RESUMO
Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.
Assuntos
Benzoatos/farmacologia , Ciclopropanos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Repressoras/metabolismo , Animais , Benzoatos/uso terapêutico , Linhagem Celular Tumoral , Ciclopropanos/uso terapêutico , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/genética , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR mAb, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs, and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore, APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells.
Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Apelina , Receptores de Apelina , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Endoderma/fisiologia , Perfilação da Expressão Gênica , Hemangioblastos/efeitos dos fármacos , Hemangioblastos/metabolismo , Hemangioblastos/fisiologia , Hematopoese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Mesoderma/fisiologia , Análise em Microsséries , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Mesonefro/citologia , Mesonefro/embriologia , Neovascularização Fisiológica/fisiologia , Aorta/citologia , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Gônadas/citologia , Gônadas/embriologia , Gônadas/crescimento & desenvolvimento , Células-Tronco Hematopoéticas/fisiologia , Humanos , Mesonefro/crescimento & desenvolvimentoRESUMO
The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ß-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
Assuntos
Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , Células-Tronco Pluripotentes/metabolismo , Transgenes , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Células-Tronco Pluripotentes/citologia , Nucleases dos Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Leucemia de Células T/fisiopatologia , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Animais , Western Blotting , Imunoprecipitação da Cromatina , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Técnicas Histológicas , Proteínas de Homeodomínio/imunologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Janus Quinases/metabolismo , Estimativa de Kaplan-Meier , Cariotipagem , Luciferases , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-7/metabolismo , Proteínas Repressoras/imunologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5(GFP) was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5(GFP/w) human embryonic stem cells (hESCs). Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5(pos)CD34(pos) population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5(pos)SIRPA(pos) to NKX2-5(pos)SIRPA(pos)VCAM1(pos). Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.
Assuntos
Antígenos CD34/metabolismo , Antígenos de Diferenciação/metabolismo , Sistema Cardiovascular/crescimento & desenvolvimento , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores Imunológicos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Miócitos Cardíacos/metabolismoRESUMO
The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34(+)KDR(+) endothelial progenitor cells after 6days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.
Assuntos
Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células Endoteliais/citologia , Lisofosfolipídeos/farmacologia , Óxido Nítrico Sintase Tipo III/análise , Fator de Ativação de Plaquetas/farmacologia , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno/química , Combinação de Medicamentos , Células-Tronco Embrionárias/citologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Laminina/química , Óxido Nítrico Sintase Tipo III/metabolismo , Proteoglicanas/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
This unit describes a series of technical procedures to form clonal human embryonic stem cell (hESC) lines that are genetically modified by homologous recombination. To develop a reporter knock-in hESC line, a vector is configured to contain a reporter gene adjacent to a positive selection cassette. These core elements are flanked by homologous sequences that, following electroporation into hESCs, promote the integration of the vector into the appropriate genomic locus. The positive selection cassette facilitates the enrichment and isolation of genetically modified hESC colonies that are then screened by PCR to identify correctly targeted lines. The selection cassette, flanked by loxP sites, is subsequently excised from the positively targeted hESCs via the transient expression of Cre recombinase. This is necessary because the continued presence of the cassette may interfere with the regulation of the reporter or neighboring genes. Finally, these genetically modified hESCs are clonally isolated using single-cell deposition flow cytometry. Reporter knock-in hESC lines are valuable tools that allow easy and rapid identification and isolation of specific hESC derivatives.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Técnicas Genéticas , Recombinação Genética , Transgenes/genética , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Eletroporação , Citometria de Fluxo/métodos , Genes Reporter , Vetores Genéticos , Humanos , Modelos Genéticos , Mutagênese Insercional , Reação em Cadeia da PolimeraseRESUMO
This unit describes a protocol for the large-scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Animais , Proliferação de Células , Células Cultivadas , Humanos , CamundongosRESUMO
Differentiating human embryonic stem cells (HESCs) represent an experimental platform for establishing the relationships between the earliest lineages that emerge during human development. Here we report the targeted insertion in HESCs of sequences encoding green fluorescent protein (GFP) into the locus of MIXL1, a gene transiently expressed in the primitive streak during embryogenesis.(1,2) GFP fluorescence in MIXL1(GFP/)(w) HESCs differentiated in the presence of BMP4 reported the expression of MIXL1, permitting the identification of viable human primitive streak-like cells. The use of GFP as a reporter for MIXL1 combined with cell surface staining for platelet-derived growth factor receptor alpha (PDGFRalpha) enabled the isolation of a cell population that was highly enriched in primitive hematopoietic precursors, the earliest derivatives of the primitive streak. These experiments demonstrate the utility of MIXL1(GFP/w) HESCs for analyzing the previously inaccessible events surrounding the development of human primitive streak-like cells and their subsequent commitment to hematopoiesis.
Assuntos
Células-Tronco Embrionárias/fisiologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/genética , Animais , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Humanos , Modelos AnimaisRESUMO
The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.
Assuntos
Resistência Microbiana a Medicamentos/genética , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo/métodos , Técnicas de Transferência de Genes , Mutagênese Insercional/métodos , Vetores Genéticos/genética , HumanosRESUMO
The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.
Assuntos
Eletroporação/métodos , Células-Tronco Embrionárias/citologia , Linhagem Celular , Marcação de Genes/métodos , Engenharia Genética/métodos , Vetores Genéticos , HumanosRESUMO
Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.