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1.
Annu Rev Immunol ; 39: 583-609, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637019

RESUMO

Understanding tumor immune microenvironments is critical for identifying immune modifiers of cancer progression and developing cancer immunotherapies. Recent applications of single-cell RNA sequencing (scRNA-seq) in dissecting tumor microenvironments have brought important insights into the biology of tumor-infiltrating immune cells, including their heterogeneity, dynamics, and potential roles in both disease progression and response to immune checkpoint inhibitors and other immunotherapies. This review focuses on the advances in knowledge of tumor immune microenvironments acquired from scRNA-seq studies across multiple types of human tumors, with a particular emphasis on the study of phenotypic plasticity and lineage dynamics of immune cells in the tumor environment. We also discuss several imminent questions emerging from scRNA-seq observations and their potential solutions on the horizon.


Assuntos
Neoplasias , Análise de Célula Única , Animais , Humanos , Imunoterapia , Neoplasias/terapia , Análise de Sequência de RNA , Microambiente Tumoral
2.
Cell ; 187(18): 4905-4925.e24, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38971151

RESUMO

Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to poly (ADP-ribose) polymerase (PARP) inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T cell receptor profiles, along with validatory multimodal datasets from >100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor (PARPi) niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v.1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTregs) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC class II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.


Assuntos
Terapia Neoadjuvante , Neoplasias Ovarianas , Piperidinas , Inibidores de Poli(ADP-Ribose) Polimerases , Linfócitos T Reguladores , Microambiente Tumoral , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/imunologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Humanos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Camundongos , Terapia Neoadjuvante/métodos , Microambiente Tumoral/efeitos dos fármacos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Indazóis/uso terapêutico , Indazóis/farmacologia , Recombinação Homóloga , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral
3.
Cell ; 187(1): 166-183.e25, 2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-38181739

RESUMO

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.


Assuntos
Melanoma , Humanos , Redes Reguladoras de Genes , Imunoterapia , Melanócitos , Melanoma/tratamento farmacológico , Melanoma/genética , Fator de Transcrição 4/genética , Microambiente Tumoral
4.
Cell ; 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39454576

RESUMO

Differential expression analysis of single-cell RNA sequencing (scRNA-seq) data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remain challenging. We introduce Memento, a tool for robust and efficient differential analysis of mean expression, variability, and gene correlation from scRNA-seq data, scalable to millions of cells and thousands of samples. We applied Memento to 70,000 tracheal epithelial cells to identify interferon-responsive genes, 160,000 CRISPR-Cas9 perturbed T cells to reconstruct gene-regulatory networks, 1.2 million peripheral blood mononuclear cells (PBMCs) to map cell-type-specific quantitative trait loci (QTLs), and the 50-million-cell CELLxGENE Discover corpus to compare arbitrary cell groups. In all cases, Memento identified more significant and reproducible differences in mean expression compared with existing methods. It also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by perturbations.

5.
Cell ; 186(21): 4676-4693.e29, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37729907

RESUMO

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.


Assuntos
Evolução Biológica , Invertebrados , Neurônios , Animais , Ctenóforos/genética , Expressão Gênica , Neurônios/fisiologia , Filogenia , Análise de Célula Única , Invertebrados/citologia , Invertebrados/genética , Invertebrados/metabolismo , Comunicação Parácrina
6.
Cell ; 186(1): 194-208.e18, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36580914

RESUMO

The diversity and complex organization of cells in the brain have hindered systematic characterization of age-related changes in its cellular and molecular architecture, limiting our ability to understand the mechanisms underlying its functional decline during aging. Here, we generated a high-resolution cell atlas of brain aging within the frontal cortex and striatum using spatially resolved single-cell transcriptomics and quantified changes in gene expression and spatial organization of major cell types in these regions over the mouse lifespan. We observed substantially more pronounced changes in cell state, gene expression, and spatial organization of non-neuronal cells over neurons. Our data revealed molecular and spatial signatures of glial and immune cell activation during aging, particularly enriched in the subcortical white matter, and identified both similarities and notable differences in cell-activation patterns induced by aging and systemic inflammatory challenge. These results provide critical insights into age-related decline and inflammation in the brain.


Assuntos
Envelhecimento , Substância Branca , Camundongos , Animais , Envelhecimento/genética , Encéfalo/metabolismo , Neuroglia , Longevidade , Transcriptoma , Análise de Célula Única
7.
Cell ; 186(6): 1179-1194.e15, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36931245

RESUMO

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.


Assuntos
Células-Tronco Neurais , Camundongos , Animais , Humanos , Células-Tronco Neurais/metabolismo , Neurônios , Diferenciação Celular/fisiologia , Neuroglia/metabolismo , Encéfalo , Astrócitos
8.
Cell ; 185(25): 4756-4769.e13, 2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36493754

RESUMO

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.


Assuntos
Células-Tronco Adultas , Eucariotos , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Linhagem da Célula , Células-Tronco Pluripotentes/fisiologia , Eucariotos/classificação , Eucariotos/citologia
9.
Cell ; 184(12): 3281-3298.e22, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34019796

RESUMO

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.


Assuntos
Anatomia Artística , Atlas como Assunto , Desenvolvimento Embrionário , Endoderma/embriologia , Modelos Biológicos , Organoides/embriologia , Fator de Transcrição CDX2/metabolismo , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Feminino , Gastrulação , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Intestinos/embriologia , Masculino , Mesoderma/embriologia , Pessoa de Meia-Idade , Neuregulina-1/metabolismo , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologia
10.
Cell ; 184(12): 3222-3241.e26, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34004146

RESUMO

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.


