RESUMO
DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.
Assuntos
Dano ao DNA , Animais , Reparo do DNA , Replicação do DNA , Células-Tronco Hematopoéticas/metabolismo , Mamíferos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are little clinical data to support this practice, and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune-related mRNA compared with adult platelets. In this study, we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. METHODS: Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined. RESULTS: Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6Chi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on monocytes limited the adult platelet-induced monocyte trafficking phenotype, as well as adult platelet-induced monocyte migration in vitro. Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not. CONCLUSIONS: These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.
Assuntos
Monócitos , Trombocitopenia , Camundongos , Animais , Animais Recém-Nascidos , Plaquetas , Transfusão de Plaquetas/efeitos adversos , Transfusão de Plaquetas/métodos , Trombocitopenia/genéticaRESUMO
RATIONALE: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived ß2M (ß-2 microglobulin) and TGF-ß (transforming growth factor ß) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. OBJECTIVE: To determine the molecular mechanisms and signal transduction pathways by which ß2M and TGF-ß regulate monocyte responses both in vitro and in vivo. METHODS AND RESULTS: Wild-type- (WT) and platelet-specific ß2M knockout mice were treated intravenously with either ß2M or TGF-ß to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma ß2M increased proinflammatory monocytes, while increased plasma TGFß increased proreparative monocytes. TGF-ßR (TGF-ß receptor) inhibition blunted monocyte responses to both ß2M and TGF-ß in vivo. Using imaging flow cytometry, we found that ß2M decreased monocyte SMAD2/3 nuclear localization, while TGF-ß promoted SMAD nuclear translocation but decreased noncanonical/inflammatory (JNK [jun kinase] and NF-κB [nuclear factor-κB] nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. ß2M, but not TGF-ß, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a proreparative monocyte response. CONCLUSIONS: Our findings indicate that elevated plasma ß2M and TGF-ß dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor but induce SMAD-dependent canonical signaling (TGF-ß) versus noncanonical SMAD-independent signaling (ß2M) in a ubiquitin ligase dependent manner. This work has broad implications as ß2M is increased in several inflammatory conditions, while TGF-ß is increased in fibrotic diseases. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Monócitos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Microglobulina beta-2/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Smad/metabolismo , Células THP-1 , Microglobulina beta-2/farmacologiaRESUMO
Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta-gonad-mesonephros (AGM) niche. In-depth analysis of loss-of-function and transgenic reporter mouse models show that Kitl-deficient embryos harbor decreased numbers of yolk sac erythro-myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)-derived erythropoiesis, and a reduction in tissue-resident macrophages. Pre-HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss-of-function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.
Assuntos
Desenvolvimento Embrionário/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Fator de Células-Tronco/genética , Animais , Aorta/crescimento & desenvolvimento , Linhagem da Célula/genética , Proliferação de Células/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/crescimento & desenvolvimento , Mesonefro/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Nicho de Células-Tronco/genética , Saco Vitelino/crescimento & desenvolvimentoRESUMO
The paradigm that all blood cells are derived from hematopoietic stem cells (HSCs) has been challenged by two findings. First, there are tissue-resident hematopoietic cells, including subsets of macrophages that are not replenished by adult HSCs, but instead are maintained by self-renewal of fetal-derived cells. Second, during embryogenesis, there is a conserved program of HSC-independent hematopoiesis that precedes HSC function and is required for embryonic survival. The presence of waves of HSC-independent hematopoiesis as well as fetal HSCs raises questions about the origin of fetal-derived adult tissue-resident macrophages. In the murine embryo, historical examination of embryonic macrophage and monocyte populations combined with recent reports utilizing genetic lineage-tracing approaches has led to a model of macrophage ontogeny that can be integrated with existing models of hematopoietic ontogeny. The first wave of hematopoiesis contains primitive erythroid, megakaryocyte and macrophage progenitors that arise in the yolk sac, and these macrophage progenitors are the source of early macrophages throughout the embryo, including the liver. A second wave of multipotential erythro-myeloid progenitors (EMPs) also arises in the yolk sac. EMPs colonize the fetal liver, initiating myelopoiesis and forming macrophages. Lineage tracing indicates that this second wave of macrophages are distributed in most fetal tissues, although not appreciably in the brain. Thus, fetal-derived adult tissue-resident macrophages, other than microglia, appear to predominately derive from EMPs. While HSCs emerge at midgestation and colonize the fetal liver, the relative contribution of fetal HSCs to tissue macrophages at later stages of development is unclear. The inclusion of macrophage potential in multiple waves of hematopoiesis is consistent with reports of their functional roles throughout development in innate immunity, phagocytosis, and tissue morphogenesis and remodeling. Understanding the influences of developmental origin, as well as local tissue-specific signals, will be necessary to fully decode the diverse functions and responses of tissue-resident macrophages.
