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Purpose of Review Hematopoietic stem cells (HSCs) sit at the top of the hierarchy that meets the daily burden of blood production. HSC maintenance relies on extrinsic cues from the bone marrow (BM) microenvironment to balance stem cell self-renewal and cell fate decisions. In this brief review, we will highlight the studies and model systems that define the centralized role of BM vascular endothelium in modulating HSC activity in health and stress. Recent Findings The BM microenvironment is composed of a diverse array of intimately associated vascular and perivascular cell types. Recent dynamic imaging studies, coupled with single-cell RNA sequencing (scRNA-seq) and functional readouts, have advanced our understanding of the HSC-supportive cell types and their cooperative mechanisms that govern stem cell fate during homeostasis, regeneration, and aging. These findings have established complex and discrete vascular microenvironments within the BM that express overlapping and unique paracrine signals that modulate HSC fate. Summary Understanding the spatial and reciprocal HSC-niche interactions and the molecular mechanisms that govern HSC activity in the BM vascular microenvironment will be integral in developing therapies aimed at ameliorating hematological disease and supporting healthy hematopoietic output.

The molecular complexity of the bone marrow (BM) microenvironment and its response to stress are incompletely understood, despite its key role in the regulation of hematopoiesis. Here we map the transcriptional landscape of BM vascular, perivascular, and osteoblast niche populations at single-cell resolution at both homeostasis and under stress hematopoiesis. This analysis revealed a previously unappreciated level of cellular heterogeneity within the BM niche, identified novel cellular subsets, and resolved cellular sources of pro-hematopoietic growth factors, chemokines, and membrane-bound ligands. Under conditions of stress, our studies revealed a significant transcriptional remodeling of these niche elements, including an adipocytic skewing of the perivascular cells. Among the stress-induced changes, we observed that vascular Notch ligand delta-like ligands (Dll1,4) were downregulated. In the absence of vascular Dll4, hematopoietic stem cells (HSC) prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the BM niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress, and illustrate the utility of single cell transcriptomic data in systematically evaluating the regulation of hematopoiesis by discrete niche populations.

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.

Abnormally low level of interstitial oxygen, or hypoxia, is a hallmark of tumor microenvironment and a known promoter of cancer chemoresistance. Inside a solid tumor mass, the hypoxia stems largely from inadequate supply of oxygenated blood through sparse or misshapen tumor vasculature whilst oxygen utilization rates are low in typical tumor's glycolytic metabolism. In acute leukemias, however, markers of intracellular hypoxia such as increased pimonidazole adduct staining and HIF-1α stabilization are observed in advanced leukemic bone marrows (BM) despite an increase in BM vasculogenesis. We utilized intravital fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) in a BCR-ABL B-ALL mouse model to image the extracellular oxygen concentrations (pO2) in leukemic BM, and we related the extracellular oxygen levels to intracellular hypoxia, vascular markers and local leukemia burden. We observed a transient increase in BM pO2 in initial disease stages with intermediate leukemia BM burden, which correlated with an expansion of blood-carrying vascular network in the BM. Yet, we also observed increased formation of intracellular pimonidazole adducts in leukemic BM at the same time. This intermediate stage was followed by a significant decrease of extracellular pO2 and further increase of intracellular hypoxia as leukemia cellularity overwhelmed BM in disease end-stage. Remarkably, treatment of leukemic mice with IACS-010759, a pharmacological inhibitor of mitochondrial Complex I, substantially increased pO2 in the BM with advanced B-ALL, and it alleviated intracellular hypoxia reported by pimonidazole staining. High rates of oxygen consumption by B-ALL cells were confirmed by Seahorse assay including in ex vivo cells. Our results suggest that B-ALL expansion in BM is associated with intense oxidative phosphorylation (OxPhos) leading to the onset of metabolic BM hypoxia despite increased BM vascularization. Targeting mitochondrial respiration may be a novel approach to counteract BM hypoxia in B-ALL and, possibly, tumor hypoxia in other OxPhos-reliant malignancies.

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation., Myelosuppressive injuries lead to chronic MAPK activation and impair blood reconstitution. Here, the authors show that chronic activation endothelial MAPK impairs hematopoietic stem cell (HSC) function through NFkB signaling, and that post-myelosuppressive HSC defects can be reversed by administration of Stem Cell Growth Factor SCGFa.

Purpose of review In this review, we aim to discuss the role of the bone marrow microenvironment in supporting hematopoiesis, with particular focus on the contribution of the endothelial niche in dictating hematopoietic stem cell (HSC) fate. Recent findings Evidence gathered in the past two decades revealed that specific cell types within the bone marrow niche influence the hematopoietic system. Endothelial cells have emerged as a key component of the HSC niche, directly affecting stem cell quiescence, self-renewal, and lineage differentiation. Physiological alterations of the bone marrow niche occurring in aging have been described to be sufficient to promote functional aging of young HSCs. Furthermore, a growing body of evidence suggests that aberrant activation of endothelial-derived signaling pathways can aid or trigger neoplastic transformation. Summary Several groups have contributed to the characterization of the different cell types that comprise the complex bone marrow environment, whose function was long perceived as an undiscernible sum of many parts. Further studies will need to uncover niche cell-type-specific pathways, in order to provide new targets and therapeutic options that aim at withdrawing the microenvironmental support to malignant cells while sparing normal HSCs.

Interest in human brown fat as a novel therapeutic target to tackle the growing obesity and diabetes epidemic has increased dramatically in recent years. While much insight into brown fat biology has been gained from murine cell lines and models, few resources are available to study human brown fat in vitro, which makes the need for new ways to derive and study human brown adipocytes imperative. Human ES cell based reporter systems present an excellent tool to identify, mark, and purify cell populations of choice. In this study, we detail the derivation and characterization of a novel human ES UCP1 reporter cell line that marks UCP1 positive adipocytes in vitro. We targeted a mCherry reporter to the UCP1 stop codon via CRISPR-Cas9 based gene targeting. The brown adipocytes derived from reporter cells express UCP1, display high mitochondrial content, multi-locular lipid morphology, and exhibit functional properties such as lipolysis. The mCherry positive cells purified after cell sorting show elevated expression of brown fat marker genes and a high similarity to isolated human brown fat via RNA-seq analysis. Finally, we demonstrate the utility of this reporter to real time monitor UCP1 expression upon stimulation. This reporter cell line thus presents new opportunities to study human brown fat biology by enabling future work to understand early human brown fat development, perform disease modeling, and facilitate drug screening.

Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.

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.

