilineage hematopoietic engraftment in primary and secondary mouse recipients. We have used these repopulating hematopoietic stem cells to model therapeutic transplantation from ES cells. We treated an immunodeficient Rag2(-/-) mouse by therapeutic cloning, that is, isogenic ES cell generation by somatic cell nuclear transfer, gene correction, and cell replacement therapy. Comparable approaches with human ES cells are being developed to lay the foundation for cellular therapies in patients with a variety of bone marrow diseases.

The extent to which primitive embryonic blood progenitors contribute to definitive lymphoid-myeloid hematopoiesis in the adult remains uncertain. In an effort to characterize factors that distinguish the definitive adult hematopoietic stem cell (HSC) and primitive progenitors derived from yolk sac or embryonic stem (ES) cells, we examined the effect of ectopic expression of HoxB4, a homeotic selector gene implicated in self-renewal of definitive HSCs. Expression of HoxB4 in primitive progenitors combined with culture on hematopoietic stroma induces a switch to the definitive HSC phenotype. These progenitors engraft lethally irradiated adults and contribute to long-term, multilineage hematopoiesis in primary and secondary recipients. Our results suggest that primitive HSCs are poised to become definitive HSCs and that this transition can be promoted by HoxB4 expression. This strategy for blood engraftment enables modeling of hematopoietic transplantation from ES cells.

Control of intracellular bacterial infections requires interferon-gamma (IFN-gamma) both for establishing a Th1 T-cell response and for activating macrophages to kill the bacteria. Exposure of mice deficient in IFN-gamma to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death. We report here that IFN-gamma-deficient mice infected with mycobacteria also undergo a dramatic remodeling of the hematopoietic system. Myeloid cell proliferation proceeds unchecked throughout the course of mycobacterial infection, resulting in a transition to extramedullary hematopoiesis. The splenic architecture of infected IFN-gamma-deficient mice is completely effaced by expansion of macrophages, granulocytes, and extramedullary hematopoietic tissue. These features coincide with splenomegaly, an increase in splenic myeloid colony-forming activity, and marked granulocytosis in the peripheral blood. Systemic levels of cytokines are elevated, particularly interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). These results suggest that in addition to its central role in cellular immunity, IFN-gamma may be a key cytokine in coordinate regulation of immune effector cells and myelopoiesis. This model should be valuable for deciphering the cross-talk between the immune response and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections.