RESUMEN
The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.
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Autorrenovación de las Células , Células Madre Hematopoyéticas , Proteínas Nucleares , Animales , Femenino , Humanos , Masculino , Ratones , Células Cultivadas , Endocitosis , Endosomas/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Sangre Fetal/citología , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/embriología , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Developmental hematopoiesis consists of multiple, partially overlapping hematopoietic waves that generate the differentiated blood cells required for embryonic development while establishing a pool of undifferentiated hematopoietic stem cells (HSCs) for postnatal life. This multilayered design in which active hematopoiesis migrates through diverse extra and intraembryonic tissues has made it difficult to define a roadmap for generating HSCs vs non-self-renewing progenitors, especially in humans. Recent single-cell studies have helped in identifying the rare human HSCs at stages when functional assays are unsuitable for distinguishing them from progenitors. This approach has made it possible to track the origin of human HSCs to the unique type of arterial endothelium in the aorta-gonad-mesonephros region and document novel benchmarks for HSC migration and maturation in the conceptus. These studies have delivered new insights into the intricate process of HSC generation and provided tools to inform the in vitro efforts to replicate the physiological developmental journey from pluripotent stem cells via distinct mesodermal and endothelial intermediates to HSCs.
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Embrión de Mamíferos , Células Madre Hematopoyéticas , Femenino , Embarazo , Humanos , Hematopoyesis/fisiología , MesonefroRESUMEN
The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.
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Células Endoteliales , Células Madre Hematopoyéticas , Diferenciación Celular , Endotelio , Femenino , Hematopoyesis , Humanos , Mesonefro , EmbarazoRESUMEN
Atkins et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20211924) create a PSC differentiation model for human yolk sac hematopoiesis and discover multipotent progenitors with erythro-myeloid and T lymphoid potential. The multipotent progenitors emerge via hemogenic endothelium and share origin with primitive erythroid wave in KDR+CD235a/b+ mesoderm.
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Hematopoyesis , Saco Vitelino , Humanos , MesodermoRESUMEN
Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet ß-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic ß-cells via the αvß3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.
RESUMEN
Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation1. Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or progenitor cells (HSPCs) in culture, whereas stabilizing MLLT3 expression in culture enabled more than 12-fold expansion of transplantable HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Similar to endogenous MLLT3, overexpressed MLLT3 localized to active promoters in HSPCs, sustained levels of H3K79me2 and protected the HSC transcriptional program in culture. MLLT3 thus acts as HSC maintenance factor that links histone reader and modifying activities to modulate HSC gene expression, and may provide a promising approach to expand HSCs for transplantation.
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Autorrenovación de las Células , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Proteínas Nucleares/genética , Unión ProteicaRESUMEN
Hematopoietic stem cells (HSCs) are multipotent cells responsible for the maintenance of the hematopoietic system throughout life. Dysregulation of the balance in HSC self-renewal, death, and differentiation can have serious consequences such as myelodysplastic syndromes or leukemia. All-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A/RA, has been shown to have pleiotropic effects on hematopoietic cells, enhancing HSC self-renewal while also increasing differentiation of more mature progenitors. Furthermore, ATRA has been shown to have key roles in regulating the specification and formation of hematopoietic cells from pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Here, we summarize the known roles of vitamin A and RA receptors in the regulation of hematopoiesis from HSCs, ES, and iPSCs.
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Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Retinoides/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Humanos , Modelos Biológicos , Receptores de Ácido Retinoico/fisiología , Transducción de SeñalRESUMEN
BACKGROUND: Genetic diversity and the heterogeneous nature of cardiac fibroblasts (CFbs) have hindered characterization of the molecular mechanisms that regulate cardiac fibrosis. The Hybrid Mouse Diversity Panel offers a valuable tool to examine genetically diverse cardiac fibroblasts and their role in fibrosis. METHODS: Three strains of mice (C57BL/6J, C3H/HeJ, and KK/HlJ) were selected from the Hybrid Mouse Diversity Panel and treated with either isoproterenol (ISO) or saline by an intraperitoneally implanted osmotic pump. After 21 days, cardiac function and levels of fibrosis were measured by echocardiography and trichrome staining, respectively. Activation and proliferation of CFbs were measured by in vitro and in vivo assays under normal and injury conditions. RNA sequencing was done on isolated CFbs from each strain. Results were analyzed by Ingenuity Pathway Analysis and validated by reverse transcription-qPCR, immunohistochemistry, and ELISA. RESULTS: ISO treatment in C57BL/6J, C3H/HeJ, and KK/HlJ mice resulted in minimal, moderate, and extensive levels of fibrosis, respectively (n=7-8 hearts per condition). Isolated CFbs treated with ISO exhibited strain-specific increases in the levels of activation but showed comparable levels of proliferation. Similar results were found in vivo, with fibroblast activation, and not proliferation, correlating with the differential levels of cardiac fibrosis after ISO treatment. RNA sequencing revealed that CFbs from each strain exhibit unique gene expression changes in response to ISO. We identified Ltbp2 as a commonly upregulated gene after ISO treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure patients. CONCLUSIONS: This study highlights the importance of genetic variation in cardiac fibrosis by using multiple inbred mouse strains to characterize CFbs and their response to ISO treatment. Our data suggest that, although fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. In addition, by comparing CFbs from multiple strains, we identified pathways as potential therapeutic targets and LTBP2 as a marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.
