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Our previous studies demonstrated the capacity of a stroma layer consisting of AGM-derived myrAKT-transduced endothelial cells (AGM-EC) to support the in vitro conversion of hemogenic endothelium (HE) to hematopoietic stem cells (HSCs) that functionally reconstitute long-term multilineage hematopoiesis in adult recipients following transplantation. Here we present our protocol for an engineered stromal-free niche developed using complementary single cell transcriptomic analyses of HE/HSC and the AGM-EC niche to define ligand-receptor interactions sufficient to support HSC formation from embryonic hemogenic precursors in vitro. This protocol will be useful to identify and characterize additional niche-derived signaling ligands/pathways that may further enhance HSC generation from HE in vitro in a well-defined platform, and provides a basis for further translational efforts toward HSC production in vitro such as from embryonic or pluripotent stem cells.

Fusion oncoproteins are the initiating event in AML pathogenesis, although they are thought to require additional cooperating mutations for leukemic transformation. CBFA2T3-GLIS2 (C/G) fusion occurs exclusively in infants and is associated with highly aggressive disease1-4. Here we report that lentiviral transduction of C/G fusion is sufficient to induce malignant transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) that fully recapitulates C/G AML. Engineered CB HSPCs co-cultured with endothelial cells undergo complete malignant transformation with identical molecular, morphologic, phenotypic and disease characteristics observed in primary C/G AML. Interrogating the transcriptome of engineered cells identified a library of C/G fusion-specific targets that are candidates for chimeric antigen receptor (CAR) T cell therapy. We developed CAR-T cells directed against one of the targets, FOLR1, and demonstrated the pre-clinical efficacy against C/G AML while sparing normal hematopoiesis. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Moreover, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

The human fetal liver is a critical organ for prenatal hematopoiesis, the study of which offers insights into niche signals that regulate the fates of hematopoietic stem and progenitor cells (HSPCs) during fetal development. Here, we demonstrate that human fetal liver endothelium uniquely supports the maturation and expansion of multilineage HSPCs. Specifically, co-culture of fetal liver-derived immature CD43+CD45− hematopoietic cells with human fetal liver endothelial cells (ECs) led to a profound increase in the numbers of phenotypic CD45+CD34+ HSPCs and multilineage colony-forming progenitors generated in vitro, when compared to co-culture with ECs derived from other organs. We further identified a supportive role for EC-derived WNT5A in this process via gain- and loss-of-function studies within ECs. Our study emphasizes the importance of the organ-specific endothelial niche in supporting hematopoietic development and provides novel insight into signals that may facilitate HSPC expansion in vitro for clinical applications.

Summary Precursors of hematopoietic stem cells (pre-HSCs) have been identified as intermediate precursors during the maturation process from hemogenic endothelial cells to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic day 10.5. Although pre-HSCs acquire an efficient adult-repopulating ability after ex vivo co-culture, their native hematopoietic capacity remains unknown. Here, we employed direct transplantation assays of CD45−VE-cadherin(VC)+KIT+(V+K+) cells (containing pre-HSCs) into immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that freshly isolated V+K+ cells exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multilineage repopulating capacity. Additionally, B cell colony-forming assays demonstrated the predominant B-1 progenitor colony-forming ability of these cells; however, increased B-2 progenitor colony-forming ability emerged after co-culture with Akt-expressing AGM endothelial cells, conditions that support pre-HSC maturation into HSCs. Our studies revealed an unexpected B-1 lymphocyte bias of the V+K+ population and acquisition of B-2 potential during commitment to the HSC fate., Graphical Abstract, Highlights • Majority of E10.5 EPCR+VC+KIT+ cells are B-1- (not B-2)-biased repopulating cells • E10.5 CD45−VC+KIT+ cells contain the first B-1a repopulating cells • E10.5 CD45−VC+KIT+ cells acquire B-2 potential after ex vivo co-culture, Yoshimoto and colleagues demonstrate an unexpected inherent B-1-biased repopulation ability of E10.5 pre-HSCs in immunodeficient neonatal mice. The B-1-biased pre-HSCs acquire B-2 potential following ex vivo co-culture, reflecting the developmental process of hemogenic endothelial cells to HSCs through B-1-biased state in vivo. Notably, the E10.5 pre-HSCs are the first B-1a repopulating cells upon direct transplantation without co-culture.

SUMMARYDuring embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSC). Although previous studies have shown that yolk sac-derived erythromyeloid progenitors and HSC emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSC originate from common HE. Here we demonstrate by clonal assays and single cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSC. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSC arise independently from HE with distinct phenotypic and transcriptional properties.

Haematopoietic stem cells (HSCs) develop from haemogenic endothelium (HE) within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, we used single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from HE to HSC, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering revealed dynamics of gene expression during the HE to HSC transition, identifying surface receptors specifically expressed on developing HSCs. Transcriptional profiles of niche endothelial cells enabled identification of corresponding ligands, including those signaling to Notch receptors, VLA-4 integrin, and CXCR4, which, when integrated in an engineered platform, were sufficient to support the generation of engrafting HSCs. These studies provide a transcriptional map of the signaling interactions necessary for the development of HSCs and advance the goal of engineering HSC for therapeutic applications.

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSC). Although previous studies have shown that yolk sac-derived erythromyeloid progenitors and HSC emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSC originate from common HE. Here we demonstrate by clonal assays and single cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSC. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSC arise independently from HE with distinct phenotypic and transcriptional properties.

During embryonic development, sequential waves of hematopoiesis give rise to blood-forming cells with diverse lineage potentials and self-renewal properties. This process must accomplish two important yet divergent goals: the rapid generation of differentiated blood cells to meet the needs of the developing embryo and the production of a reservoir of hematopoietic stem cells to provide for life-long hematopoiesis in the adult. Vascular beds in distinct anatomical sites of extraembryonic tissues and the embryo proper provide the necessary conditions to support these divergent objectives, suggesting a critical role for specialized vascular niche cells in regulating disparate blood cell fates during development. In this review, we will examine the current understanding of how organ- and stage-specific vascular niche specialization contributes to the development of the hematopoietic system.

During murine embryonic development, the first hematopoietic stem cells (HSCs) emerge within the major arterial vasculature, including the aorta-gonad-mesonephros (AGM) region. Throughout their emergence and subsequent maturation, HSCs retain a close physical association with the surrounding endothelial cell layer, suggesting that signaling interactions between HSC and the surrounding vascular niche may play an integral role in HSC development. Indeed, we have previously shown that co-culture with AGM-derived endothelial cells (AGM EC) engineered to constitutively express Akt (AGM Akt-EC) is sufficient to mature non-engrafting HSC precursors from hemogenic endothelium to fully functional HSCs1-3. Here, we describe how to generate these AGM Akt-EC cells for use in co-culture experiments, providing detailed instructions from the isolation of AGM EC from embryonic tissues, to their infection with the PGK.myr-AKT lentivirus and subsequent characterization by flow cytometry.

Engineered spiral microvessels reveal unique contributions of curvature to EC responses to flow and support tissue perfusion., Vascularization remains a long-standing challenge in engineering complex tissues. Particularly needed is recapitulating 3D vascular features, including continuous geometries with defined diameter, curvature, and torsion. Here, we developed a spiral microvessel model that allows precise control of curvature and torsion and supports homogeneous tissue perfusion at the centimeter scale. Using this system, we showed proof-of-principle modeling of tumor progression and engineered cardiac tissue vascularization. We demonstrated that 3D curvature induced rotation and mixing under laminar flow, leading to unique phenotypic and transcriptional changes in endothelial cells (ECs). Bulk and single-cell RNA-seq identified specific EC gene clusters in spiral microvessels. These mark a proinflammatory phenotype associated with vascular development and remodeling, and a unique cell cluster expressing genes regulating vascular stability and development. Our results shed light on the role of heterogeneous vascular structures in differential development and pathogenesis and provide previously unavailable tools to potentially improve tissue vascularization and regeneration.

Tober et al. show that hematopoietic stem cells (HSCs) that mature from pre-HSCs in vivo in the fetal liver, versus ex vivo, are not molecularly equivalent. Although both express cell surface programmed death ligand 1 (PD-L1), it is not required for the engraftment of fetal HSCs into adult recipients., Hematopoietic stem cells (HSCs) mature from pre-HSCs that originate in the major arteries of the embryo. To identify HSCs from in vitro sources, it will be necessary to refine markers of HSCs matured ex vivo. We purified and compared the transcriptomes of pre-HSCs, HSCs matured ex vivo, and fetal liver HSCs. We found that HSC maturation in vivo or ex vivo is accompanied by the down-regulation of genes involved in embryonic development and vasculogenesis, and up-regulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that ex vivo–matured and fetal liver HSCs express programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on HSCs matured ex vivo. PD-L1 signaling is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for, HSC maturation.

Summary Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes., Highlights • Index sorting and stromal co-culture identifies clonal embryonic pre-HSCs • Clonal pre-HSCs have both B-1a and B-2 lymphocyte potential • Clonal pre-HSCs express distinctive levels of Delta-like-4, In this article, Hadland and colleagues use index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays to characterize pre-HSCs at the single-cell level, revealing stage-specific properties of pre-HSCs and a common origin for B-1a and B-2 lymphocytes at the emergence of HSC development.

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin+ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin+CD61+EPCR+ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro. Disclosures Bernstein: Lyell Immunopharma: Current equity holder in publicly-traded company, Other: Laboratory Support; Deverra Therapeutics: Current equity holder in publicly-traded company.

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSC), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. Prior studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSC originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent and multipotent progenitors originate from the same population of HE that gives rise to HSC. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We found that expression of arterial marker CXCR4 specifically enriches for functional HSC potential in hemogenic precursors in the para-aortic splanchnopleura (P-Sp), where the first HSC precursors are detected at around E9 in the murine embryo. In contrast, we detected clonal HE with lymphoid and multilineage hematopoietic potential at the same stage that are distinct from HSC precursors and largely lack CXCR4 expression. Single-cell RNA sequencing demonstrated that Cxcr4 is uniquely expressed in P-Sp-derived cells enriched for arterial-specific genes and genes essential for HSC maintenance and self-renewal, whereas expression of these genes is decreased in the majority of Runx1-expressing HE. Altogether, our studies suggest a model in which the earliest lymphoid and multipotent progenitors emerge from HE that is phenotypically and transcriptionally distinct from HSC precursors, and that co-expression of arterial endothelial and self-renewal factors uniquely defines HE with HSC competence.

Hematopoietic stem cell (HSC) transplantation is curative for malignant and genetic blood disorders, but is limited by donor availability and immune-mismatch. Deriving HSCs from patient-matched embryonic/induced-pluripotent stem cells (ESCs/iPSCs) could address these limitations. Prior efforts in murine models exploited ectopic HoxB4 expression to drive self-renewal and enable multi-lineage reconstitution, yet fell short in delivering robust lymphoid engraftment. Here, by titrating exposure of HoxB4-ESC-HSC to Notch ligands, we report derivation of engineered HSCs that self-renew, repopulate multi-lineage hematopoiesis in primary and secondary engrafted mice, and endow adaptive immunity in immune-deficient recipients. Single-cell analysis shows that following engraftment in the bone marrow niche, these engineered HSCs further specify to a hybrid cell type, in which distinct gene regulatory networks of hematopoietic stem/progenitors and differentiated hematopoietic lineages are co-expressed. Our work demonstrates engineering of fully functional HSCs via modulation of genetic programs that govern self-renewal and lineage priming.

We studied 232 consecutive children transplanted between 1990 and 2011 with relapse after 1(st) hematopoietic cell transplant (HCT). Kaplan-Meier survival and hazard ratios for mortality were calculated for factors known at time of relapse using Cox proportional hazards models. The median (range) age at time of 1(st) HCT was 10.9 (0.5–20.9) years, time to relapse was 6.1 (0.2–89.5) months after HCT, and age at relapse was 11.7 (0.7–23.6) yrs. The 3-year overall survival (OS) after relapse was 13% (95% Confidence Interval (CI): 9%, 18%).The median (range) follow-up for the 18 surviving patients was 7.2 (3.0–24.4) years after relapse. The remaining 214 died after a median of 3 months (0.02–190.4). OS was not significantly different for patients with ALL as compared to AML. Fifty-one patients proceeded to 2(nd) transplant of whom 9 survive. Factors associated with improved survival included late relapse (greater than 12 months), ALL in first CR at the time of first transplant and chemotherapy-based first conditioning regimens. These results can be used to counsel patients at the time of relapse after first transplant and as a baseline for comparison as to the effectiveness of newer therapies which are greatly needed for treatment of post-transplant relapse.

SUMMARY During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties., Graphical Abstract, In brief Integrating multiple single-cell approaches, Dignum et al. reveal immunophenotypic and transcriptional properties of hemogenic endothelium (HE) that are precursors to the first HSCs. They identify a population of HE with clonal multipotent hematopoietic potential distinct from HSC-competent HE and uncover arterial-associated transcriptional programs linked to HSC competence.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43−CD73−DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs., It is unclear whether arterial specification is required for hematopoietic stem cell formation. Here, the authors use a chemically defined human pluripotent stem cell (hPSC) differentiation system to show the role of NOTCH signaling in forming arterial-type hemogenic endothelial cells.

In adult hematopoiesis, the hematopoietic stem cell (HSC) sits at the top of a hierarchy of hematopoietic progenitors responsible for generating the diverse repertoire of blood and immune cells. During embryonic development, however, the initial waves of hematopoiesis provide the first functioning blood cells of the developing embryo, such as primitive erythrocytes arising in the yolk sac, independently of HSCs. In the field of developmental immunology, it has been recognized that some components of the immune system, such as B-1a lymphocytes, are uniquely produced during the embryonic and neonatal period, suggesting a "layered" development of immunity. Several recent studies have shed new light on the developmental origin of the layered immune system, suggesting complex and sometimes multiple contributions to unique populations of innate-like immune cells from both fetal HSCs and earlier HSC-independent progenitors. In this review, we will attempt to synthesize these studies to provide an integrated model of developmental hematopoiesis and layered immunity that may offer new insights into the origin of HSCs.

To the editor: Cell surface markers of adult hematopoietic stem cells (HSCs) are well established, but fewer markers are available for embryonic HSCs and their immediate precursors (pre-HSCs). HSCs differentiate from hemogenic endothelial cells in the major arteries of the embryo [dorsal aorta

Different concentrations of activin A and BMP4 are used to polarize stem cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into >90% CD31+/VE-cadherin+ definitive ECs. To test the functionality of ECs at different stages of differentiation, we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge, this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential.

During adulthood and embryogenesis, fate decisions of hematopoietic stem and progenitor cells (HSPCs), such as specification, self-renewal, and differentiation are tightly regulated by their neighboring niche cells. Moreover, distinct types of niches supply differential cues to direct alternative cell fates for HSPCs. Yet, currently the intrinsic mechanisms balancing HSPC response obliqueness to microenvironmental signals are unknown. Friend Leukemia integration-1 (Fli-1), is an ETS transcription factor expressed by vascular beds and hematopoietic lineages. Fli-1 belongs to the "heptad factors" which are hypothesized to specify and sustain a hematopoietic cell fate. While Fli-1 overexpression is linked to leukemia, the functional role Fli-1 plays in HSPC specification and maintenance remains undefined. We show that inducible deletion of Fli-1 using a Rosa-CreERT2 transgenic adult mice (Fli-1ROSAΔ), results in a rapid thrombocytopenia-associated mortality. Transplantation of Fli-1ROSAΔ bone marrow (BM) cells into WT recipients, to exclude vascular-mediated defects, followed by induction of Fli-1 deletion, resulted with the same phenotype. In a set of modulated competitive transplantation experiments (differential induction time points pre- or post-transplant), we observed defective ability of Fli-1ROSAΔ HSPCs to lodge, engraft, and to sustain hematopoiesis post repopulation. Fli-1 deficient HSPCs exhibited reduced quiescent cell cycling status, a hallmark of stemness, and displayed enhanced apoptosis. Thus, Fli-1 is essential for previously unrecognized cell-autonomous HSPC functions. To determine whether Fli-1 modulates HSPC specification, Fli-1 was conditionally deleted using a developmental VE-cadherin (CDH5)-Cre transgenic model (Fli-1CDH5Δ). This resulted with premature mortality of Fli-1CDH5Δ embryos, accompanied with a hemorrhagic phenotype. Reduced numbers of hematopoietic cells were still detected in the AGM of e10.5 Fli-1CDH5Δ embryos. Conditional Fli-1 deletion using a developmental hematopoietic Vav-1 Cre transgenic model (Fli-1Vav-1Δ) resulted again with premature mortality. Reduced presence of embryonic Fli-1Vav-1Δ liver HSPCs was observed at e12.5. We also applied two in vitro co-culture systems, to study Fli-1 in endothelial to hematopoietic transition (EHT). First, isolated hemogenic endothelial cells (HEC) from WT and Fli-1ROSAΔ embryos were co-cultured with AGM-derived vascular niche. HECs isolated from Fli-1ROSAΔ AGM were still able to convert to CD45+ cells, however these cells did not expand on a vascular niche. Secondly, we have applied an endothelial to hematopoietic reprogramming system in which isolated lung ECs are virally introduced with DOX inducible FosB, Gfi1, Runx1, and Spi1 (FGRS) factors and co-cultured with vascular niche cells. Both WT and Fli-1ROSAΔ ECs were able to acquire a hemogenic like state resulting with a final capacity to convert into hematopoietic cells. Again, Fli-1ROSAΔ cells displayed lesser numbers of CD45+ cells at the end point, presumably due to impaired interaction with the vascular niche. Indeed, reduced expansion capacity was observed both for mature CD45+ and for HSPC derived from Fli-1CDH5Δ AGM region. Adult Fli-1ROSAΔ HSPCs exhibited the same niche-dependent expansion defect. Induction of Fli-1 deletion in vitro in adult HSPCs revealed loss of dependency on vascular niche inductive signals, as no additive expansion effect was observed for Fli-1ROSAΔ HSPCs in the presence of a vascular niche. Hence, Fli-1 is essential for HSPC expansion rather than hematopoietic specification. Differential RNA-seq analysis combined with epigenetic studies of expanding WT and Fli-1ROSAΔ HSPCs, revealed dysregulation of Fli-1-controlled pathways involved in transduction of microenvironmental signals for self-renewal. Unexpectedly, H3K27Ac analysis, a marker for transcriptional priming, revealed increased global acetylation of Fli-1ROSAΔ HSPCs' chromatin. Therefor, Fli-1 may not only perform as transcription activator, but foremostly as a genomic suppressor via modulation of histone acetylation status. Decrypting the mechanism(s) by which Fli-1 orchestrates HSPC self-renewal, may promote an improved expansion protocol of human HSPC pre-transplantation, and provide additional insights for microenvironmental sensing by Fli-1-dependent leukemic cells. Disclosures No relevant conflicts of interest to declare.