Assuntos
Hematopoese , Macrófagos/citologia , Animais , Linhagem da Célula , Humanos , Modelos ImunológicosRESUMO
The mechanisms regulating the sequential steps of terminal erythroid differentiation remain largely undefined, yet are relevant to human anemias that are characterized by ineffective red cell production. Erythroid Krüppel-like Factor (EKLF/KLF1) is a master transcriptional regulator of erythropoiesis that is mutated in a subset of these anemias. Although EKLF's function during early erythropoiesis is well studied, its role during terminal differentiation has been difficult to functionally investigate due to the impaired expression of relevant cell surface markers in Eklf(-/-) erythroid cells. We have circumvented this problem by an innovative use of imaging flow cytometry to investigate the role of EKLF in vivo and have performed functional studies using an ex vivo culture system that enriches for terminally differentiating cells. We precisely define a previously undescribed block during late terminal differentiation at the orthochromatic erythroblast stage for Eklf(-/-) cells that proceed beyond the initial stall at the progenitor stage. These cells efficiently decrease cell size, condense their nucleus, and undergo nuclear polarization; however, they display a near absence of enucleation. These late-stage Eklf(-/-) cells continue to cycle due to low-level expression of p18 and p27, a new direct target of EKLF. Surprisingly, both cell cycle and enucleation deficits are rescued by epistatic reintroduction of either of these 2 EKLF target cell cycle inhibitors. We conclude that the cell cycle as regulated by EKLF during late stages of differentiation is inherently critical for enucleation of erythroid precursors, thereby demonstrating a direct functional relationship between cell cycle exit and nuclear expulsion.
Assuntos
Núcleo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Eritroblastos/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Animais , Sítios de Ligação , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Eritroblastos/citologia , Eritropoese/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Adult-repopulating hematopoietic stem cells (HSCs) emerge in low numbers in the midgestation mouse embryo from a subset of arterial endothelium, through an endothelial-to-hematopoietic transition. HSC-producing arterial hemogenic endothelium relies on the establishment of embryonic blood flow and arterial identity, and requires ß-catenin signaling. Specified prior to and during the formation of these initial HSCs are thousands of yolk sac-derived erythro-myeloid progenitors (EMPs). EMPs ensure embryonic survival prior to the establishment of a permanent hematopoietic system, and provide subsets of long-lived tissue macrophages. While an endothelial origin for these HSC-independent definitive progenitors is also accepted, the spatial location and temporal output of yolk sac hemogenic endothelium over developmental time remain undefined. We performed a spatiotemporal analysis of EMP emergence, and document the morphological steps of the endothelial-to-hematopoietic transition. Emergence of rounded EMPs from polygonal clusters of Kit(+) cells initiates prior to the establishment of arborized arterial and venous vasculature in the yolk sac. Interestingly, Kit(+) polygonal clusters are detected in both arterial and venous vessels after remodeling. To determine whether there are similar mechanisms regulating the specification of EMPs with other angiogenic signals regulating adult-repopulating HSCs, we investigated the role of embryonic blood flow and Wnt/ß-catenin signaling during EMP emergence. In embryos lacking a functional circulation, rounded Kit(+) EMPs still fully emerge from unremodeled yolk sac vasculature. In contrast, canonical Wnt signaling appears to be a common mechanism regulating hematopoietic emergence from hemogenic endothelium. These data illustrate the heterogeneity in hematopoietic output and spatiotemporal regulation of primary embryonic hemogenic endothelium.
Assuntos
Endotélio Vascular/metabolismo , Hematopoese Extramedular/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Wnt/metabolismo , Saco Vitelino/metabolismo , Animais , Endotélio Vascular/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Transgênicos , Saco Vitelino/irrigação sanguínea , Saco Vitelino/citologiaRESUMO
In this issue of Blood, Toda et al present a shift in the paradigm of erythroid enucleation and provide novel tools to further study and optimize terminal erythroid maturation in vitro.
Assuntos
Eritroblastos/metabolismo , Eritrócitos/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Humanos , c-Mer Tirosina QuinaseRESUMO
Megakaryocyte (MK) development in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in the maturational chemotaxis of MKs toward sinusoidal vessels. Here, we demonstrate that both IV administration of SDF-1 and stabilization of endogenous SDF-1 acutely increase MK-vasculature association and thrombopoiesis with no change in MK number. In the setting of radiation injury, we find dynamic fluctuations in marrow SDF-1 distribution that spatially and temporally correlate with variations in MK niche occupancy. Stabilization of altered SDF-1 gradients directly affects MK location. Importantly, these SDF-1-mediated changes have functional consequences for platelet production, as the movement of MKs away from the vasculature decreases circulating platelets, while MK association with the vasculature increases circulating platelets. Finally, we demonstrate that manipulation of SDF-1 gradients can improve radiation-induced thrombocytopenia in a manner additive with earlier TPO treatment. Taken together, our data support the concept that SDF-1 regulates the spatial distribution of MKs in the marrow and consequently circulating platelet numbers. This knowledge of the microenvironmental regulation of the MK lineage could lead to improved therapeutic strategies for thrombocytopenia.
Assuntos
Movimento Celular , Quimiocina CXCL12/fisiologia , Megacariócitos/citologia , Megacariócitos/fisiologia , Lesões Experimentais por Radiação , Nicho de Células-Tronco/genética , Trombopoese/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Células da Medula Óssea/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/efeitos da radiação , Células Cultivadas , Quimiocina CXCL12/administração & dosagem , Feminino , Células Progenitoras de Megacariócitos/citologia , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Células Progenitoras de Megacariócitos/fisiologia , Células Progenitoras de Megacariócitos/efeitos da radiação , Megacariócitos/efeitos dos fármacos , Megacariócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/patologia , Receptores CXCR4/administração & dosagem , Receptores CXCR4/metabolismo , Nicho de Células-Tronco/efeitos dos fármacos , Nicho de Células-Tronco/efeitos da radiação , Trombopoese/efeitos dos fármacos , Trombopoese/efeitos da radiaçãoRESUMO
Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.
Assuntos
Células Eritroides/metabolismo , Eritropoese/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Animais , Aquaporina 1/genética , Aquaporina 3/genética , Aquaporinas/genética , Linhagem da Célula/genética , Células Cultivadas , Eritroblastos/metabolismo , Eritrócitos/metabolismo , Feminino , Sistema Hematopoético/citologia , Sistema Hematopoético/embriologia , Sistema Hematopoético/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Life-threatening thrombocytopenia can develop following bone marrow injury due to decreased platelet production from megakaryocytes (MKs). However, the study of primary MKs has been complicated by their low frequency in the bone marrow and by technical challenges presented by their unique maturation properties. More accurate and efficient methods for the analysis of in vivo MKs are needed to enhance our understanding of megakaryopoiesis and ultimately develop new therapeutic strategies for thrombocytopenia. Imaging flow cytometry (IFC) combines the morphometric capabilities of microscopy with the high-throughput analyses of flow cytometry (FC). Here, we investigate the application of IFC on the ImageStream(X) platform to the analysis of primary MKs isolated from murine bone marrow. Our data highlight and address technical challenges for conventional FC posed by the wide range of cellular size within the MK lineage as well as the shared surface phenotype with abundant platelet progeny. We further demonstrate that IFC can be used to reproducibly and efficiently quantify the frequency of primary murine MKs in the marrow, both at steady-state and in the setting of radiation-induced bone marrow injury, as well as assess their ploidy distribution. The ability to accurately analyze the full spectrum of maturing MKs in the bone marrow now allows for many possible applications of IFC to enhance our understanding of megakaryopoiesis and platelet production.