Assuntos
Hipocampo/citologia , Neocórtex/citologia , Transcriptoma/genética , Animais , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
11.
Cell ; 184(26): 6243-6261.e27, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34914922

RESUMO

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.


Assuntos
COVID-19/patologia , COVID-19/virologia , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/virologia , Macrófagos/patologia , Macrófagos/virologia , SARS-CoV-2/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , COVID-19/diagnóstico por imagem , Comunicação Celular , Estudos de Coortes , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/diagnóstico por imagem , Fibrose Pulmonar Idiopática/genética , Células-Tronco Mesenquimais/patologia , Fenótipo , Proteoma/metabolismo , Receptores de Superfície Celular/metabolismo , Síndrome do Desconforto Respiratório/diagnóstico por imagem , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/virologia , Tomografia Computadorizada por Raios X , Transcrição Gênica
12.
Cell ; 184(16): 4168-4185.e21, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216539

RESUMO

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.


Assuntos
Autoimunidade/imunologia , Modelos Biológicos , Células Th17/imunologia , Acetiltransferases/metabolismo , Trifosfato de Adenosina/metabolismo , Aerobiose/efeitos dos fármacos , Algoritmos , Animais , Autoimunidade/efeitos dos fármacos , Cromatina/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Citocinas/metabolismo , Eflornitina/farmacologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Epigenoma , Ácidos Graxos/metabolismo , Glicólise/efeitos dos fármacos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos Endogâmicos C57BL , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Oxirredução/efeitos dos fármacos , Putrescina/metabolismo , Análise de Célula Única , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Células Th17/efeitos dos fármacos , Transcriptoma/genética
13.
Cell ; 184(3): 741-758.e17, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33484631

RESUMO

Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.


Assuntos
Encéfalo/crescimento & desenvolvimento , Genoma , Sensação/genética , Transcrição Gênica , Alelos , Animais , Animais Recém-Nascidos , Linhagem da Célula/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Loci Gênicos , Impressão Genômica , Camundongos , Família Multigênica , Neuroglia/metabolismo , Neurônios/metabolismo , Transcriptoma/genética , Córtex Visual/metabolismo
14.
Cell ; 184(7): 1895-1913.e19, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33657410

RESUMO

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.


Assuntos
COVID-19/imunologia , Megacariócitos/imunologia , Monócitos/imunologia , RNA Viral , SARS-CoV-2/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , China , Estudos de Coortes , Citocinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , RNA Viral/isolamento & purificação , Análise de Célula Única , Transcriptoma/imunologia , Adulto Jovem
15.
Annu Rev Cell Dev Biol ; 37: 495-517, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34416113

RESUMO

With the discovery of the incredible diversity of neurons, Cajal and coworkers laid the foundation of modern neuroscience. Neuron types are not only structural units of nervous systems but also evolutionary units, because their identities are encoded in the genome. With the advent of high-throughput cellular transcriptomics, neuronal identities can be characterized and compared systematically across species. The comparison of neurons in mammals, reptiles, and birds indicates that the mammalian cerebral cortex is a mosaic of deeply conserved and recently evolved neuron types. Using the cerebral cortex as a case study, this review illustrates how comparing neuron types across species is key to reconciling observations on neural development, neuroanatomy, circuit wiring, and physiology for an integrated understanding of brain evolution.


Assuntos
Evolução Biológica , Córtex Cerebral , Animais , Encéfalo/fisiologia , Córtex Cerebral/anatomia & histologia , Mamíferos , Neurogênese , Neurônios/metabolismo
16.
Cell ; 176(6): 1407-1419.e14, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30827680

RESUMO

The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.


Assuntos
Encéfalo/fisiologia , Fatores Etários , Animais , Encéfalo/citologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese , Nicho de Células-Tronco
17.
Cell ; 179(2): 527-542.e19, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585086

RESUMO

Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Software , Transcriptoma , Animais , Humanos , Rim/citologia , Pâncreas/citologia , Análise de Célula Única , Peixe-Zebra/anatomia & histologia
18.
Cell ; 174(4): 999-1014.e22, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096314

RESUMO

The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Sistema Nervoso/metabolismo , Análise de Célula Única/métodos , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/crescimento & desenvolvimento
19.
Cell ; 175(4): 1156-1167.e15, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30270040

RESUMO

The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterium-rich environment. It is unknown whether these tasks are spatially zonated along the villus axis. Here, we extracted a large panel of landmark genes characterized by transcriptomics of laser capture microdissected villus segments and utilized it for single-cell spatial reconstruction, uncovering broad zonation of enterocyte function along the villus. We found that enterocytes at villus bottoms express an anti-bacterial gene program in a microbiome-dependent manner. They next shift to sequential expression of carbohydrates, peptides, and fat absorption machineries in distinct villus compartments. Finally, they induce a Cd73 immune-modulatory program at the villus tips. Our approach can be used to uncover zonation patterns in other organs when prior knowledge of landmark genes is lacking.


Assuntos
Enterócitos/metabolismo , Transcriptoma , Animais , Diferenciação Celular , Movimento Celular , Enterócitos/citologia , Enterócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Célula Única
20.
Cell ; 174(4): 843-855.e19, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30017245

RESUMO

Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasia Residual/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor X Retinoide gama/antagonistas & inibidores , Animais , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos SCID , Mutação , Neoplasia Residual/metabolismo , Neoplasia Residual/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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