During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for pediatric patients with hematologic malignancies. Historically, matched sibling donors (MSD) have been the preferred donor source given ease of availability and lower rates of graft-versus-host-disease (GVHD). However, only 30% of patients have a MSD and relapse rates are high after MSD HSCT, raising the question of best donor choice. As conditioning regimens evolve, GVHD management improves and supportive care advances, it is important to evaluate the role of donor source on short and long-term clinical outcomes to inform donor selection. We performed a single-center retrospective analysis comparing post-HSCT outcomes in a cohort of pediatric patients undergoing MSD, matched unrelated donor (MUD), and umbilical cord blood (CB) transplants from 2006 to 2018. Methods: A retrospective analysis was performed on an IRB-approved protocol through Fred Hutchinson Cancer Research Center. 232 patients were included who received MSD (n=56), MUD (n=89) or CB (n=87) transplants. Of note, 24 CB patients received expanded CB cells in addition to unmanipulated unit(s). GVHD prophylaxis in all patients consisted of a calcineurin inhibitor and MMF or methotrexate. The vast majority received a high-intensity conditioning regimen (86%, 96%, and 82% respectively for MSD, MUD and CB). Overall survival (OS) and disease-free survival (DFS) were evaluated using the Kaplan-Meier method. Probabilities of non-relapse mortality (NRM), relapse, and acute GVHD were evaluated using cumulative incidence (CI) estimates with appropriate competing risks. The Cox regression model was used for adjusted analysis for age, year of transplant, sex, CMV status, MRD status, disease risk, and conditioning regimen. Results: Patient/treatment/donor demographics are shown in Table 1. Median follow-up was 2.6, 3.7 and 3.1 years for MSD, MUD and CB respectively. Patient diagnosis, disease risk, gender, age, and CMV serology were balanced between groups. CI of engraftment was similar as well, with only one graft failure in the MUD group (Fig 1). Median time to platelet recovery was significantly faster in MUD and MSD groups as compared to the CB group (p Conclusions: Our data demonstrate no difference in unadjusted OS between MSD, MUD and CB recipients. Importantly, despite this equivalence, 5-year DFS was significantly better in the CB v. MSD group, reflecting the lower relapse rate observed in CB patients and seen previously by us and others. CB continues to be viewed as an "alternative" donor for HSCT due to the low stem cell dose in a CB graft resulting in delayed neutrophil recovery, primary graft failure and increased NRM. However, this was not observed herein, supporting the use of CB for pediatric HSCT perhaps especially in patients at high risk of post-transplant relapse. Disclosures Milano: ExCellThera: Research Funding; Amgen: Research Funding. Delaney:Nohla Therapeutics: Employment, Equity Ownership; Biolife Solutions: Membership on an entity's Board of Directors or advisory committees. Bleakley:HighPass Biotherapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34 + CD45 + myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34 + CD38 − cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.