Assuntos
Citometria de Fluxo/métodos , Megacariócitos/citologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Erythropoiesis is a robust process of cellular expansion and maturation occurring in murine bone marrow and spleen. We previously determined that sublethal irradiation, unlike bleeding or hemolysis, depletes almost all marrow and splenic erythroblasts but leaves peripheral erythrocytes intact. To better understand the erythroid stress response, we analyzed progenitor, precursor, and peripheral blood compartments of mice post-4 Gy total body irradiation. Erythroid recovery initiates with rapid expansion of late-stage erythroid progenitors-day 3 burst-forming units and colony-forming units, associated with markedly increased plasma erythropoietin (EPO). Although initial expansion of late-stage erythroid progenitors is dependent on EPO, this cellular compartment becomes sharply down-regulated despite elevated EPO levels. Loss of EPO-responsive progenitors is associated temporally with a wave of maturing erythroid precursors in marrow and with emergence of circulating erythroid progenitors and subsequent reestablishment of splenic erythropoiesis. These circulating progenitors selectively engraft and mature in irradiated spleen after short-term transplantation, supporting the concept that bone marrow erythroid progenitors migrate to spleen. We conclude that sublethal radiation is a unique model of endogenous stress erythropoiesis, with specific injury to the extravascular erythron, expansion and maturation of EPO-responsive late-stage progenitors exclusively in marrow, and subsequent reseeding of extramedullary sites.
Assuntos
Medula Óssea/patologia , Células Precursoras Eritroides/citologia , Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Lesões Experimentais por Radiação/prevenção & controle , Baço/citologia , Animais , Medula Óssea/efeitos da radiação , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Transfusão de Eritrócitos , Células Precursoras Eritroides/efeitos da radiação , Eritropoese/efeitos da radiação , Eritropoetina/sangue , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/patologia , Baço/efeitos da radiação , Células-Tronco , Irradiação Corporal TotalRESUMO
Hematopoietic stem and progenitor cells (HSPCs), which continuously maintain all mature blood cells, are regulated within the marrow microenvironment. We previously reported that pharmacologic treatment of naïve mice with prostaglandin E2 (PGE2) expands HSPCs. However, the cellular mechanisms mediating this expansion remain unknown. Here, we demonstrate that PGE2 treatment in naïve mice inhibits apoptosis of HSPCs without changing their proliferation rate. In a murine model of sublethal total body irradiation (TBI), in which HSPCs are rapidly lost, treatment with a long-acting PGE2 analog (dmPGE2) reversed the apoptotic program initiated by TBI. dmPGE2 treatment in vivo decreased the loss of functional HSPCs following radiation injury, as demonstrated both phenotypically and by their increased reconstitution capacity. The antiapoptotic effect of dmPGE2 on HSPCs did not impair their ability to differentiate in vivo, resulting instead in improved hematopoietic recovery after TBI. dmPGE2 also increased microenvironmental cyclooxygenase-2 expression and expanded the α-smooth muscle actin-expressing subset of marrow macrophages, thus enhancing the bone marrow microenvironmental response to TBI. Therefore, in vivo treatment with PGE2 analogs may be particularly beneficial to HSPCs in the setting of injury by targeting them both directly and also through their niche. The current data provide rationale for in vivo manipulation of the HSPC pool as a strategy to improve recovery after myelosuppression.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Dinoprostona/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Lesões Experimentais por Radiação/tratamento farmacológico , Protetores contra Radiação/farmacologia , Actinas/genética , Actinas/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células da Medula Óssea/patologia , Células da Medula Óssea/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/efeitos da radiação , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Dinoprostona/análogos & derivados , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Macrófagos/patologia , Macrófagos/efeitos da radiação , Masculino , Camundongos , Camundongos Transgênicos , Lesões Experimentais por Radiação/imunologia , Lesões Experimentais por Radiação/patologia , Irradiação Corporal TotalRESUMO
The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient numbers of in vitro-derived red blood cells (RBC) for clinical purposes. We and others have determined that BMI1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts (SREs). However, the mechanisms of BMI1 action remain poorly understood. BMI1 overexpression led to 10 billion-fold increase BMI1-induced (i)SRE self-renewal. Despite prolonged culture and BMI1 overexpression, human iSREs can terminally mature and agglutinate with typing reagent monoclonal antibodies against conventional RBC antigens. BMI1 and RING1B occupancy, along with repressive histone marks, were identified at known BMI1 target genes, including the INK-ARF locus, consistent with an altered cell cycle following BMI1 inhibition. We also identified upregulated BMI1 target genes with low repressive histone modifications, including key regulator of cholesterol homeostasis. Functional studies suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. These findings support the hypothesis that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand the pool of immature erythroid precursors for eventual clinical uses.
