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1.
Nat Immunol ; 25(6): 1007-1019, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38816617

RESUMO

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.


Assuntos
Plaquetas , Diferenciação Celular , Células-Tronco Hematopoéticas , Megacariócitos , Plaquetas/imunologia , Plaquetas/metabolismo , Animais , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Diferenciação Celular/imunologia , Megacariócitos/citologia , Linhagem da Célula , Camundongos Endogâmicos C57BL , Hematopoese , Trombopoese , Camundongos Knockout , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/imunologia
2.
Nat Neurosci ; 26(5): 891-901, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37095395

RESUMO

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.


Assuntos
Ependimoma , Células-Tronco Neurais , Criança , Feminino , Gravidez , Humanos , Medula Espinal , Ependimoma/genética , Ependimoma/metabolismo , Diferenciação Celular/genética , Células-Tronco Neurais/fisiologia , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética
3.
Nature ; 597(7875): 196-205, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497388

RESUMO

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.


Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologia
4.
Front Immunol ; 11: 594243, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362776

RESUMO

The pathological mechanisms that lead to the onset and reactivation of celiac disease (CD) remain largely unknown. While gluten free diet (GFD) improves the intestinal damage and associated clinical symptoms in majority of cases, it falls short of providing full recovery. Additionally, late or misdiagnosis is also common as CD presents with a wide range of symptoms. Clear understanding of CD pathogenesis is thus critical to address both diagnostic and treatment concerns. We aimed to study the molecular impact of short gluten exposure in GFD treated CD patients, as well as identify biological pathways that remain altered constitutively in CD regardless of treatment. Using RNAseq profiling of PBMC samples collected from treated CD patients and gluten challenged patient and healthy controls, we explored the peripheral transcriptome in CD patients following a short gluten exposure. Short gluten exposure of just three days was enough to alter the genome-wide PBMC transcriptome of patients. Pathway analysis revealed gluten-induced upregulation of mainly immune response related pathways, both innate and adaptive, in CD patients. We evaluated the perturbation of biological pathways in sample-specific manner. Compared to gluten exposed healthy controls, pathways related to tight junction, olfactory transduction, metabolism of unsaturated fatty acids (such as arachidonic acid), metabolism of amino acids (such as cysteine and glutamate), and microbial infection were constitutively altered in CD patients regardless of treatment, while GFD treatment appears to mostly normalize immune response pathways to "healthy" state. Upstream regulator prediction analysis using differentially expressed genes identified constitutively activated regulators relatively proximal to previously reported CD associated loci, particularly SMARCA4 on 19p13.2 and CSF2 on 5q31. We also found constitutively upregulated genes in CD that are in CD associated genetic loci such as MEF2BNB-MEF2B (BORCS8-MEF2B) on 19p13.11 and CSTB on 21q22.3. RNAseq revealed strong effects of short oral gluten challenge on whole PBMC fraction and constitutively altered pathways in CD PBMC suggesting important factors other than gluten in CD pathogenesis.


Assuntos
Doença Celíaca/etiologia , Glutens/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Transcriptoma , Adulto , Idoso , Doença Celíaca/metabolismo , Doença Celíaca/terapia , Biologia Computacional/métodos , Dietoterapia , Dieta Livre de Glúten , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
Nature ; 587(7834): 377-386, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32894860

RESUMO

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Atenção à Saúde/métodos , Atenção à Saúde/tendências , Medicina/métodos , Medicina/tendências , Patologia , Análise de Célula Única , Inteligência Artificial , Atenção à Saúde/ética , Atenção à Saúde/normas , Diagnóstico Precoce , Educação Médica , Europa (Continente) , Feminino , Saúde , Humanos , Legislação Médica , Masculino , Medicina/normas
6.
Nat Commun ; 10(1): 4137, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515492

RESUMO

Developmental cell death plays an important role in the construction of functional neural circuits. In vertebrates, the canonical view proposes a selection of the surviving neurons through stochastic competition for target-derived neurotrophic signals, implying an equal potential for neurons to compete. Here we show an alternative cell fitness selection of neurons that is defined by a specific neuronal heterogeneity code. Proprioceptive sensory neurons that will undergo cell death and those that will survive exhibit different molecular signatures that are regulated by retinoic acid and transcription factors, and are independent of the target and neurotrophins. These molecular features are genetically encoded, representing two distinct subgroups of neurons with contrasted functional maturation states and survival outcome. Thus, in this model, a heterogeneous code of intrinsic cell fitness in neighboring neurons provides differential competitive advantage resulting in the selection of cells with higher capacity to survive and functionally integrate into neural networks.


Assuntos
Modelos Biológicos , Células Receptoras Sensoriais/citologia , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Camundongos Endogâmicos C57BL , Propriocepção/efeitos dos fármacos , Receptor trkC/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia
7.
Nat Commun ; 9(1): 3647, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194434

RESUMO

Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5 h on-chip processing time) and library preparation cost (~81¢ per cell) compared to prior microfluidic implementations. We apply this method to measure regulatory variation in peripheral blood mononuclear cells (PBMCs) and show robust, de novo clustering of single cells by hematopoietic cell type.


