RESUMEN
Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.
Asunto(s)
Anemia/inmunología , Enfermedades de los Peces/inmunología , Factor de Transcripción GATA1/metabolismo , Inflamasomas/metabolismo , Inflamación/inmunología , Neutrófilos/inmunología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Caspasa 1/genética , Caspasa 1/metabolismo , Diferenciación Celular , Células Eritroides/citología , Factor de Transcripción GATA1/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Humanos , Inflamasomas/genética , Células K562 , Masculino , Ratones , Ratones Endogámicos C57BL , Proteolisis , Proteínas de Pez Cebra/genéticaRESUMEN
JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.
Asunto(s)
Deficiencias de Hierro , Trastornos Mieloproliferativos , Policitemia Vera , Trombocitemia Esencial , Ratones , Animales , Hierro , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/diagnóstico , Policitemia Vera/genética , Janus Quinasa 2/genética , Trombocitemia Esencial/genética , Mutación , Fenotipo , Hemoglobinas/genéticaRESUMEN
We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.
Asunto(s)
Interferón-alfa/uso terapéutico , Janus Quinasa 2/genética , Megacariocitos/metabolismo , Trastornos Mieloproliferativos/genética , Animales , Técnicas de Sustitución del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Megacariocitos/citología , Ratones , Ratones Transgénicos , Trastornos Mieloproliferativos/tratamiento farmacológico , Glicoproteína IIb de Membrana Plaquetaria/genética , Mutación Puntual/efectos de los fármacosRESUMEN
The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Factor de Transcripción GATA1/metabolismo , Células Mieloides/citología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Eritrocitos/citología , Retroalimentación Fisiológica , Femenino , Genes Reporteros , Granulocitos/citología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Masculino , Megacariocitos/citología , Ratones , Modelos Biológicos , Monocitos/citología , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Procesos EstocásticosRESUMEN
The molecular mechanisms governing the transition from hematopoietic stem cells (HSCs) to lineage-committed progenitors remain poorly understood. Transcription factors (TFs) are powerful cell intrinsic regulators of differentiation and lineage commitment, while cytokine signaling has been shown to instruct the fate of progenitor cells. However, the direct regulation of differentiation-inducing hematopoietic TFs by cell extrinsic signals remains surprisingly difficult to establish. PU.1 is a master regulator of hematopoiesis and promotes myeloid differentiation. Here we report that tumor necrosis factor (TNF) can directly and rapidly upregulate PU.1 protein in HSCs in vitro and in vivo. We demonstrate that in vivo, niche-derived TNF is the principal PU.1 inducing signal in HSCs and is both sufficient and required to relay signals from inflammatory challenges to HSCs.
Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/metabolismo , Mielopoyesis , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Madre Hematopoyéticas/patología , Inflamación/metabolismo , Inflamación/patología , Ratones , Nicho de Células MadreRESUMEN
Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.
Asunto(s)
Rastreo Celular/métodos , Células Madre Hematopoyéticas/citología , Dispositivos Laboratorio en un Chip , Células Madre Embrionarias de Ratones/citología , Análisis de la Célula Individual/métodos , Animales , Adhesión Celular , Células Cultivadas , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/metabolismo , Prueba de Estudio Conceptual , Reproducibilidad de los Resultados , Imagen de Lapso de TiempoRESUMEN
Liquid handling robots have the potential to automate many procedures in life sciences. However, they are not in widespread use in academic settings, where funding, space and maintenance specialists are usually limiting. In addition, current robots require lengthy programming by specialists and are incompatible with most academic laboratories with constantly changing small-scale projects. Here, we present the Pipetting Helper Imaging Lid (PHIL), an inexpensive, small, open-source personal liquid handling robot. It is designed for inexperienced users, with self-production from cheap commercial and 3D-printable components and custom control software. PHIL successfully automates pipetting (incl. aspiration) for e.g. tissue immunostainings and stimulations of live stem and progenitor cells during time-lapse microscopy using 3D printed peristaltic pumps. PHIL is cheap enough to put a personal pipetting robot within the reach of most labs and enables users without programming skills to easily automate a large range of experiments.
Asunto(s)
Disciplinas de las Ciencias Biológicas , Robótica , Microscopía , Robótica/métodos , Programas InformáticosRESUMEN
Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.
