RESUMEN
During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and identifies NO as a conserved downstream regulator of HSC development.
Asunto(s)
Fenómenos Fisiológicos Sanguíneos , Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Pez CebraRESUMEN
Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery.
Asunto(s)
Dinoprostona/metabolismo , Desarrollo Embrionario , Células Madre Hematopoyéticas/metabolismo , Proteínas Wnt/metabolismo , Pez Cebra/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Células Madre Embrionarias/metabolismo , Hígado/fisiología , Ratones , Regeneración , Transducción de Señal , Pez Cebra/embriología , beta Catenina/metabolismoRESUMEN
All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.
Asunto(s)
Células Madre Embrionarias/citología , Silenciador del Gen , Hematopoyesis , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Células Madre Multipotentes/citología , Complejo Represivo Polycomb 2/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Cromatina/genética , Cromatina/metabolismo , Desarrollo Embrionario , Femenino , Humanos , Linfocitos/metabolismo , Ratones , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/deficiencia , Complejo Represivo Polycomb 2/genéticaRESUMEN
Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-Ï activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Transducción de Señal , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Proliferación Celular/genética , Células Cultivadas , Citocinas/inmunología , Embrión de Mamíferos , Embrión no Mamífero , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/genética , Inflamación/inmunología , Interferones/genética , Interferones/metabolismo , Ratones , Pez Cebra/embriologíaRESUMEN
BACKGROUND AND AIMS: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions. APPROACH AND RESULTS: Exposure of zebrafish embryos to 17ß-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b-/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development. CONCLUSIONS: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.
Asunto(s)
Sistema Biliar/embriología , Estradiol/metabolismo , Receptor beta de Estrógeno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hígado/embriología , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Sistema Biliar/citología , Sistema Biliar/metabolismo , Diferenciación Celular/genética , Línea Celular , Embrión no Mamífero , Estradiol/administración & dosificación , Receptor beta de Estrógeno/genética , Femenino , Técnicas de Silenciamiento del Gen , Hepatocitos/fisiología , Hígado/citología , Hígado/metabolismo , Masculino , Modelos Animales , Morfolinos/administración & dosificación , Morfolinos/genética , Transducción de Señal/genética , Células Madre/fisiología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proto-Oncogenes/fisiología , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Aorta/metabolismo , Proteínas de Unión al ADN/genética , Diaminas/farmacología , Embrión no Mamífero , Células Endoteliales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proto-Oncogenes/genética , Receptores Notch/metabolismo , Tiazoles/farmacología , Factores de Transcripción/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND & AIMS: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. METHODS: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein-coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. RESULTS: Exposure to 17ß-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. CONCLUSIONS: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. TRANSCRIPT PROFILING: The accession number in the Gene Expression Omnibus is GSE92544.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Neoplasias Hepáticas/metabolismo , Hígado/crecimiento & desarrollo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Pez Cebra/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinogénesis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Hepatocitos , Humanos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/patología , Regeneración Hepática , Masculino , Tamaño de los Órganos/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Receptores Acoplados a Proteínas G/genética , Factores Sexuales , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Carga Tumoral/efectos de los fármacos , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.
Asunto(s)
Hígado/embriología , Hígado/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Cannabinoides/metabolismo , Recuento de Células , Proliferación Celular/efectos de los fármacos , Cisteína/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Metabolómica , Metionina/metabolismo , Mutación/genética , Tamaño de los Órganos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.
Asunto(s)
Células Acinares/citología , Hepatocitos/citología , Hepatopáncreas/embriología , Hígado/embriología , Páncreas Exocrino/embriología , Receptores Citoplasmáticos y Nucleares/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Diferenciación Celular/genética , Endodermo/citología , Proteínas de Unión a Ácidos Grasos/metabolismo , Técnicas de Silenciamiento del Gen , Factor Nuclear 4 del Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Morfolinos/genética , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Transactivadores/genética , Factores de Transcripción/genética , Tripsina/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismoRESUMEN
Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.
Asunto(s)
Dinoprostona/metabolismo , Células Madre Hematopoyéticas/metabolismo , Selectina-P/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Células Madre Hematopoyéticas/citología , Transducción de Señal/fisiologíaRESUMEN
Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.
Asunto(s)
Análisis Mutacional de ADN/métodos , Programas Informáticos , Pez Cebra/genética , Alelos , Animales , Mapeo Cromosómico/métodos , Cromosomas/genética , Cruzamientos Genéticos , Femenino , Frecuencia de los Genes , Genómica/métodos , Homocigoto , Masculino , Cadenas de Markov , Ratones , Ratones Endogámicos C57BL , Mutación , Polimorfismo de Nucleótido Simple , Recombinación Genética , Factores de TiempoRESUMEN
Many pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrial-derived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability.
Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Inducción Embrionaria , Glucosa/metabolismo , Células Madre Hematopoyéticas/fisiología , Animales , Animales Modificados Genéticamente , Metabolismo de los Hidratos de Carbono/genética , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero , Inducción Embrionaria/efectos de los fármacos , Inducción Embrionaria/genética , Regulación del Desarrollo de la Expresión Génica , Glucosa/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Glucólisis/fisiología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Fosforilación Oxidativa , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants.
Asunto(s)
16,16-Dimetilprostaglandina E2/farmacología , Plaquetas/efectos de los fármacos , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Sangre Fetal/efectos de los fármacos , Supervivencia de Injerto/inmunología , Neoplasias Hematológicas/terapia , Adulto , Anciano , Plaquetas/citología , Plaquetas/inmunología , Células Cultivadas , Criopreservación , Femenino , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/trasplante , Perfilación de la Expresión Génica , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/patología , Humanos , Masculino , Persona de Mediana Edad , Quimera por Trasplante , Trasplante Homólogo , Resultado del TratamientoRESUMEN
Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo, and human hepatocyte sourcing has limited many fields of research for decades. Here we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. The first class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted the differentiation of induced pluripotent stem cell-derived hepatocytes toward a more mature phenotype than what was previously obtainable. The identification of these small molecules can help address a major challenge affecting many facets of liver research and may lead to the development of new therapeutics for liver diseases.