Assuntos
Montagem e Desmontagem da Cromatina , Ensaios de Triagem em Larga Escala , Imagem Óptica/métodos , Análise de Célula Única/métodos , Animais , Linhagem Celular , Epigênese Genética , Humanos , Camundongos
8.
PLoS Biol ; 16(6): e2006387, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29912866

RESUMO

Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously described classes unexpectedly found to be identical. A division into discrete classes, however, was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.


Assuntos
Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Neurônios GABAérgicos/classificação , Neurônios GABAérgicos/metabolismo , Potenciais de Ação , Algoritmos , Animais , Quimiocinas CXC/genética , Dendritos/metabolismo , Neurônios GABAérgicos/citologia , Interneurônios/citologia , Interneurônios/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Neurológicos , Células Piramidais/citologia , Células Piramidais/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Análise de Sequência de RNA , Análise de Célula Única , Transmissão Sináptica , Transcriptoma , Peptídeo Intestinal Vasoativo/genética
9.
BMC Cancer ; 18(1): 174, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29433456

RESUMO

BACKGROUND: Tumor heterogeneity in breast cancer tumors is today widely recognized. Most of the available knowledge in genetic variation however, relates to the primary tumor while metastatic lesions are much less studied. Many studies have revealed marked alterations of standard prognostic and predictive factors during tumor progression. Characterization of paired primary- and metastatic tissues should therefore be fundamental in order to understand mechanisms of tumor progression, clonal relationship to tumor evolution as well as the therapeutic aspects of systemic disease. METHODS: We performed full exome sequencing of primary breast cancers and their metastases in a cohort of ten patients and further confirmed our findings in an additional cohort of 20 patients with paired primary and metastatic tumors. Furthermore, we used gene expression from the metastatic lesions and a primary breast cancer data set to study the gene expression of the AKAP gene family. RESULTS: We report that somatic mutations in A-kinase anchoring proteins are enriched in metastatic lesions. The frequency of mutation in the AKAP gene family was 10% in the primary tumors and 40% in metastatic lesions. Several copy number variations, including deletions in regions containing AKAP genes were detected and showed consistent patterns in both investigated cohorts. In a second cohort containing 20 patients with paired primary and metastatic lesions, AKAP mutations showed an increasing variant allele frequency after multiple relapses. Furthermore, gene expression profiles from the metastatic lesions (n = 120) revealed differential expression patterns of AKAPs relative to the tumor PAM50 intrinsic subtype, which were most apparent in the basal-like subtype. This pattern was confirmed in primary tumors from TCGA (n = 522) and in a third independent cohort (n = 182). CONCLUSION: Several studies from primary cancers have reported individual AKAP genes to be associated with cancer risk and metastatic relapses as well as direct involvement in cellular invasion and migration processes. Our findings reveal an enrichment of mutations in AKAP genes in metastatic breast cancers and suggest the involvement of AKAPs in the metastatic process. In addition, we report an AKAP gene expression pattern that consistently follows the tumor intrinsic subtype, further suggesting AKAP family members as relevant players in breast cancer biology.


Assuntos
Proteínas de Ancoragem à Quinase A/genética , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Família Multigênica , Mutação , Proteínas de Ancoragem à Quinase A/metabolismo , Neoplasias da Mama/metabolismo , Estudos de Coortes , Variações do Número de Cópias de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Humanos , Perda de Heterozigosidade , Metástase Neoplásica/genética , Estadiamento de Neoplasias , Sequenciamento do Exoma
10.
PLoS One ; 12(12): e0188772, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29228002

RESUMO

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.


Assuntos
Ciclo Celular/genética , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Algoritmos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/patologia
11.
Mol Syst Biol ; 13(5): 930, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28495919

RESUMO

Alternative transcription start sites (TSSs) have been extensively studied genome-wide for many cell types and have been shown to be important during development and to regulate transcript abundance between cell types. Likewise, single-cell gene expression has been extensively studied for many cell types. However, how single cells use TSSs has not yet been examined. In particular, it is unknown whether alternative TSSs are independently expressed, or whether they are co-activated or even mutually exclusive in single cells. Here, we use a previously published single-cell RNA-seq dataset, comprising thousands of cells, to study alternative TSS usage. We find that alternative TSS usage is a regulated process, and the correlation between two TSSs expressed in single cells of the same cell type is surprisingly high. Our findings indicate that TSSs generally are regulated by common factors rather than being independently regulated or stochastically expressed.


Assuntos
Encéfalo/citologia , Sítio de Iniciação de Transcrição , Animais , Sequência de Bases , Cistatina C/genética , Expressão Gênica , Camundongos , Análise de Célula Única
12.
Cancer Res ; 77(7): 1741-1752, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28087597

RESUMO

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.


Assuntos
Aminoácidos/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/fisiologia , Glioma/tratamento farmacológico , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Transporte Biológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Morte Celular , Linhagem Celular Tumoral , Di-Hidropiridinas/farmacologia , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Micotoxinas/farmacologia , Células-Tronco Neoplásicas/patologia , Proteômica , Sódio/metabolismo
13.
Nat Neurosci ; 19(10): 1331-40, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27571008

RESUMO

Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development.