The generation of hematopoietic stem cells (HSCs) from pluripotent stem cell (PSC) sources is a long-standing goal that will require a comprehensive understanding of the molecular and cellular factors that determine HSC fate during embryogenesis. A precise interplay between niche components, such as the vascular, mesenchymal, primitive myeloid cells, and the nervous system provides the unique signaling milieu for the emergence of functional HSCs in the aorta-gonad-mesonephros (AGM) region. Over the last several years, the interrogation of these aspects in the embryo model and in the PSC differentiation system has provided valuable knowledge that will continue educating the design of more efficient protocols to enable the differentiation of PSCs intobona fide, functionally transplantable HSCs. Herein, we provide a synopsis of early hematopoietic development, with particular focus on the recent discoveries and remaining questions concerning AGM hematopoiesis. Moreover, we acknowledge the recent advances towards the generation of HSCsin vitroand discuss possible approaches to achieve this goal in light of the current knowledge.

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow. Stress signals from cancer and other conditions promote HSPC mobilization into circulation and subsequent homing to tissue microenvironments. HSPC infiltration into tissue microenvironments can influence disease progression; notably, in cancer, HSPCs encourage tumor growth. Here we have uncovered a mutually exclusive distribution of EPHB4 receptors in bone marrow sinusoids and ephrin B2 ligands in hematopoietic cells. We determined that signaling interactions between EPHB4 and ephrin B2 control HSPC mobilization from the bone marrow. In mice, blockade of the EPHB4/ephrin B2 signaling pathway reduced mobilization of HSPCs and other myeloid cells to the circulation. EPHB4/ephrin B2 blockade also reduced HSPC infiltration into tumors as well as tumor progression in murine models of melanoma and mammary cancer. These results identify EPHB4/ephrin B2 signaling as critical to HSPC mobilization from bone marrow and provide a potential strategy for reducing cancer progression by targeting the bone marrow.

Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Chronic inflammation within the bone marrow (BM) microenvironment has been proposed as a key driver of aging-associated hematopoietic defects including loss of hematopoietic stem cell (HSC) self-renewal, myeloid-biased HSC differentiation, and a predisposition towards development of myeloid neoplasms. In support of this idea, we recently discovered that constitutive activation of the NFKB signaling pathway within endothelial cells (ECs) of young mice leads to an inflammatory stress within the BM, resulting in loss of HSC self-renewal and a myeloid-biased output at the expense of lymphopoiesis. Since chronic vascular inflammation has emerged as a key component of the systemic aging process, we hypothesized that suppression of NFKB dependent vascular inflammation during physiological aging could temporize BM inflammation and preserve HSC health. To test this hypothesis, we utilized the Tie2.IkBss mouse model wherein the canonical NFKB signaling pathway is suppressed specifically within ECs of adult mice, and physiologically aged these mice to assess their hematopoietic function. Our findings demonstrate that aged Tie2.IkBss mice displayed a significant decrease in their peripheral blood myeloid biased output as compared to their aged littermate controls. HSC transplantation assays revealed that HSCs derived from aged Tie2.IkBss mice demonstrated a significant decrease in their myeloid-biased differentiation and displayed enhanced lymphopoiesis. Furthermore, serial transplantation revealed that HSCs from aged Tie2.IkBss mice displayed balanced multi-lineage reconstitution ability during both primary as well as secondary transplantation assays. Collectively, these results suggest that inflammatory cytokines derived from the aged vascular niche play a crucial role in promoting aging-associated myeloid bias of HSCs, and strategies directed towards suppression of chromic vascular inflammation will provide novel therapeutic interventions towards prevention of aging-associated myeloid malignancies and improving overall healthspan.

Summary Hematopoietic stem cells (HSCs) inhabit distinct microenvironments within the adult bone marrow (BM), which govern the delicate balance between HSC quiescence, self-renewal, and differentiation. Previous reports have proposed that HSCs localize to the vascular niche, comprised of endothelium and tightly associated perivascular cells. Herein, we examine the capacity of BM endothelial cells (BMECs) to support ex vivo and in vivo hematopoiesis. We demonstrate that AKT1-activated BMECs (BMEC-Akt1) have a unique transcription factor/cytokine profile that supports functional HSCs in lieu of complex serum and cytokine supplementation. Additionally, transplantation of BMEC-Akt1 cells enhanced regenerative hematopoiesis following myeloablative irradiation. These data demonstrate that BMEC-Akt1 cultures can be used as a platform for the discovery of pro-HSC factors and justify the utility of BMECs as a cellular therapy. This technical advance may lead to the development of therapies designed to decrease pancytopenias associated with myeloablative regimens used to treat a wide array of disease states., Graphical Abstract, Highlights • Reproducible isolation of murine bone marrow-derived endothelial and stromal cells • Cultured endothelial cells that support the ex vivo maintenance of repopulating LT-HSCs • Vascular and stromal platforms for the discovery of pro-HSC growth factors • Transplantation of niche-specific endothelium that promotes hematopoietic recovery, Compound niches within the bone marrow microenvironment modulate hematopoietic stem cell (HSC) function, including quiescence, self-renewal, and differentiation. In this article, Butler and colleagues describe a robust and reproducible method to isolate and culture murine bone marrow-specific endothelial and stromal cells that provide an ex vivo and in vivo platform to explore the cross-talk that regulates HSC activity.

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34 + CD45 + hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence.

Hematopoietic stem cells (HSCs) predominantly reside either in direct contact or in close proximity to the vascular endothelium throughout their lifespan. From the moment of HSC embryonic specification from hemogenic endothelium, endothelial cells (ECs) act as a critical cellular-hub that regulates a vast repertoire of biological processes crucial for HSC maintenance throughout its lifespan. In this review, we will discuss recent findings in endothelial niche-mediated regulation of HSC function during development, aging and regenerative conditions.Studies employing genetic vascular models have unequivocally confirmed that ECs provide the essential instructive cues for HSC emergence during embryonic development as well as adult HSC maintenance during homeostasis and regeneration. Aging of ECs may impair their ability to maintain HSC function contributing to the development of aging-associated hematopoietic deficiencies. These findings have opened up new avenues to explore the therapeutic application of ECs. ECs can be adapted to serve as an instructive platform to expand bona fide HSCs and also utilized as a cellular therapy to promote regeneration of the hematopoietic system following myelosuppressive and myeloablative injuries.ECs provide a fertile niche for maintenance of functional HSCs throughout their lifecycle. An improved understanding of the EC-HSC cross-talk will pave the way for development of EC-directed strategies for improving HSC function during aging.