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Cardiomiopatías/genética , Cardiomiopatías/patología , Proliferación Celular , Fibroblastos/patología , Variación Genética , Proteínas de Unión a TGF-beta Latente/genética , Animales , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibrosis , Predisposición Genética a la Enfermedad , Isoproterenol , Proteínas de Unión a TGF-beta Latente/metabolismo , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fenotipo , Especificidad de la Especie , TranscriptomaRESUMEN
The cellular mechanisms driving cardiac tissue formation remain poorly understood, largely due to the structural and functional complexity of the heart. It is unclear whether newly generated myocytes originate from cardiac stem/progenitor cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Here, we identify the source of new cardiomyocytes during mouse development and after injury. Our findings suggest that cardiac progenitors maintain proliferative potential and are the main source of cardiomyocytes during development; however, the onset of αMHC expression leads to reduced cycling capacity. Single-cell RNA sequencing reveals a proliferative, "progenitor-like" population abundant in early embryonic stages that decreases to minimal levels postnatally. Furthermore, cardiac injury by ligation of the left anterior descending artery was found to activate cardiomyocyte proliferation in neonatal but not adult mice. Our data suggest that clonal dominance of differentiating progenitors mediates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late embryonic development and shortly after birth.
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Lesiones Cardíacas/patología , Corazón/crecimiento & desarrollo , Miocitos Cardíacos/citología , Animales , Animales Recién Nacidos , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Madre Embrionarias/citología , Femenino , Corazón Fetal/citología , Corazón Fetal/crecimiento & desarrollo , Lesiones Cardíacas/genética , Masculino , Ratones , Ratones Transgénicos , Mioblastos Cardíacos/citología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocitos Cardíacos/metabolismo , Pericardio/citología , Pericardio/embriología , Pericardio/crecimiento & desarrollo , Embarazo , Análisis de Secuencia de ARNRESUMEN
The transcription factor hepatic leukemia factor (HLF) is strongly expressed in hematopoietic stem cells (HSCs) and is thought to influence both HSC self-renewal and leukemogenesis. However, the physiological role of HLF in hematopoiesis and HSC function is unclear. Here, we report that mice lacking Hlf are viable with essentially normal hematopoietic parameters, including an intact HSC pool during steady-state hematopoiesis. In contrast, when challenged through transplantation, Hlf-deficient HSCs showed an impaired ability to reconstitute hematopoiesis and became gradually exhausted upon serial transplantation. Transcriptional profiling of Hlf-deficient HSCs revealed changes associated with enhanced cellular activation, and cell-cycle analysis demonstrated a significant reduction of quiescent HSCs. Accordingly, toxic insults targeting dividing cells completely eradicated the HSC pool in Hlf-deficient mice. In summary, our findings point to HLF as a critical regulator of HSC quiescence and as an essential factor for maintaining the HSC pool during regeneration.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proliferación Celular , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Animales , Apoptosis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células Cultivadas , Daño del ADN , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , TranscriptomaRESUMEN
The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.
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Células Madre Embrionarias/citología , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/metabolismo , Mesonefro/citología , Mesonefro/embriología , Neovascularización Fisiológica/fisiología , Aorta/citología , Aorta/embriología , Aorta/crecimiento & desarrollo , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/fisiología , Gónadas/citología , Gónadas/embriología , Gónadas/crecimiento & desarrollo , Células Madre Hematopoyéticas/fisiología , Humanos , Mesonefro/crecimiento & desarrolloRESUMEN
The contribution of the different waves and sites of developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre. Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%-40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1-/- concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5-13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization.
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Linaje de la Célula , Células Eritroides/citología , Células Eritroides/metabolismo , Hemangioblastos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Saco Vitelino/metabolismo , Envejecimiento , Animales , Eritropoyesis , Femenino , Feto/citología , Eliminación de Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Integrasas/metabolismo , Hígado/embriología , Ratones Endogámicos C57BL , Embarazo , Factores de TiempoRESUMEN
DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.