Asunto(s)
Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/metabolismo , Epistasis Genética , Eritropoyesis/genética , Evolución Molecular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Sistema Hematopoyético/embriología , Sistema Hematopoyético/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Pez Cebra/embriología , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
Developmental signals determine organ morphology and position during embryogenesis. To discover novel modifiers of liver development, we performed a chemical genetic screen in zebrafish and identified retinoic acid as a positive regulator of hepatogenesis. Knockdown of the four RA receptors revealed that all receptors affect liver formation, however specific receptors exert differential effects. Rargb knockdown results in bilateral livers but does not impact organ size, revealing a unique role for Rargb in conferring left-right positional information. Bilateral populations of hepatoblasts are detectable in rargb morphants, indicating Rargb acts during hepatic specification to position the liver, and primitive endoderm is competent to form liver on both sides. Hearts remain at the midline and gut looping is perturbed in rargb morphants, suggesting Rargb affects lateral plate mesoderm migration. Overexpression of Bmp during somitogenesis similarly results in bilateral livers and midline hearts, and inhibition of Bmp signaling rescues the rargb morphant phenotype, indicating Rargb functions upstream of Bmp to regulate organ sidedness. Loss of rargb causes biliary and organ laterality defects as well as asplenia, paralleling symptoms of the human condition right atrial isomerism. Our findings uncover a novel role for RA in regulating organ laterality and provide an animal model of one form of human heterotaxia.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Receptores de Ácido Retinoico/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Embrión no Mamífero/metabolismo , Hígado/embriología , Hígado/metabolismo , Modelos Animales , Proteína Nodal/metabolismo , Fenotipo , Receptores de Ácido Retinoico/genética , Transducción de Señal , Tretinoina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Receptor de Ácido Retinoico gammaRESUMEN
Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes.
Asunto(s)
Dinoprostona/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Homeostasis/efectos de los fármacos , Vertebrados , Animales , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Dinoprostona/agonistas , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Proteínas Proto-Oncogénicas c-myb/genética , Vertebrados/embriología , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
Acetaminophen (APAP) toxicity is the most common drug-induced cause of acute liver failure in the United States. The only available treatment, N-acetylcysteine (NAC), has a limited time window of efficacy, indicating a need for additional therapeutic options. Zebrafish have emerged as a powerful tool for drug discovery. Here, we developed a clinically relevant zebrafish model of APAP toxicity. APAP depleted glutathione stores, elevated aminotransferase levels, increased apoptosis, and caused dose-dependent hepatocyte necrosis. These outcomes were limited by NAC and conserved in zebrafish embryos. In a targeted embryonic chemical screen, prostaglandin E2 (PGE2) was identified as a potential therapeutic agent; in the adult, PGE2 similarly decreased APAP-associated toxicity. Significantly, when combined with NAC, PGE2 extended the time window for a successful intervention, synergistically reducing apoptosis, improving liver enzymes, and preventing death. Use of a wnt reporter zebrafish line and chemical genetic epistasis showed that the effects of PGE2 are mediated through the wnt signaling pathway. Zebrafish can be used as a clinically relevant toxicological model amenable to the identification of additional therapeutics and biomarkers of APAP injury; our data suggest combinatorial PGE2 and NAC treatment would be beneficial for patients with APAP-induced liver damage.
Asunto(s)
Acetaminofén/toxicidad , Acetilcisteína , Enfermedad Hepática Inducida por Sustancias y Drogas , Dinoprostona/metabolismo , Fallo Hepático Agudo , Transducción de Señal/fisiología , Pez Cebra , Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Analgésicos no Narcóticos/toxicidad , Animales , Animales Modificados Genéticamente , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Genes Reporteros , Glutatión/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Fallo Hepático Agudo/tratamiento farmacológico , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Proteoma/análisis , Pez Cebra/anatomía & histología , Pez Cebra/fisiologíaRESUMEN
BACKGROUND: Liver function tests (LFTs) are elevated in >50% of hospitalized individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), with increased enzyme levels correlating with a more severe COVID-19 course. Despite these observations, evaluations of viral presence within liver parenchyma and viral impact on liver function remain controversial. METHODS AND RESULTS: Our work is a comprehensive immunopathological evaluation of liver tissue from 33 patients with severe, and ultimately fatal, cases of SARS-CoV-2 infection. Coupled with clinical data, we reveal the absence of SARS-CoV-2 infection in cholangiocytes and hepatocytes despite dramatic systemic viral presence. Critically, we identify significant focal viral sinusoidal aggregates in 2/33 patients and single viral RNA molecules circulating in the hepatic sinusoids of 15/33 patients. Utilizing co-immunofluorescence, focal viral liver aggregates in patients with COVID-19 were colocalized to platelet and fibrin clots, indicating the presence of virus-containing sinusoidal microthrombi. Furthermore, this patient cohort, from the initial months of the COVID-19 pandemic, demonstrates a general downtrend of LFTs over the course of the study timeline and serves as a remarkable historical time point of unattenuated viral replication within patients. CONCLUSIONS: Together, our findings indicate that elevated LFTs found in our patient cohort are not due to direct viral parenchymal infection with SARS-CoV-2 but rather likely a consequence of systemic complications of COVID-19. This work aids in the clinical treatment considerations of patients with SARS-CoV-2 as therapies for these patients may be considered in terms of their direct drug hepatotoxity rather than worsening hepatic function due to direct infection.