Recent studies have identified a specialized subset of CD31hiendomucinhi (CD31hiEMCNhi) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3-/- mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

NO produced by inducible NO synthase (iNOS) contributes to ischemic brain injury, but the cell types expressing iNOS and mediating tissue damage have not been elucidated. To examine the relative contribution of iNOS in resident brain cells and peripheral leukocytes infiltrating the ischemic brain, we used bone marrow (BM) chimeric mice in which the middle cerebral artery was occluded and infarct volume was determined 3 d later. iNOS−/− mice engrafted with iNOS+/+ BM exhibited larger infarcts (44 ± 2 mm3; n = 13; mean ± SE) compared with autologous transplanted iNOS−/− mice (24 ± 3 mm3; n = 10; p < 0.01), implicating blood-borne leukocytes in the damage. Furthermore, iNOS+/+ mice transplanted with iNOS−/− BM had large infarcts (39 ± 6 mm3; n = 13), similar to those of autologous transplanted iNOS+/+ mice (39 ± 4 mm3; n = 14), indicating the resident brain cells also play a role. Flow cytometry and cell sorting revealed that iNOS is highly expressed in neutrophils and endothelium but not microglia. Surprisingly, postischemic iNOS expression was enhanced in the endothelium of iNOS+/+ mice transplanted with iNOS−/− BM and in leukocytes of iNOS−/− mice with iNOS+/+ BM, suggesting that endothelial iNOS suppresses iNOS expression in leukocytes and vice versa. To provide independent evidence that neutrophils mediate brain injury, neutrophils were isolated and transferred to mice 24 h after stroke. Consistent with the result in chimeric mice, transfer of iNOS+/+, but not iNOS−/−, neutrophils into iNOS−/− mice increased infarct volume. The findings establish that iNOS in both neutrophils and endothelium mediates tissue damage and identify these cell types as putative therapeutic targets for stroke injury.

Generating engraftable human haematopoietic cells from autologous tissues is a potential route to new therapies for blood diseases. However, directed differentiation of pluripotent stem cells yields haematopoietic cells that engraft poorly. Here, we have devised a method to phenocopy the vascular-niche microenvironment of haemogenic cells, thereby enabling reprogramming of human endothelial cells into engraftable haematopoietic cells without transition through a pluripotent intermediate. Highly purified non-haemogenic human umbilical vein endothelial cells or adult dermal microvascular endothelial cells were transduced with the transcription factors FOSB, GFI1, RUNX1 and SPI1 (hereafter referred to as FGRS), and then propagated on serum-free instructive vascular niche monolayers to induce outgrowth of haematopoietic colonies containing cells with functional and immunophenotypic features of multipotent progenitor cells (MPPs). These endothelial cells that have been reprogrammed into human MPPs (rEC-hMPPs) acquire colony-forming-cell potential and durably engraft into immune-deficient mice after primary and secondary transplantation, producing long-term rEC-hMPP-derived myeloid (granulocytic/monocytic, erythroid, megakaryocytic) and lymphoid (natural killer and B cell) progenies. Conditional expression of FGRS transgenes, combined with vascular induction, activates endogenous FGRS genes, endowing rEC-hMPPs with a transcriptional and functional profile similar to that of self-renewing MPPs. Our approach underscores the role of inductive cues from the vascular niche in coordinating and sustaining haematopoietic specification and may prove useful for engineering autologous haematopoietic grafts to treat inherited and acquired blood disorders.

EGFL7 is a secreted angiogenic factor produced by embryonic endothelial cells. To understand its role in placental development, we established a novel Egfl7 knockout mouse. The mutant mice have gross defects in chorioallantoic branching morphogenesis and placental vascular patterning. Microangiography and 3D imaging revealed patchy perfusion of Egfl7 −/− placentas marked by impeded blood conductance through sites of narrowed vessels. Consistent with poor feto-placental perfusion, Egfl7 knockout resulted in reduced placental weight and fetal growth restriction. The placentas also showed abnormal fetal vessel patterning and over 50% reduction in fetal blood space. In vitro , placental endothelial cells were deficient in migration, cord formation and sprouting. Expression of genes involved in branching morphogenesis, Gcm1 , Syna and Synb , and in patterning of the extracellular matrix, Mmrn1 , were temporally dysregulated in the placentas. Egfl7 knockout did not affect expression of the microRNA embedded within intron 7. Collectively, these data reveal that Egfl7 is crucial for placental vascularization and embryonic growth, and may provide insight into etiological factors underlying placental pathologies associated with intrauterine growth restriction, which is a significant cause of infant morbidity and mortality.

Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. Key points Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens., The complex microenvironmental signalling pathways that govern haematopoietic stem cell (HSC) activity remain poorly defined. Here, the authors identify endothelial NF-κB signalling as regulating regenerative HSC function, accelerating haematopoietic recovery following myelosuppressive injury in mice.

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.

Aging leads to a drastic decline in hematopoietic stem cell (HSC) function. In addition to a loss of their self-renewal potential, old HSCs exhibit a myeloid biased differentiation and an increased propensity to develop hematologic malignancies. While some of these age-related changes reflect cell-intrinsic alterations within HSCs, recent findings suggest that signals from the bone marrow (BM) microenvironment, in particular the BM vascular niche, might play crucial roles in regulating HSC aging. In support of this idea, we recently discovered that physiological aging of BM endothelial cells leads to an altered molecular crosstalk between the endothelial niche and HSCs that instructs young HSCs to behave as aged HSCs. We have previously demonstrated that AKT/mTOR activation within young BM endothelial cells supports balanced HSC self-renewal and differentiation. Based on these observations, we hypothesized that deficient AKT/mTOR signaling within endothelium will deprive the hematopoietic system of proper endothelial-derived instructive signals resulting in HSC aging phenotypes. We have generated preliminary data demonstrating that aged endothelial cells, in contrast to aged HSCs, display decreased AKT/mTOR signaling. Furthermore, our data indicates that pharmacological inhibition of mTOR signaling using Rapamycin, a widely regarded rejuvenating agent and clinically utilized immunosuppressant, has deleterious effects on the hematopoietic system, in part due to its adverse impact on endothelium by abolishing both mTORC1 and mTORC2 signaling, in homeostatic conditions and following myelosuppressive therapy (i.e. radiation and chemotherapy). To formally determine whether endothelial-specific inhibition of mTOR signaling can promote hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that HSCs from mTOR(ECKO)mice displayed all the phenotypic and functional attributes of an aged HSC including loss of cell polarity, increased DNA damage, myeloid biased output and loss of engraftment potential during serial transplantation. Gene expression profiling of HSCs isolated from young mTOR(ECKO)mice revealed that their intrinsic gene expression signature resembled aged HSCs, both at steady state and following transplantation. Notably, these aging-like phenotypic and functional HSC alterations are only maintained in secondary transplants that were performed in an mTOR(ECKO)microenvironment confirming the role of EC-derived instructive signals in governing HSC aging. Furthermore, we assessed the function of BM endothelial cells from mTOR(ECKO)mice and revealed severe defects in overall endothelial health which included alterations in proliferation, wound healing ability, and their metabolic profile that displayed a hyperglycolytic phenotype at steady state as well as increased glycolytic capacity under stress conditions, compared to control BMECs. Moreover, mTOR(ECKO)BMECs showed high respiratory capacity, thus phenocopying the metabolic profile of aged and tumor-associated BMECs. Furthermore, the BM microenvironment of mTOR(ECKO)mice manifested an increase in a NF-kB dependent inflammatory signature. Interestingly, chronic inflammation driven by NF-kB signaling is the leading cause of vascular dysfunction and aging. In summary, we demonstrate that deletion of mTOR signaling within young endothelium drives premature aging of HSCs and the hematopoietic system. Utilizing our model system, we have now identified candidate pro-aging and pro-rejuvenation factors that will lay the foundation for designing therapeutic strategies to rejuvenate the HSC microenvironment, thereby restoring the functional properties of the hematopoietic system to youthful levels and improving overall health. Disclosures No relevant conflicts of interest to declare.