RESUMO
Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes.
Assuntos
Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/fisiologia , Hematopoese/fisiologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/fisiologia , Animais , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , HumanosRESUMO
In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (10(2)- to 10(5)-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (10(6)- to 10(60)-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.
Assuntos
Diferenciação Celular , Eritroblastos/citologia , Feto/citologia , Mamíferos/embriologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Dexametasona/farmacologia , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Saco Vitelino/citologia , Saco Vitelino/efeitos dos fármacos , Saco Vitelino/metabolismoRESUMO
In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian ß-globin gene loci, and determined that within the murine locus, neither the ß-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic ß-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ε-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine ß-globin locus results in significant decrease in the expression of the embryonic ß-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for ß-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Globinas beta/genética , Acetilação , Animais , Imunoprecipitação da Cromatina , Embrião de Mamíferos , Células Eritroides/metabolismo , Expressão Gênica , Histonas/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult ß-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of "embryonic" ßH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with ßH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human ß-globin locus, like human fetal erythroblasts, express predominately human γ-, low ß-, and no ε-globin transcripts. Significantly, E9.5 yolk sac-derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only ß-globin transcripts. We conclude that yolk sac-derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.
Assuntos
Células Eritroides/citologia , Eritropoese/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Hematopoéticas/citologia , Fígado , Globinas beta/genética , Animais , Animais não Endogâmicos , Linhagem da Célula/fisiologia , Eritroblastos/citologia , Eritrócitos/citologia , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fígado/citologia , Fígado/embriologia , Fígado/fisiologia , Mamíferos , Camundongos , Camundongos Transgênicos , Saco Vitelino/fisiologiaRESUMO
Since the discovery that CXCR7 binds to CXCL12/SDF-1α, the role of CXCR7 in CXCL12-mediated biological processes has been under intensive scrutiny. However, there is no consensus in the literature on the expression of CXCR7 protein by peripheral blood cells. In this study we analyzed human and mouse leukocytes and erythrocytes for CXCR7 protein expression, using a competitive CXCL12 binding assay as well as by flow cytometry and immunohistochemistry using multiple CXCR7 Abs. CXCR7(-/-) mice were used as negative controls. Together, these methods indicate that CXCR7 protein is not expressed by human peripheral blood T cells, B cells, NK cells, or monocytes, or by mouse peripheral blood leukocytes. CXCR7 protein is, however, expressed on mouse primitive erythroid cells, which supply oxygen to the embryo during early stages of development. These studies therefore suggest that, whereas CXCR7 protein is expressed by primitive RBCs during murine embryonic development, in adult mammals CXCR7 protein is not expressed by normal peripheral blood cells.
Assuntos
Eritrócitos/metabolismo , Leucócitos/metabolismo , Receptores CXCR/biossíntese , Adulto , Animais , Separação Celular , Embrião de Mamíferos , Citometria de Fluxo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.