Assuntos
Neurônios Motores/fisiologia , Músculo Liso/fisiologia , Neurônios/fisiologia , Mamilos/fisiologia , Piloereção/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Gânglios Simpáticos/fisiologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Supressoras de Tumor/metabolismo
14.
Science ; 353(6294): 78-82, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27365449

RESUMO

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Encéfalo/metabolismo , Neoplasias da Mama/metabolismo , DNA Complementar/biossíntese , Feminino , Humanos , Camundongos , Especificidade de Órgãos , RNA Mensageiro/metabolismo
15.
EMBO J ; 35(18): 1963-78, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27354364

RESUMO

Pre-B-cell leukemia homeobox (PBX) transcription factors are known to regulate organogenesis, but their molecular targets and function in midbrain dopaminergic neurons (mDAn) as well as their role in neurodegenerative diseases are unknown. Here, we show that PBX1 controls a novel transcriptional network required for mDAn specification and survival, which is sufficient to generate mDAn from human stem cells. Mechanistically, PBX1 plays a dual role in transcription by directly repressing or activating genes, such as Onecut2 to inhibit lateral fates during embryogenesis, Pitx3 to promote mDAn development, and Nfe2l1 to protect from oxidative stress. Notably, PBX1 and NFE2L1 levels are severely reduced in dopaminergic neurons of the substantia nigra of Parkinson's disease (PD) patients and decreased NFE2L1 levels increases damage by oxidative stress in human midbrain cells. Thus, our results reveal novel roles for PBX1 and its transcriptional network in mDAn development and PD, opening the door for new therapeutic interventions.


Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Neurônios Dopaminérgicos/fisiologia , Redes Reguladoras de Genes , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas/metabolismo , Substância Negra/patologia , Humanos , Fator de Transcrição 1 de Leucemia de Células Pré-B
16.
Nat Immunol ; 17(7): 797-805, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27135602

RESUMO

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.


Assuntos
Sistema Nervoso Central/imunologia , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/fisiologia , Microglia/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Monócitos/imunologia , Parabiose , Proteínas Proto-Oncogênicas/genética , Transativadores/genética
17.
J Biol Chem ; 290(13): 8348-59, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25697355

RESUMO

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid ß-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.


Assuntos
Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Glutamina/fisiologia , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Linhagem Celular Tumoral , Ciclosporinas/farmacologia , Sinergismo Farmacológico , Enoil-CoA Hidratase/metabolismo , Ácidos Graxos/biossíntese , Glicólise , Humanos , Leucemia , Metaboloma , Oxirredução , Ácido Pantotênico/metabolismo , Perexilina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Racemases e Epimerases/metabolismo , Transcriptoma
18.
Genomics ; 105(3): 150-8, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25543032

RESUMO

Cell-free DNA has been used for fetal rhesus factor and sex determination, fetal aneuploidy screening, cancer diagnostics and monitoring, and other applications. However current methods of using cell free DNA require amplification, which leads to allelic dropout and bias especially when starting with small amounts of DNA. Here we describe an amplification-free method for sequencing of cell-free DNA, even from low levels of starting material. We evaluated this method in the context of prenatal diagnosis of fetal aneuploidy and compared it with a PCR-based library preparation method as well as a recently described method using unique molecular identifiers (UMI). All methods performed well, however coverage was increased by the amplification-free method and GC-induced bias was reduced by both the amplification-free method and the UMI method. Future diagnostic applications including whole genome sequencing of cell-free DNA will benefit from amplification-free sequencing.


Assuntos
Aneuploidia , Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA/métodos , Feminino , Humanos , Reação em Cadeia da Polimerase , Gravidez
19.
PLoS One ; 9(12): e115698, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25531110

RESUMO

Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells, including the ability to self-renew and differentiate, commonly referred to as stemness. In addition, such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs), transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin, revealed that NSC-proximal GICs were more sensitive, corroborating the transcriptome data. Furthermore, Ca2+ drug sensitivity was reduced in GICs after differentiation, with most potent effect in the NSC-proximal GIC, supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium, sodium and Ca2+. Conversely, a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+, were expressed in NSC-distal GICs. In particular, expression of the AMPA glutamate receptor subunit GRIA1, was found to associate with Ca2+ sensitive NSC-proximal GICs, and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP, brain lipid-binding protein) in this extended analysis. In summary, NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis, providing a potential target mechanism for eradication of an immature population of malignant cells.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/tratamento farmacológico , Cálcio/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunofluorescência , Perfilação da Expressão Gênica , Glioma/genética , Glioma/patologia , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
20.
Cancer Cell ; 25(6): 794-808, 2014 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-24835589

RESUMO

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.


Assuntos
Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Animais , Antígenos CD/biossíntese , Antígenos CD/imunologia , Deleção Cromossômica , Cromossomos Humanos Par 5 , Citometria de Fluxo , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Mutação , Síndromes Mielodisplásicas/imunologia , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Prognóstico
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