Bone marrow endothelial cells (BMECs) are an indispensable component for the hematopoietic stem cell (HSC) niche. In particular, Akt-activation endows ECs with the instructive capacity to support the self-renewal of HSCs to maintain homeostasis and promote the regeneration of the hematopoietic system following myeloablative stress, whereas MAPK signaling promotes HSC differentiation into lineage committed progenitors. Our group has recently demonstrated that in both in vitro and in vivo settings, aged BMECs can instruct an aged phenotype in young HSCs. Notably, we have generated preliminary data demonstrating that young BMECs maintain high levels of Akt signaling, whereas aged BMECs preferential signaling through MAPK. These data suggest that aged BMECs could be primed to negatively affect HSC function, which may lead to aged-related hematopoietic disorders, including the development of hematopoietic malignancies. Acute myeloid leukemia (AML) is the most common acute leukemia in adults, which incidence is predicted to rise with the expected increase in the elderly population. Disease recurrence is driven by a rare population of tumor cells, capable of self-renewal and expansion similarly to normal HSCs. These cells, referred as leukemia initiating cells (LIC), have the ability to persist during chemotherapy, and to repopulate disease. Notably, AML is known to alter the normal bone marrow vascular architecture, leading to increased vascular leakiness and to secrete a number of angiogenic factors, including VEGF-A, which has been suggested to support leukemic growth. As a consequence, the vascular niche might actively contribute to the maintenance and survival of LICs during the course of the disease and after induction of remission by chemotherapeutic agents. To this end we have generated data where we demonstrate that transplantation of AML LICs resulted in significant increases in the phosphorylation states of AKT and MAPK signaling. This data confirms that leukemic cells hijack the pro-HSC EC-specific Akt signaling pathway to enable their progression, but also over activate signaling pathways that have been demonstrated to support the differentiation of normal HSCs and support survival of AML. To formally test if endothelial MAPK signaling impacts the ability of BMECs to support functional hematopoiesis, we generated a mouse model where mice carrying a Rosa26 Stop/Floxed MEK1DD (mutant MAPKK1 that renders MAPK constitutively activate) were crossed to a tamoxifen inducible EC-specific VEcadherin Cre transgenic mouse to generate MAPKVCC mice. Analysis of these mice revealed that MAPKVCC mice manifested a profound decline in their HSC and progenitor potential resulting from loss of vascular integrity and enhanced inflammation within their BM microenvironment. We next isolated primary BMECs from MAPKVCC mice or littermate controls, and co-cultured them in an ex vivo platform with putative MLL-AF9 AML LICs (cKit+CD34+CD11b+). We observed that MAPK pathway activation conferred BMECs the ability to significantly expand putative LICs ex vivo. Moreover, transplantation of LICs first expanded on MAPKVCC BMECs resulted in notably shorter survival of recipient mice, compared to mice receiving control BMEC-expanded LICs. We further characterized MapkVCC BMEC metabolic profile by Seahorse XFe96 Extracellular Flux Analyzer. MapkVCC BMECs displayed a hyperglycolytic phenotype at steady state as well as increased glycolytic capacity under stress conditions, compared to control BMECs. Moreover, MapkVCC BMECs showed high respiratory capacity, thus phenocopying the metabolic profile of aged BMECs and tumor-associated ECs. Notably, MAPKVCC BMECs induced a metabolic switch in MLL-AF9 LICs ex vivo, towards increased glycolysis in both steady and stressed state and retained oxidative respiration potential under stress. These results suggest that age-associated aberrant MAPK signaling in the BM endothelial niche enhances the acquisition of the Warburg's effect in LICs, providing a selective microenvironment for the emergence of malignant clones at the expenses of normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem and progenitor cells (HSPCs) balance the physiological demands of maintaining peripheral leukocytes, erythrocytes, and platelets, while maintaining a potent stem cell reserve. These characteristics have made HSPC transplantation the only curative option for treating many hematological disorders. The regenerative potential of hematopoietic stem cells (HSCs) reside in their ability to home to a supportive niche, allowing for both HSC self-renewal and reconstitution of the hematopoietic system. The bone marrow (BM) microenvironment regulates HSC quiescence, self-renewal, and differentiation. The BM niche is composed of a number of cell types, including Lepr+ cells, Nestin+ cells, and endothelial cells. Collectively, the HSC niche modulates HSC fate decisions through the expression of paracrine factors, including Cxcl12 and Kitl. The BM microenvironment also plays a critical role in the reestablishment of hematopoiesis following myeloablative injury. Vegfr2-mediated vascular repair is critical for hematopoietic reconstitution following chemotherapeutic and radiation-mediated insult, while BM granulocyte production of Tnfa supports the regeneration of sinusoidal endothelium and subsequent hematopoietic recovery. While the importance of an HSC-supportive microenvironment during hematopoietic homeostasis and during regenerative conditions is coming into focus, poorly defined BM niche cells have limited the precise mechanistic insights necessary to elucidate new regenerative factors and strategies. Current methodology used to examine cellular subsets within the BM microenvironment rely on immunophenotypic fractionation, localization, and genetic lineage tracing. Ambiguous BM niche cellular immunophenotypes and gene expression have limited cellular resolution and confounded the interpretation of cre-mediated genetic deletion models. Herein, we aim to resolve the identities of distinct BM endothelial cell (BMEC) subpopulations to ultimately develop genetic tools to elucidate the paracrine requirements of the HSC-supportive endothelial niche. To this end, we sort-purified murine BMECs (VECAD+CD31+CD45-TER119-) for single cell RNA sequencing (scRNA-Seq). scRNA-Seq revealed the emergence of distinct BM arteriole, sinusoidal, and transitional endothelial populations, with arteriole BMECs significantly enriched for Kitl and Cxcl12. To confirm an arteriole enrichment in Kitl and Cxcl12 expression, we performed scRNA-Seq transcriptional analysis of sort-purified BM cells from KitlGFP and Cxcl12DsRed reporter mice. KitlGFP BM cells identified a distinct arteriole, but not sinusoidal, BMEC population. Cxcl12DsRed BM cells identified both arteriole and sinusoidal BMEC cell populations, but confirmed an increase in Cxcl12 expression in arterioles. We next examined candidate genes to generate BMEC subset-specific inducible cre mice. Analysis revealed that Vegfr3 (Flt4) expression was specific to sinusoidal BMECs, while Bmx1 appeared enriched in arterioles. We utilized a previously described Vegfr3YFP transgenic reporter mouse and found sinusoidal BMEC restricted expression. We then generated an inducible Vegfr3creERT2 line that directs efficient recombination to sinusoidal endothelium, with no detectable off-target activity. We next examined a previously described Bmx1creERT2 mouse line by generating Bmx1creERT2;ROSA26tdTomato reporter mice. In contrast to a recent report, Bmx1creERT2 activity was not spatially confined to the BM arteriole niche, but also labeled additional niche components, making it an unsuitable for arteriole-specific deletion. Moreover, previously reported constitutive Eporcre mice used to delete Kitl in sinusoids displays detectable cre activity in both arteriole and erythrocyte populations. More refined genetic models will need to be generated to test current and future candidate factors in the BM niche. Using our transcriptional data set, we have generated and validated a new inducible Vegfr3creERT2 mouse line that displays sinusoidal-restricted expression in the BM. Arteriole-specific creERT2 lines are currently being evaluated. These models will be used to systematically evaluate novel candidate arteriole- and sinusoidal-specific hematopoietic paracrine factors identified in our transcriptional analysis. Disclosures No relevant conflicts of interest to declare.

