RESUMO
ETS transcription factors ETV2, FLI1, and ERG1 specify pluripotent stem cells into induced vascular endothelial cells (iVECs). However, iVECs are unstable and drift toward nonvascular cells. We show that human midgestation c-Kit(-) lineage-committed amniotic cells (ACs) can be reprogrammed into vascular endothelial cells (rAC-VECs) without transitioning through a pluripotent state. Transient ETV2 expression in ACs generates immature rAC-VECs, whereas coexpression with FLI1/ERG1 endows rAC-VECs with a vascular repertoire and morphology matching mature endothelial cells (ECs). Brief TGFß-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and nonvascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Therefore, short-term ETV2 expression and TGFß inhibition with constitutive ERG1/FLI1 coexpression reprogram mature ACs into durable rAC-VECs with clinical-scale expansion potential. Banking of HLA-typed rAC-VECs establishes a vascular inventory for treatment of diverse disorders.
Assuntos
Líquido Amniótico/citologia , Diferenciação Celular , Células Endoteliais/citologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , HumanosRESUMO
The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.
Assuntos
Medula Óssea/irrigação sanguínea , Microambiente Celular , Hematopoese , Células-Tronco Hematopoéticas , Análise de Célula Única , Nicho de Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Linhagem da Célula , Endotélio Vascular/citologia , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq , Receptores Notch/metabolismo , Nicho de Células-Tronco/genética , Estresse Fisiológico/genética , Transcriptoma/genéticaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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
Endothelial cells that line capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establishes specialized vascular niches that deploy sets of growth factors, known as angiocrine factors. These cues participate actively in the induction, specification, patterning and guidance of organ regeneration, as well as in the maintainance of homeostasis and metabolism. When upregulated following injury, they orchestrate self-renewal and differentiation of tissue-specific resident stem and progenitor cells into functional organs. Uncovering the mechanisms by which organotypic endothelium distributes physiological levels of angiocrine factors both spatially and temporally will lay the foundation for clinical trials that promote organ repair without scarring.
Assuntos
Células Endoteliais/metabolismo , Neovascularização Fisiológica , Comunicação Parácrina , Animais , Capilares/citologia , Diferenciação Celular , Autorrenovação Celular , Homeostase , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Pulmão/citologia , Pulmão/patologia , Especificidade de Órgãos , Osteogênese , RegeneraçãoRESUMO
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
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
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.
Assuntos
Microambiente Celular , Reprogramação Celular , Células Endoteliais/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/transplante , Animais , Aorta , Linhagem da Célula , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Gônadas , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/citologia , Mesonefro , Camundongos , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/transplante , Células Mieloides/citologia , Células-Tronco Pluripotentes , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transgenes/genéticaRESUMO
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
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.
Assuntos
Hematopoese Extramedular , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Citocinas/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição 1 de Leucemia de Células Pré-B , Baço/fisiologia , Estresse FisiológicoRESUMO
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 mm(3); n = 13; mean ± SE) compared with autologous transplanted iNOS(-/-) mice (24 ± 3 mm(3); n = 10; p < 0.01), implicating blood-borne leukocytes in the damage. Furthermore, iNOS(+/+) mice transplanted with iNOS(-/-) BM had large infarcts (39 ± 6 mm(3); n = 13), similar to those of autologous transplanted iNOS(+/+) mice (39 ± 4 mm(3); 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.
Assuntos
Infarto Encefálico/imunologia , Endotélio Vascular/imunologia , Neutrófilos/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico/imunologia , Acidente Vascular Cerebral/imunologia , Animais , Infarto Encefálico/genética , Infarto Encefálico/patologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Endotélio Vascular/patologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo II/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Fatores de TempoRESUMO
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.
Assuntos
Endotélio/metabolismo , Regeneração Hepática/fisiologia , Fígado/irrigação sanguínea , Fígado/citologia , Neovascularização Fisiológica/fisiologia , Transdução de Sinais , Animais , Proliferação de Células , Técnicas de Cocultura , Endotélio/citologia , Hepatectomia , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/citologia , Proteína 1 Inibidora de Diferenciação/deficiência , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Fenótipo , Regulação para Cima , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Wnt2/metabolismoRESUMO
UNLABELLED: 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.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Transdução Genética , Antígenos CD/biossíntese , Antígenos CD/genética , Caderinas/biossíntese , Caderinas/genética , Técnicas de Cocultura , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Células Alimentadoras , Células-Tronco Hematopoéticas/citologia , Humanos , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/genéticaRESUMO
Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.
Assuntos
Vasos Sanguíneos/citologia , Proliferação de Células , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Nicho de Células-Tronco/fisiologia , Alicerces Teciduais , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sangue Fetal/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Engenharia Tecidual/métodosRESUMO
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
Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs.
RESUMO
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
Hematopoietic stem cells (HSCs) develop from hemogenic endothelium within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, here we use single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from hemogenic endothelium to HSCs, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering reveals dynamics of gene expression during the hemogenic endothelium to HSC transition, identifying surface receptors specifically expressed on developing HSCs. Transcriptional profiling of niche endothelial cells identifies corresponding ligands, including those signaling to Notch receptors, VLA-4 integrin, and CXCR4, which, when integrated in an engineered platform, are 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 HSCs for therapeutic applications.