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Roturas del ADN de Doble Cadena , Hematopoyesis/fisiología , Células Precursoras de Linfocitos B/fisiología , Recombinación V(D)J/fisiología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Cromatina/metabolismo , Femenino , Fluorouracilo/farmacología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Factores de Transcripción MEF2/fisiología , Masculino , Ratones , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/efectos de la radiación , Irradiación Corporal Total/efectos adversosRESUMEN
Vascular endothelial growth factor C (VEGF-C) is a major driver of lymphangiogenesis in embryos and adults. Vegfc gene deletion in mouse embryos results in failure of lymphangiogenesis, fluid accumulation in tissues, and lethality. The VEGF-C receptors VEGFR3 and VEGFR2 are required for embryonic blood vessel formation. The related VEGF is essential for both blood vessel formation and embryonic hematopoiesis, whereas the possible involvement of VEGF-C in hematopoiesis is unknown. Here we unveil a novel hematopoietic function of VEGF-C in fetal erythropoiesis. Deletion of Vegfc in embryonic day 7.5 (E7.5) embryos in the C57BL6 mouse genetic background led to defective fetal erythropoiesis, characterized by anemia and lack of enucleated red blood cells in blood circulation. Macrophages and erythroid cells in the fetal liver (FL) were also decreased after midgestation because of decreased cell proliferation and increased apoptosis. However, the Lin(-)Sca-1(+)c-Kit(+) stem cell compartment in E14.5 FL was not affected by Vegfc deletion. VEGF-C loss did not disrupt the generation of primitive erythroid cells or erythro-myeloid progenitors (EMPs) in the yolk sac, but it decreased the expression of α4-integrin on EMPs and compromised EMP colonization of the FL. The distribution, maturation, and enucleation of primitive erythroblasts were also impaired by Vegfc deletion. In contrast, Vegfc deletion from E10.5 onward did not compromise definitive hematopoiesis in the liver, and Vegfc deletion in adult mice did not cause anemia. These results reveal an unexpected role for VEGF-C, a major lymphangiogenic growth factor, in the transition to FL erythropoiesis.
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Eritropoyesis , Feto/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Anemia/patología , Animales , Apoptosis , Linaje de la Célula , Proliferación Celular , Embrión de Mamíferos/metabolismo , Células Eritroides/metabolismo , Eliminación de Gen , Hepatocitos/metabolismo , Integrina alfa4/metabolismo , Hígado/irrigación sanguínea , Hígado/embriología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Tamaño de los ÓrganosRESUMEN
Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34(+)CD38(-/lo)CD90(+)CD45(+)GPI-80(+) fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function.
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Diferenciación Celular/fisiología , Linaje de la Célula , Genes Homeobox/genética , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/metabolismo , Células Madre Multipotentes/citología , Antígenos CD34/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica/métodos , Proteínas de Homeodominio/genética , Humanos , Antígenos Comunes de Leucocito/metabolismo , TranscriptomaRESUMEN
Reporting in Developmental Cell, Pereira et al. (2016) use in vitro lineage reprogramming insights to inform understanding of hematopoietic stem cell (HSC) development in vivo. They find Prom1(+)Sca1(+)CD34(+)CD45(-) hemogenic precursors, akin to fibroblast-derived hemato-vascular precursors, in mouse placenta and embryo. The cells mature into transplantable HSCs in culture.
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Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Reprogramación Celular , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Embrionarias de Ratones/citología , Animales , Femenino , EmbarazoRESUMEN
BACKGROUND: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication. METHODS: To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment. RESULTS: Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC. CONCLUSION: In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.
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Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Leucemia/patología , Células Madre Neoplásicas/patología , Proteínas Nucleares/antagonistas & inhibidores , Animales , Secuencia de Bases , Técnicas de Cocultivo , Cartilla de ADN , Sangre Fetal/citología , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma , Proteína Gli2 con Dedos de ZincRESUMEN
Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.
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Hematopoyesis/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Sirtuina 1/genética , Sirtuina 1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation.
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Permeabilidad Capilar , Endotelio Vascular/metabolismo , Útero/metabolismo , Animales , Endometrio/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Ratones , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genéticaRESUMEN
Stem cells ensure the maintenance of tissue homeostasis throughout life by tightly regulating their self-renewal and differentiation. In a recent study published in Nature, Nakada et al, 2014 unveil an unexpected endocrine mechanism that regulates hematopoietic stem cell (HSC) self-renewal.