Summary Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long-term hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free cocultures, ECs, through direct cellular contact, stimulated incremental expansion of repopulating CD34 − Flt3 − cKit + Lineage − Sca1 + LT-HSCs, which retained their self-renewal ability, as determined by single-cell and serial transplantation assays. Angiocrine expression of Notch ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2-deficient, mice. In transgenic notch-reporter (TNR. Gfp ) mice, regenerating TNR. Gfp + LT-HSCs were detected in cellular contact with sinusoidal ECs. Interference with angiocrine, but not perfusion, function of SECs impaired repopulation of TNR. Gfp + LT-HSCs. ECs establish an instructive vascular niche for clinical-scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.

SummaryMyelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.

Recent evidence has suggested that bone marrow derived progenitor cells may contribute to the development of intimal hyperplasia after arterial injury, a process that classically has been believed to involve extracellular matrix deposition and the migration and proliferation of cells within the arterial wall. The first studies demonstrating the existence of bone marrow derived cells in the neointima employed mouse models of arterial injury in conjunction with whole bone marrow transplant. Later studies have shown specifically that bone marrow derived hematopoietic or mesenchymal stem cells can be recruited to the neointima and differentiate into smooth muscle cells or endothelial cells. Although the data vary widely depending on different animal models of arterial injury and methods of labeling bone marrow derived cells, it appears that progenitor cells do indeed contribute to intimal hyperplasia, at least in mouse models of arterial injury. To date, signaling molecules such as c-kit and c-kit ligand, and stromal derived factor-1alpha, in addition to matrix metalloproteinase-9, have emerged as critical factors that recruit progenitor cells to sites of arterial injury. While much progress has been made, several tasks remain, including the need for a more in-depth understanding of the mechanisms underlying progenitor cell recruitment, characterization of the involved progenitor cells, and finally validation that the observations made in these mouse models of disease are also applicable to human arterial restenosis.

Adhesion of leukemic cells to vascular cells may confer resistance to chemotherapeutic agents. We hypothesized that disruption of leukemic cell cytoskeletal stability and interference with vascular cell interactions would promote leukemic cell death. We demonstrate that low and nontoxic doses of microtubule-destabilizing agent combretastatin-A4-phosphate (CA4P) inhibit leukemic cell proliferation in vitro and induce mitotic arrest and cell death. Treatment of acute myeloid leukemias (AMLs) with CA4P leads to disruption of mitochondrial membrane potential, release of proapoptotic mitochondrial membrane proteins, and DNA fragmentation, resulting in cell death in part through a caspase-dependent manner. Furthermore, CA4P increases intracellular reactive oxygen species (ROS), and antioxidant treatment imparts partial protection from cell death, suggesting that ROS accumulation contributes to CA4P-induced cytotoxicity in AML. In vivo, CA4P inhibited proliferation and circulation of leukemic cells and diminished the extent of perivascular leukemic infiltrates, prolonging survival of mice that underwent xenotransplantation without inducing hematologic toxicity. CA4P decreases the interaction of leukemic cells with neovessels by down-regulating the expression of the adhesion molecule VCAM-1 thereby augmenting leukemic cell death. These data suggest that CA4P targets both circulating and vascular-adherent leukemic cells through mitochondrial damage and down-regulation of VCAM-1 without incurring hematologic toxicities. As such, CA4P provides for an effective means to treat refractory organ-infiltrating leukemias.

Bone marrow–derived cells contribute to physiological and pathological vascular remodeling throughout ontogenesis and adult life. During tissue regeneration and tumor growth, the release of cytokines and chemokines mediates the recruitment of hematopoietic and endothelial progenitor cells that contribute to the assembly of neovessels. Current evidence implies that platelets contribute structurally and instructively to vascular remodeling. Platelets adhere almost immediately to exposed or activated endothelium, and they are major storage and delivery vehicles for pro- and antiangiogenic growth factors including VEGF-A and thrombospondin (TSP), and cytokines and chemokines, such as stromal-derived factor 1 (SDF-1). By site-specific deployment of these factors, platelets orchestrate the local angiogenic stimulus within a tissue and direct the recruitment and differentiation of circulating bone marrow–derived cells. These insights have profound clinical implications; inhibition of platelet-deployed growth factors or their receptors may be an effective strategy to block tumor growth, whereas activation of these pathways may be used to accelerate revascularization and tissue regeneration.

Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in their recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also contribute to thymic repair. Unbiased transcriptome analysis on the damage-resistant non-hematopoietic compartemtn of the thymus revealed significant upregulation of Bmp4 and its downstream signalling targets (Fig. 1a). Further interrogation revealed that while thymic expression of BMP4 was restricted to fibroblasts and endothelial cells (ECs), only ECs increase their expression of Bmp4 after damage; and specific and inducible deletion of BMP4 in ECs led to significantly worse regeneration (Fig. 1b). Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration has thus far remained largely unclear. Careful interrogation of ECs after damage revealed that, much like ILCs, ECs are extremely resistant to multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and TBI. However, whole organ imaging analysis using light sheet field microscopy suggested that even though the number of ECs remain unchanged after damage, there is considerable structural changes to the vasculature including shortening of the vessels and reduced branching. Although BMP4 receptors are widely expressed in the thymus, there was enriched expression for BMP4 receptor subunits on TECs, which is consistent with the role of BMP4 in thymus ontogeny by promoting TEC development, at least partially due to its ability to induce expression of Foxn1 (Fig. 1c), a key transcription factor for the development and maintenance of TECs. Consistent with these findings, after thymic damage we observed a significant increase in the expression of Foxn1 after damage as well as GSEA enrichment for downstream FOXN1 target genes (Fig. 1d); including Dll4, the Notch ligand critical for T cell development and whose concentration we have previously shown can directly regulate thymic size (Velardi 2014 J Exp Med 211:2341). Finally, using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function; but only when the exEC were derived from the thymus but not from heart or kidney (Fig. 1e). Consistent with endogenous regeneration, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 and Dll4 . Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic renewal. These studies thus not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. Figure 1 Figure 1. Disclosures van den Brink: PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy.

Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in BM stromal cells facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). Leukemic cells have been shown to hijack the homeostatic mechanisms of normal hematopoietic stem cells (HSCs) and take refuge within the BM niche. This mechanism is pivotal during chemotherapy and contributes to disease relapse. In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both acute lymphocytic leukemia (ALL) cells and components of the BM niche, using multiphoton intravital microscopy (MP-IVM) . We generated a transplantable, fluorescent leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene (KG Harutyunyan et al, Blood 2014 124:2396). The resulting p190-Bcr/Abl tdTomato cells caused rapid development of ALL in non-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of multiple organ and BM sites, followed by death within 14-18 days. We utilized Col2.3-GFPemd transgenic mice as recipients of leukemia to highlight the osteoblastic niche, and visualized vasculature by injection of TRITC-dextran. We showed the dynamic of homing and engraftment of ALL leukemic B cells (LBC) in OB-GFP recipient mice, with homing in the vicinity of blood vessels visualized by MP-IVM, followed by proliferation and leukemia expansion. This was accompanied by invasion of both vascular and osteoblastic components of BM microenvironment. Longitudinal assessment of hypoxia utilizing pimonidazole staining showed progressive development of BM hypoxia starting from Day 10 p.i., paralleling leukemia progression, despite the abundant vascularization of the BM. To assess the integrity of the BM vascular niche during leukemia progression the experimental mice were intravitally injected with low doses of an Alexa-Fluor-647-conjugated VE-cadherin antibody (MG. Poulos et al, Epub 2013 Sep 5) to visualize the number and morphology of the perfused vessels and to analyze the architecture of the hematopoietic compartment. We observed that at late stage of leukemia progression (day 12-14 p.i) BM vessels are disorganized as a result of expansion of leukemic B cells. Ongoing longitudinal imaging experiments will characterize the cellular origin of hypoxic niche cells and the dynamic of vasculature alteration in ALL. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves interactions between leukemia cells and BM niche cells, as well as disordered vasculature. Citation Format: Karine G. Harutyunyan, Saradhi Mallampati, Anna Zal, Mateusz Rytelewski, Michael C. Gutkin, Jason M. Butler, Tomasz Zal, Marina Konopleva. Imaging the interaction of leukemia and bone marrow microenvironment in murine model of ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2017-876

The molecular pathways and microenvironmental cues that choreograph the conversion of endothelial cells (ECs) into true engraftable hematopoietic stem cells (HSCs) remain undefined. This is due to lack of models to recreate the short-lived transition from EC to hemogenic cells and to HSCs. Extending on our previous work (Sandler V. et al, Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014), we have developed a novel, sequential, clinically-translatable in vitro model of the adult EC to hematopoietic transition (EHT). This model uses precise, conditional, on-off expression of transcription factors (FosB, Gfi1, Runx1, and Spi1 - FGRS) and an inductive vascular niche to reprogram adult mouse ECs into true HSCs (rEC-HSCs) with high efficiency. During the induction phase (days 0-8), FGRS are conditionally expressed in adult non-lymphatic ECs isolated from Runx1-IRES-GFP reporter mice and co-cultured with the supportive vascular niche cells. During the specification phase (days 8-20), FGRS-transduced VEcad+Runx1-CD45- ECs activate expression of endogenous Runx1, initiating the hematopoietic program and silencing EC fate. The VEcad+Runx1+CD45+ cells, then complete specification and full commitment to VEcad-Runx1+CD45+ hematopoietic stem and progenitor cells (rEC-HSPCs). Specified rEC-HSPCs are then expanded (days 20-28) on the vascular niche generating a large number of hematopoietic cells and, at this point, expression of exogenous FGRS is turned off. Transplantation of rEC-HSPCs (CD45.2) into lethally irradiated (CD45.1) recipient mice reconstitute both short-term and long-term hematopoiesis, and are capable of engrafting secondary and tertiary recipients (rEC-HSCs). Once engrafted, rEC-HSPCs give rise to functional myeloid and lymphoid cells with full complement of polarized T cell subsets. rEC-HSC-derived immune cells undergo T-cell receptor (TCR) rearrangement and reconstitute adaptive immune function in Rag1-/- mice. To prove the stem cell potential of rEC-HSCs, we performed clonal analyses on the VEcad+Runx1+CD45+ cells (days 8-20), in which single cells were plated in coculture with vascular niche. Notably, 7 out of 386 CD45.2+ clones gave rise to expanding colonies that were capable of 4 months primary multilineage engraftment into lethally irradiated CD45.1+ recipients. In addition, limiting dilution transplantation of isochronic VEcad-Runx1+CD45+ cells indicated that 1 out of 538 rEC-HSPCs are repopulating rEC-HSCs with primary and secondary engraftment potential. Thus, based on both clonal and limiting dilution transplantation studies, we demonstrate that rEC-HSCs represent true repopulating prototypical HSCs. In addition, since during the expansion phase there are >200 fold expansion of the SLAM-coded KLS population, these data support the notion that during the 20-28 days expansion phase there is robust proliferation of rEC-HSCs. Serial analyses of the primary, secondary and tertiary engrafted rEC-HSCs, showed no evidence of leukemias. Molecular analyses indicated that both in vitro and in vivo expanded rEC-HSCs had complete erasure of vascular signature and stable expression of hematopoietic profile. Moreover, employing Runx1-IRES-GFP reporter mice enabled deconvolution of stage-specific pathways involved in generation of engraftable rEC-HSCs. Inhibition of TGFβ signaling along with activation of BMP and CXCL12 pathways reinforced the induction phase. Active Notch and CXCL12 signaling throughout the specification and expansion phases supported self-renewal of transplantable rEC-HSCs. This stepwise reprogramming approach provides an interrogatable in vitro platform to elucidate the critical pathways involved in the transition of ECs into hemogenic-like and ultimately hematopoietic cells in vitro. Specifically, these data unequivocally support that our approach lead to generation of large number HSCs, enabling therapeutic application. Thus, our reprogramming platform that does not require transition through a pluripotent state, will facilitate devising strategies to reprogram ECs into abundant autologous long-term repopulating HSCs amenable to genetic modification for treatment of inherited and acquired hematological disorders. Disclosures Ginsberg: Angiocrine Bioscience: Employment. Butler:Angiocrine Bioscience: Research Funding. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

Nature 532, 323–328 (2016); doi:10.1038/nature17624 The authors Karin Golan, Eman Khatib and Anju Kumari were erroneously omitted from the author list of this Article. They are all associated with the affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel, and the Author Contributions section should have included the following statements: K.