Background: AML is an aggressive hematologic malignancy that remains difficult to treat. A common mutation found in AML is FLT3-ITD, occurring in 15% of childhood AML. Although chemotherapy has successfully induced remission, patients with a high FLT3 ITD:WT allelic ratio (FLT3-AR) exhibit a high relapse rate, requiring hematopoietic stem cell transplantation to increase the chance of long-term remission. In this study, we demonstrate the requirement of ECs for survival of FLT3-ITD progenitors from primary pediatric AML specimens in the presence of AC220, a potent and selective inhibitor of FLT3. We further show that the Notch pathway plays a role in EC-mediated protection amongst patient samples with high FLT3-AR, suggesting the potential therapeutic use of Notch blockade in the treatment of this high-risk subset. Results: To determine whether ECs confer protection to FLT3-ITD progenitors, we quantified the number of CFC present after 2 weeks of liquid culture or EC co-culture with AC220 (added at days 0, 3 and 7) from four AML specimens with high FLT3-AR (≥1). We used PCR to determine the presence of FLT3-ITD in individual CFC. We found that the numbers of FLT3-ITD CFC (p=0.007) and FLT3-WT CFC (p=0.044) were reduced in liquid culture compared to EC co-culture, suggesting that ECs mediate the survival of FLT3-ITD hematopoietic progenitors against the therapeutic treatment of AC220. Previously, we demonstrated that ECs are critical for the growth and expansion of hematopoietic stem cells, which is dependent on the activation of Notch signaling. We asked whether Notch plays a role in EC-mediated protection of AML progenitors against AC220, using RNA-seq analysis on three FLT3-ITD-harboring AML. Among the significantly altered genes (FDR Next, we assessed the effect of inhibiting Notch signaling on AML progenitor survival during AC220 treatment in EC co-culture, using inhibitory antibodies specific to the Negative Regulatory Region (NRR) of both Notch1 and Notch2 receptors (anti-NRR1 and anti-NRR2; kindly provided by Chris Siebel, Genentech). We co-cultured bone marrow cells from eight patient specimens with low FLT3-AR ( Conclusion: Our studies suggest that inhibiting Notch signaling may have therapeutic potential for overcoming drug resistance induced by the tumor microenvironment in a subset of AML with high FLT3-AR. We have previously shown that a high FLT3-AR is associated with the presence of FLT3-ITD in the least mature hematopoietic subset (CD34+ CD33- precursors), which is thought to contain leukemic stem cells, and this association is correlated with poorer outcome. Additionally, AML cells that give rise to CFC after long-term co-culture with bone marrow stroma or ECs are derived from the CD34+CD33- AML precursors. Ongoing studies aim to determine whether Notch signaling plays a role in the survival of AML CD34+CD33- cells with the goal of eliminating leukemic stem cells responsible for relapse. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSC) are generated during a transient window of embryonic development from endothelial-like hemogenic precursors within specific arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros region). During HSC emergence, hemogenic precursors must acquire and maintain HSC-defining properties such as the ability to self-renew, home, and provide multilineage hematopoiesis, properties which distinguish rare HSC from a multitude of other hematopoietic progenitors arising simultaneously in the developing embryo. However, the precise niche-derived signals necessary and sufficient to support the acquisition and maintenance of these properties remains poorly defined. Toward identification of these signals, we generated a platform consisting of endothelial cells from the embryonic AGM (AGM-EC) which supports the specification and self-renewal of engrafting HSC from clonal embryo-derived hemogenic precursors in vitro. Using this platform to assay functional HSC potential at the single cell level, we determined a phenotype (VE-Caderin+CD61+EPCRhigh) that encompasses the population of hemogenic precursors during their asynchronous transition to HSC between E9.5 and E11.5 in murine embryonic development. To elucidate the transcriptional changes associated with the emergence of HSC from hemogenic precursors, we analyzed the global transcriptional profiles of FACS-purified VE-Caderin+CD61+EPCRhigh cells at various stages of embryonic development by single cell RNA-sequencing and reconstructed their developmental trajectory in "pseudotime" based on incremental changes in their transcriptional profiles. Complementary analysis of AGM-EC by bulk and single cell RNA-sequencing revealed a unique transcriptional profile of niche endothelial cells supporting HSC development enriched for immune/inflammatory signals. Combining the transcriptional profiles of emerging HSC with niche AGM-EC, we have identified candidate ligand-receptor pairs regulating intercellular interactions during HSC specification and self-renewal and have begun to validate the functional importance of these interactions in supporting HSC generation from hemogenic precursors in vitro. We expect these studies will enhance our understanding of the unique signal pathways necessary for the development of functional HSC, a critical step toward engineering HSC in vitro for clinical applications in disease modeling, drug discovery, and gene modification to identify novel therapies for hematologic and immunologic disorders. Disclosures Rafii: Angiocrine Bioscience: Equity Ownership.

