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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.

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.

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.

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.

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.

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological 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.

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.

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.

SummaryMicrovascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration.

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.

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.