As part of a previously reported trial comparing granulocyte-colony stimulating factor (G-CSF) stimulated bone marrow and peripheral blood stem cells (PBSCs) in allogeneic stem cell transplantation, we included a questionnaire to compare donor morbidity and long-term complications between the two donation procedures. Bone marrow donation was associated with significantly more donors experiencing localized pain at the donation site compared to PBSC collection. However, this was not associated with any increased delay in returning to normal activity. Although a minority of bone marrow donors suffered chronic pain at the donation site, no serious long-term side effects relating to G-CSF stimulated stem cell donation were identified.

During embryogenesis, endothelial cells induce organogenesis before the development of circulation. These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis. However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3(+)CD34(-)VEGFR2(+)VE-cadherin(+)FactorVIII(+)CD45(-) endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1-3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4-8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1. Accordingly, Id1-deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1(+/+) or Id1(-/-) LSECs transduced with Wnt2 and HGF (Id1(-/-)Wnt2(+)HGF(+) LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1(-/-) mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2(+)Id1(+)Wnt2(+)HGF(+) LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

The precise mechanisms whereby anti-angiogenesis therapy blocks tumour growth or causes vascular toxicity are unknown. We propose that endothelial cells establish a vascular niche that promotes tumour growth and tissue repair not only by delivering nutrients and O2 but also through an 'angiocrine' mechanism by producing stem and progenitor cell-active trophogens. Identification of endothelial-derived instructive angiocrine factors will allow direct tumour targeting, while diminishing the unwanted side effects associated with the use of anti-angiogenic agents.

Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1α (SDF-1α) and its mobilizing effects on BM. Blocking SDF-1α activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133+CXCR4+ myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1α. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.

Bone marrow sinusoids maintain homeostasis between developing hematopoietic cells and the circulation, and they provide niches for hematopoietic progenitors. Sinusoids are damaged by chemotherapy and radiation. Hematopoietic stem cells (HSCs) have been shown to produce endothelial progenitor cells that contribute to the repair of damaged blood vessels. Because HSCs home to the marrow during bone marrow transplant, these cells may play a role in repair of marrow sinusoids. Here, we explore the role of donor HSCs in the repair of damaged sinusoids following hematopoietic stem cell transplant. We used three methods to test this role: (a) expression of platelet endothelial cell adhesion molecule to identify endothelial progenitors and the presence of the Y chromosome to identify male donor cells in female recipients; (b) presence of the Y chromosome to identify male donor cells in female recipients, and expression of the panendothelial marker mouse endothelial cell antigen-32 to identify sinusoidal endothelium; and (c) use of Tie-2/green fluorescent protein mice as donors or recipients and presence of Dil-Ac-LDL to identify sinusoids. We found that sinusoids were predominantly host-derived posttransplant. Donor cells spread along the marrow vasculature early post-transplant in a pattern that matched stromal-derived factor-1 expression. Furthermore, these engrafting progenitors were positioned to provide physical support, as well as growth and survival signals in the form of vascular-endothelial growth factor-A. Occasionally, donor cells provide cellular “patches” in the damaged sinusoids, although this occurred at a low level compared with hematopoietic engraftment. Donor support for the repair of the marrow vascular niche may be a critical first step of hematopoietic engraftment. Disclosure of potential conflicts of interest is found at the end of this article.

Bone marrow (BM) endothelial cells (BMECs) form a network of blood vessels (BVs) that regulate both leukocyte trafficking and hematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance between these dual regulatory roles and if these events occur at the same vascular site. We define the BM architecture of functionally distinct BVs, their spatial localization and association with specific stromal precursors, which cooperatively regulate HSPC fate. BM stem and progenitor cell maintenance in a metabolically non-active state and leukocyte trafficking occur at separate sites and are differentially regulated by specific BVs with distinct permeability properties. BM arteries were found to be mostly encircled by aSMA+ pericytes whereas the ensuing small-diameter endosteal and trabecular arterioles were predominantly surrounded by stem cell-niche supporting stromal precursor cells. Live imaging and flow analysis revealed that endosteal arteriole BVs exhibited high flow rate, low permeability to external plasma from the peripheral blood, and high levels of adhesion- and tight-junction molecules. Primitive HSPCs located in peri-arteriole regions were found in a non-activated, low reactive oxygen species (ROS) state. Exposure of BM HSPCs to peripheral blood plasma, enhanced their metabolic activity, exhibited by enhanced intracellular ROS levels, and glucose uptake. The same was also evident for circulating HSPCs in the blood. Plasma-exposed HSPCs displayed enhanced motility alongside with reduced long-term repopulation potential. Live imaging showed that all immature and mature leukocyte bi-directional trafficking occurred exclusively at the more permeable sinusoids, located downstream to the endosteal arterioles. Of note, BM sinusoids contained a higher prevalence of ROShigh cells in their microenvironment, including HSPCs. Rapid AMD3100-induced HSPC mobilization preferentially affected sinusoidal but not arterial BVs permeability and CXCL12 chemokine release. Endothelial specific in vivo interference with CXCL12-CXCR4 interactions, via conditional CXCR4 genetic deletion, hampered BM barrier integrity resulting in enhanced HSPC egress. In line with these results we found that during conditions favoring BM stem and progenitor cells expansion, endothelial integrity was enhanced along with reduced HSPC bi-directional trafficking. Conversely, conditional endothelial specific induced genetic or pharmacologic disruption of barrier integrity augmented ROS levels in HSPCs, enhancing their bi-directional trafficking and differentiation while reducing their BM pool size and maintenance in a metabolically non-active state. Of note, humanized mice engrafted with pre-B ALL cells exhibited reduced BM barrier permeability most probably due to BM endothelium modification via FGF-2 secretion by the leukemic clone. Interestingly, human pre-B ALL cells displayed hypersensitivity to plasmatic exposure. We hypothesize that malignant cells modify BM endothelium to provide themselves with a supportive and protective microenvironment composed of undifferentiated BM stromal progenitors and tightly sealed endothelial barrier. In conclusion, our study identifies anatomically distinct BM BVs with different barrier functions serving as systemic leukocyte trafficking or HSPC BM maintenance sites with clinical therapeutic relevance. Disclosures Rafii: Angiocrine Bioscience: Consultancy, Equity Ownership.

