RESUMO
In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.
Assuntos
Células Endoteliais , Integrases , Tamoxifeno , Animais , Camundongos , Células Endoteliais/metabolismo , Integrases/metabolismo , Integrases/genética , Tamoxifeno/farmacologia , Medula Óssea/metabolismo , Camundongos Transgênicos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , HematopoeseRESUMO
Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Camundongos , Netrina-1/genética , Células-Tronco Hematopoéticas/fisiologia , Células da Medula Óssea , Envelhecimento/genética , Nicho de Células-TroncoRESUMO
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.
RESUMO
Aging leads to a decline in hematopoietic stem and progenitor cell (HSPC) function. We recently discovered that aging of bone marrow endothelial cells (BMECs) leads to an altered crosstalk between the BMEC niche and HSPCs, which instructs young HSPCs to behave as aged HSPCs. Here, we demonstrate aging leads to a decrease in mTOR signaling within BMECs that potentially underlies the age-related impairment of their niche activity. Our findings reveal that pharmacological inhibition of mTOR using Rapamycin has deleterious effects on hematopoiesis. To formally determine whether endothelial-specific inhibition of mTOR can influence hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that their HSPCs displayed attributes of an aged hematopoietic system. Transcriptional profiling of HSPCs from mTOR(ECKO) mice revealed that their transcriptome resembled aged HSPCs. Notably, during serial transplantations, exposure of wild-type HSPCs to an mTOR(ECKO) microenvironment was sufficient to recapitulate aging-associated phenotypes, confirming the instructive role of EC-derived signals in governing HSPC aging.
Assuntos
Envelhecimento/fisiologia , Células Endoteliais/metabolismo , Hematopoese , Serina-Treonina Quinases TOR/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Microambiente Celular , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Sirolimo/farmacologia , Transcrição GênicaRESUMO
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.
Assuntos
Células Endoteliais/metabolismo , Hematopoese/fisiologia , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Animais , Antígenos CD , Medula Óssea , Caderinas , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Inflamação , Masculino , Camundongos , Transdução de Sinais , Transplante AutólogoRESUMO
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.
Assuntos
Reabsorção Óssea/patologia , Osso e Ossos/patologia , Endotélio/patologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Reabsorção Óssea/diagnóstico por imagem , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Humanos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/patologia , Ovariectomia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Sialoglicoproteínas/metabolismo , Proteínas RoundaboutRESUMO
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.
Assuntos
Células-Tronco Adultas/metabolismo , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteína Jagged-2/biossíntese , Transdução de Sinais , Aloenxertos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Deleção de Genes , Proteína Jagged-2/genética , Camundongos , Camundongos Transgênicos , Receptor Notch2/genética , Receptor Notch2/metabolismo , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismoRESUMO
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.
Assuntos
Células Endoteliais/fisiologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Envelhecimento , Animais , Medula Óssea/irrigação sanguínea , Transplante de Medula Óssea , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/transplante , Camundongos Endogâmicos C57BL , Microvasos/patologia , Lesões Experimentais por Radiação/prevenção & controle , Tolerância a RadiaçãoRESUMO
PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.
Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Nicho de Células-Tronco , Envelhecimento , Animais , Diferenciação Celular , Autorrenovação Celular , Hematopoese , Humanos , Transdução de SinaisRESUMO
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 TGFß 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.
Assuntos
Diferenciação Celular , Reprogramação Celular , Endotélio/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Imunidade Adaptativa , Envelhecimento/genética , Animais , Linhagem Celular , Linhagem da Célula , Autorrenovação Celular , Células Clonais/citologia , Células Clonais/transplante , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Linfócitos B/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos CD/metabolismo , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/citologia , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Feminino , Citometria de Fluxo , Fatores de Troca do Nucleotídeo Guanina/genética , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Neovascularização Fisiológica , Perfusão , Placenta/irrigação sanguínea , Placenta/embriologia , Placentação , Proteínas/metabolismo , Animais , Sequência de Bases , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Padronização Corporal , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Regulação para Baixo/genética , Família de Proteínas EGF , Células Endoteliais/metabolismo , Feminino , Sangue Fetal/metabolismo , Feto/embriologia , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão , Placenta/metabolismo , GravidezRESUMO
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. Stem Cells Translational Medicine 2017;6:864-876.
Assuntos
Células da Medula Óssea/citologia , Células Endoteliais/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/metabolismo , Linhagem da Célula , Proliferação de Células , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Humanos , Primatas , Fatores de TempoRESUMO
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.
Assuntos
Células Endoteliais/metabolismo , Hematopoese , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Pancitopenia/terapia , Nicho de Células-TroncoRESUMO
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.
Assuntos
Medula Óssea/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Receptor EphB4/metabolismo , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Linhagem Celular , Efrina-B2/genética , Efrina-B2/metabolismo , Camundongos , Receptor EphB4/genéticaRESUMO
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.
Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/fisiologia , Medula Óssea/irrigação sanguínea , Hematopoese , Animais , Antígenos Ly/metabolismo , Artérias/citologia , Artérias/fisiologia , Células da Medula Óssea/citologia , Diferenciação Celular , Movimento Celular , Autorrenovação Celular , Sobrevivência Celular , Quimiocina CXCL12/metabolismo , Células Endoteliais/fisiologia , Feminino , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Leucócitos/citologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nestina/metabolismo , Pericitos/fisiologia , Permeabilidade , Plasma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores CXCR4/metabolismoRESUMO
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.
Assuntos
Células Progenitoras Endoteliais/citologia , Hematopoese , Nicho de Células-Tronco , Animais , Citocinas/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/transplanteRESUMO
Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-cadherin-expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-cadherinâºCD45⺠AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications.
Assuntos
Aorta/embriologia , Células Endoteliais/fisiologia , Endotélio Vascular/embriologia , Gônadas/embriologia , Sistema Hematopoético/embriologia , Mesonefro/embriologia , Receptor Notch1/fisiologia , Receptor Notch2/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Antígenos CD/análise , Caderinas/análise , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Endotélio Vascular/citologia , Feminino , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Antígenos Comuns de Leucócito/análise , Masculino , Proteínas de Membrana/fisiologia , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Transdução de Sinais , Células Estromais/fisiologiaRESUMO
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.