Asunto(s)
Diferenciación Celular/genética , Células Madre Hematopoyéticas/metabolismo , Homeostasis/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Regulación hacia Arriba/genética , Animales , Células Cultivadas , Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente/métodos , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Imagen de Lapso de Tiempo/métodos , Transactivadores/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Inflamación/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Estrés Fisiológico/fisiología , Transactivadores/metabolismo , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Homeostasis/fisiología , Inmunidad Innata/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
E-cadherin is a calcium-dependent cell-cell adhesion molecule extensively studied for its involvement in tissue formation, epithelial cell behavior, and suppression of cancer. However, E-cadherin expression in the hematopoietic system has not been fully elucidated. Combining single-cell RNA-sequencing analyses and immunophenotyping, we revealed that progenitors expressing high levels of E-cadherin and contained within the granulocyte-monocyte progenitors (GMPs) fraction have an enriched capacity to differentiate into basophils and mast cells. We detected E-cadherin expression on committed progenitors before the expression of other reported markers of these lineages. We named such progenitors pro-BMPs (pro-basophil and mast cell progenitors). Using RNA sequencing, we observed transcriptional priming of pro-BMPs to the basophil and mast cell lineages. We also showed that GATA-2 directly regulates E-cadherin expression in the basophil and mast cell lineages, thus providing a mechanistic connection between the expression of this cell surface marker and the basophil and mast cell fate specification.
Asunto(s)
Cadherinas/genética , Factor de Transcripción GATA2/metabolismo , Células Madre Hematopoyéticas/fisiología , Animales , Basófilos/fisiología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , Mastocitos/fisiología , Ratones , Cultivo Primario de Células , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Cell fates are controlled by environmental stimuli that rapidly change the activity of intracellular signaling. Studying these processes requires rapid manipulations of micro-environmental conditions while continuously observing single cells over long periods of time. Current microfluidic devices are unable to simultaneously i) efficiently capture and concentrate rare cells, ii) conduct automated rapid media exchanges via diffusion without displacing non-adherent cells, and iii) allow sensitive high-throughput long-term time-lapse microscopy. Hematopoietic stem and progenitor cells pose a particular challenge for these types of experiments as they are impossible to obtain in very large numbers and are displaced by the fluid flow usually used to change culture media, thus preventing cell tracking. Here, we developed a programmable automated system composed of a novel microfluidic device for efficient capture of rare cells in independently addressable culture chambers, a custom incubation system, and user-friendly control software. The chip's culture chambers are optimized for efficient and sensitive fluorescence microscopy and their media can be individually and quickly changed by diffusion without non-adherent cell displacement. The chip allows efficient capture, stimulation, and sensitive high-frequency time-lapse observation of rare and sensitive murine and human primary hematopoietic stem cells. Our 3D-printed humidification and incubation system minimizes gas consumption, facilitates chip setup, and maintains stable humidity and gas composition during long-term cell culture. This approach now enables the required continuous long-term single-cell quantification of rare non-adherent cells with rapid environmental manipulations, e.g. of rapid signaling dynamics and the later stem cell fate choices they control.
Asunto(s)
Técnicas de Cultivo de Célula , Microfluídica , Animales , Rastreo Celular , Humanos , Dispositivos Laboratorio en un Chip , Ratones , Células MadreRESUMEN
The transcription factor (TF) GATA2 plays a key role in organ development and cell fate control in the central nervous, urogenital, respiratory, and reproductive systems, and in primitive and definitive hematopoiesis. Here, we generate a knockin protein reporter mouse line expressing a GATA2VENUS fusion from the endogenous Gata2 genomic locus, with correct expression and localization of GATA2VENUS in different organs. GATA2VENUS expression is heterogeneous in different hematopoietic stem and progenitor cell populations (HSPCs), identifies functionally distinct subsets, and suggests a novel monocyte and mast cell lineage bifurcation point. GATA2 levels further correlate with proliferation and lineage outcome of hematopoietic progenitors. The GATA2VENUS mouse line improves the identification of specific live cell types during embryonic and adult development and will be crucial for analyzing GATA2 protein dynamics in TF networks.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Genes Reporteros , Células Madre Hematopoyéticas/metabolismo , Envejecimiento/genética , Animales , Linaje de la Célula , Proliferación Celular , Embrión de Mamíferos/metabolismo , Factor de Transcripción GATA2/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Hematopoyesis , Mastocitos/citología , Ratones , Modelos Biológicos , Monocitos/citología , Neutrófilos/citología , Especificidad de Órganos , Factores de Transcripción/metabolismoRESUMEN
Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.