The molecular mechanisms regulating the aging of hematopoietic stem cells (HSCs) are poorly understood. To date, most studies describing age-related alterations have focused on HSC-intrinsic alterations, showing that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential, self-renewing capacity, altered transciptomes, cell-cycling and responses to cellular stress and DNA damage. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, which is thought to predispose the aging hematopoietic to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately addressed the effects of the aging microenvironment. A large body of evidence has demonstrated functional interactions between the HSC and its niche, suggesting that local and systemic factors may regulate HSC function; however, the role of the bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding the relationship between the BM microenvironment and the HSC during aging may aid in efforts to prevent or reverse the age-related functional decline observed in the hematopoietic system. We have shown that Akt-activated endothelial cells (ECs) within the hematopoietic microenvironment, are indispensable for supporting HSC self-renewal during both steady-state and regenerative hematopoiesis and that EC-specific Mapk signaling drives the differentiation of HSCs into lineage-committed progeny. Here, we demonstrate that young BMECs maintain high levels of Akt signaling, whereas aged ECs exhibit preferential signaling through Mapk. Utilizing a novel HSC/EC co-culture system, we demonstrate that aged BMECs co-cultured with hematopoietic stem and progenitor cells (HSPCs) isolated from young mice inhibit the expansion of repopulating HSCs and are unable to expand aged HSPCs that give rise to long-term, multilineage engraftment. Of note, when we co-cultured aged HSPCs with young BMECs we found that we were able to maintain their functional capacity when assessed by competitive repopulation assays. These data suggest that BMECs play an important role in regulating HSC function. Based on these observations, we set out to test if endothelial Mapk inhibits the vascular niche from supporting functional hematopoiesis. We generated a mouse model in which Mapk was selectively overexpressed in ECs (Mapk VCC) and demonstrated that these mice exhibit a defect in phenotypic and functional HSCs, resembling phenotypes similar to aged HSCs. In particular, transplanted HSCs from Mapk VCC mice lead to diminished engraftment ability with an in increase in myeloid contribution at the expense of B and T cells. To directly test if the functional defects in the HSCs were due to the Mapk-activated vascular niche, we isolated BMECs from these mice and found that Mapk-activated ECs have a decreased ability to support the ex vivo expansion of functional HSCs, with less HSCs in quiescence and more differentiation into granulocytic myeloid cells. Transcriptome and proteomic analyses revealed that aged and Mapk-activated BMECs have similar defects in their pro-HSC angiocrine repertoire, suggesting a possible mechanism for their diminished capacity to instruct and maintain a balanced and healthy hematopoietic system. Furthermore, we utilized an endothelial-based cellular therapy approach to rejuvenate the BM microenvironment and demonstrated that transplantation of young BMECs can enhance hematopoietic recovery and restore HSC function following myeloablative injury in aged mice. Taken together, our in vivo animal model and EC/HSC co-culture system will allow us to screen for angiocrine factors that support the functional attributes of the HSC. Additionally, we have unlocked a potential therapeutic application for the transplantation of ECs following myeloablative treatment. Transplantation of BMECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSPCs following BM transplantation and accelerates the rate of hematopoietic recovery following radiation or chemotherapeutic regimens decreasing the morbidity/mortality associated with life threatening pancytopenias in the elderly. Disclosures No relevant conflicts of interest to declare.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC3) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also play a role. Unlike other lymphoid cells, ILC3 were extremely radio-resistant with little if any depletion of cells after even lethal doses of total body irradiation (TBI). However, comprehensive analysis of all thymus-resident cell subsets revealed that ILCs were not the only damage-resistant population in the thymus as endothelial cells (ECs) were also remarkably resistant to damage in multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and sublethal total body irradiation (SL-TBI, 550cGy) (Fig. 1a). Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration remains largely unclear. Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic repair. Using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function (Fig. 1b). Intriguingly, this trophic effect was only observed when exEC were derived from the thymus but not when they were derived from heart or kidney (Fig. 1b). Mechanistically, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 (Fig. 1c), a key transcription factor required for thymus organogenesis, maintenance and regeneration. In vitro co-culture assays revealed that conditioned media (CM) from exEC derived from the thymus, but not the heart or the kidney, could induce Foxn1 expression by TECs (Fig. 1d), in addition to the FOXN1 target genes Kitl and Dll4; a Notch ligand itself critical for T cell development. These findings suggest that thymus-derived exEC produce a soluble factor that contributes toward thymic regeneration via activation of Foxn1. Transcriptome analysis of highly purified thymic ECs after SL-TBI identified that, among other things, expression of Bmp4 was significantly increased, offering a potential mechanism by which thymic ECs mediate their regeneration. Consistent with this hypothesis, not only could recombinant BMP4 promote the expression of Foxn1 by TECs in vitro, induction of Foxn1 by CM from exEC(Thy) was abrogated by Noggin, an inhibitor of BMP4 signaling (Fig. 1e). Moreover, exEC(Thy) produced significantly more BMP4 compared to exEC derived from the heart or kidney; and silencing Bmp4 expression by shRNA within exEC(Thy) limited their capacity to mediate exogenous thymic regeneration and failed to induce expression of Foxn1 and Dll4. Finally, strengthening its role in endogenous regeneration, administration of a pharmacologic BMP inhibitor inhibited thymic regeneration after SL-TBI; and inducible deletion of Bmp4 specifically in ECs reduced thymic regeneration after SL-TBI (Fig. 1f). These studies not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. Disclosures Rafii: Angiocrine Bioscience: Other: Founder of Angiocrine Biosceince, which is developing the technology behind endothelial cell propagation. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Merck: Honoraria; Tobira Therapeutics: Other: Advisory board attendee; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Haematopoietic stem and progenitor cells move from the bone marrow into the circulation to replenish normal blood-cell levels. Inhibiting a prostaglandin-mediated signalling pathway may promote this process. See Letter p.365 The haematopoietic stem cell (HSC) microenvironment supports progenitor cells producing new circulating blood cells yet prevents the inappropriate exit of immature cells into the bloodstream. This study identifies endogenous prostaglandin E2 (PGE2) as a component in the maintenance of this delicate balance. Louis M. Pelus and colleagues show that inhibition of endogenous PGE2 signalling by nonsteroidal anti-inflammatory drugs (NSAIDs) significantly increases the release of functional haematopoietic stem and progenitors from bone marrow into the peripheral blood, with additive effects when used in conjunction with granulocyte colony-stimulating factor. Tests in primates and human volunteers suggest a strategy that might be used therapeutically to enhance transplantation survival: administration of NSAIDs (aspirin, ibuprofen and meloxicam) enhances stem-cell mobilization, with faster haematologic recovery and enhanced long-term stem-cell repopulation of haematopoietic grafts.