The peritoneal B-1 cells are innate-like B cells, secreting natural antibodies without T cell help. Cell transfer experiments suggest that CD5+ B-1a cells are of fetal origin because adult bone marrow stem/progenitors do not repopulate B-1a cells efficiently, while fetal liver cells do. We have previously reported the B-1 cell potential of extra-embryonic yolk sac (YS) and para-aortic region of E9.5 embryos appearing before the emergence of hematopoietic stem cells (HSCs), and that B-1 progenitors develop in the absence of HSCs. These data imply that at least a part of B-1a cells are HSC independent and derived from early YS/embryos. In addition, it has been reported that fetal liver HSCs failed to repopulate B-1a cells after transplantation. This raises a question whether HSCs at any stage of the embryo or post-natal life produce B-1a cells. Our recent report has demonstrated that E11.5 HSC precursors repopulated both B-1a and B-2 cells at a clonal level after co-culturing with Akt-overexpressing endothelial cells. In order to test whether the first HSC repopulates B-1a cells, we directly transplanted Ter119− VE-cadherin+ c-kit+ “HSC precursor cells” from E10.5 embryos into sublethally irradiated immune-deficient neonatal mice. Our precise analysis of transplantation assays revealed that “HSC precursor cells” contained both neonatal HSCs that repopulated multi-lineage blood cells and B-1 specific precursors that repopulated only peritoneal B-1a, B-1b, and splenic marginal zone B cells. Surprisingly, the frequency of B-1 precursors is more than that of neonatal HSCs. Thus, the earliest transplantable B-1 specific precursor cells are present in E10.5 embryos showing the same surface markers with cells display HSC activity.

Erythropoietin-stimulating agents (ESAs) were originally designed to replace endogenous erythropoietin in patients with anemia secondary to renal failure. Their use has subsequently been expanded to include patients with anemia of other causes, including cancer patients, in whom deficiency of erythropoietin, per se, is not the primary cause of anemia. Although early studies showed promise of ESA administration in reducing the need for transfusions and improving the quality of life in cancer patients, several large randomized clinical trials have recently shown a potential detrimental effect of ESA administration on tumor progression and survival in these patients. These studies have called into question the safety of ESAs as supportive therapy in patients being treated for oncologic conditions. However, numerous questions remain to be addressed regarding the design of these studies, the effect of various targeted hemoglobin levels, and the potential biologic mechanisms proposed to explain promotion of tumor progression and reduced survival.

The four highly conserved Notch receptors receive short-range signals that control many biological processes during development and in adult vertebrate tissues. The involvement of Notch1 signaling in tissue self-renewal is less clear, however. We developed a novel genetic approach N1IP-CRE(Notch1 Intramembrane Proteolysis) to follow, at high resolution, the descendents of cells experiencing Notch1 activation in the mouse. By combining N1IP-CRE with loss-of-function analysis, Notch activation patterns were correlated with function during development, self-renewal and malignancy in selected tissues. Identification of many known functions of Notch1 throughout development validated the utility of this approach. Importantly,novel roles for Notch1 signaling were identified in heart, vasculature, retina and in the stem cell compartments of self-renewing epithelia. We find that the probability of Notch1 activation in different tissues does not always indicate a requirement for this receptor and that gradients of Notch1 activation are evident within one organ. These findings highlight an underappreciated layer of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a general strategy applicable for monitoring proteolysis-dependent signaling in vivo.

Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-cadherin–expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-cadherin+CD45+ AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications.

During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/β-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior axis of the embryo led to differential activity of Wnt/β-catenin signaling and specified distinct anterior-like (high activin/low BMP) and posterior-like (low activin/high BMP) mesodermal populations. Cardiogenic mesoderm was generated under conditions specifying anterior-like mesoderm, whereas blood-forming endothelium was generated from posterior-like mesoderm, and vessel-forming CD31+ endothelial cells were generated from all mesoderm origins. Surprisingly, inhibition of β-catenin signaling led to the highly efficient respecification of anterior-like endothelium into beating cardiomyocytes. Cardiac respecification was not observed in posterior-derived endothelial cells. Thus, activin/BMP gradients specify distinct mesodermal subpopulations that generate cell derivatives with unique angiogenic, hemogenic and cardiogenic properties that should be useful for understanding embryogenesis and developing therapeutics., Summary: The manipulation of signals that control embryonic patterning allows human pluripotent stem cells to be differentiated into endothelial subpopulations with distinct haematopoietic, angiogenic and cardiogenic potential.