RESUMO
Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Análise Espacial , Transcriptoma/genética , Microambiente Tumoral , Proteômica , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.
Assuntos
Células-Tronco Embrionárias , Gastrulação , Animais , Diferenciação Celular/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Endoderma , Mamíferos , CamundongosRESUMO
Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.
Assuntos
Glicoproteínas de Membrana/metabolismo , Neoplasias/patologia , RNA Citoplasmático Pequeno/química , Receptores Imunológicos/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/metabolismo , RNA Citoplasmático Pequeno/metabolismo , Receptores Imunológicos/genética , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Transcrição/metabolismo , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Immune cells residing in white adipose tissue have been highlighted as important factors contributing to the pathogenesis of metabolic diseases, but the molecular regulators that drive adipose tissue immune cell remodeling during obesity remain largely unknown. Using index and transcriptional single-cell sorting, we comprehensively map all adipose tissue immune populations in both mice and humans during obesity. We describe a novel and conserved Trem2+ lipid-associated macrophage (LAM) subset and identify markers, spatial localization, origin, and functional pathways associated with these cells. Genetic ablation of Trem2 in mice globally inhibits the downstream molecular LAM program, leading to adipocyte hypertrophy as well as systemic hypercholesterolemia, body fat accumulation, and glucose intolerance. These findings identify Trem2 signaling as a major pathway by which macrophages respond to loss of tissue-level lipid homeostasis, highlighting Trem2 as a key sensor of metabolic pathologies across multiple tissues and a potential therapeutic target in metabolic diseases.
Assuntos
Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Dieta Hiperlipídica , Intolerância à Glucose , Humanos , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Metabolismo dos Lipídeos/genética , Lipídeos/análise , Macrófagos/citologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais , Análise de Célula ÚnicaRESUMO
Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans.
Assuntos
Evolução Molecular , Redes Reguladoras de Genes , Microglia/metabolismo , Doenças Neurodegenerativas/genética , Análise de Célula Única , Transcriptoma , Animais , Galinhas , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Primatas , Répteis , Roedores , Ovinos , Suínos , Peixe-ZebraRESUMO
A major challenge in the field of neurodegenerative diseases and brain aging is to identify the body's intrinsic mechanism that could sense the central nervous system (CNS) damage early and protect the brain from neurodegeneration. Accumulating evidence suggests that disease-associated microglia (DAM), a recently identified subset of CNS resident macrophages found at sites of neurodegeneration, might play such a protective role. Here, we propose that microglia are endowed with a dedicated sensory mechanism, which includes the Trem2 signaling pathway, to detect damage within the CNS in the form of neurodegeneration-associated molecular patterns (NAMPs). Combining data from transcriptional analysis of DAM at single-cell level and from human genome-wide association studies (GWASs), we discuss potential function of different DAM pathways in the diseased brain and outline how manipulating DAM may create new therapeutic opportunities.
Assuntos
Microglia/metabolismo , Doenças Neurodegenerativas/patologia , Animais , Sistema Nervoso Central/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/metabolismo , Receptores Imunológicos/metabolismo , Transdução de SinaisRESUMO
Alzheimer's disease (AD) is a detrimental neurodegenerative disease with no effective treatments. Due to cellular heterogeneity, defining the roles of immune cell subsets in AD onset and progression has been challenging. Using transcriptional single-cell sorting, we comprehensively map all immune populations in wild-type and AD-transgenic (Tg-AD) mouse brains. We describe a novel microglia type associated with neurodegenerative diseases (DAM) and identify markers, spatial localization, and pathways associated with these cells. Immunohistochemical staining of mice and human brain slices shows DAM with intracellular/phagocytic Aß particles. Single-cell analysis of DAM in Tg-AD and triggering receptor expressed on myeloid cells 2 (Trem2)-/- Tg-AD reveals that the DAM program is activated in a two-step process. Activation is initiated in a Trem2-independent manner that involves downregulation of microglia checkpoints, followed by activation of a Trem2-dependent program. This unique microglia-type has the potential to restrict neurodegeneration, which may have important implications for future treatment of AD and other neurodegenerative diseases. VIDEO ABSTRACT.
Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Microglia/patologia , Fagócitos/patologia , Doença de Alzheimer/genética , Animais , Humanos , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/patologia , Fagócitos/metabolismo , Receptores Imunológicos/metabolismo , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.
Assuntos
Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Dendríticas/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismoRESUMO
Innate lymphoid cells (ILCs) are critical modulators of mucosal immunity, inflammation, and tissue homeostasis, but their full spectrum of cellular states and regulatory landscapes remains elusive. Here, we combine genome-wide RNA-seq, ChIP-seq, and ATAC-seq to compare the transcriptional and epigenetic identity of small intestinal ILCs, identifying thousands of distinct gene profiles and regulatory elements. Single-cell RNA-seq and flow and mass cytometry analyses reveal compartmentalization of cytokine expression and metabolic activity within the three classical ILC subtypes and highlight transcriptional states beyond the current canonical classification. In addition, using antibiotic intervention and germ-free mice, we characterize the effect of the microbiome on the ILC regulatory landscape and determine the response of ILCs to microbial colonization at the single-cell level. Together, our work characterizes the spectrum of transcriptional identities of small intestinal ILCs and describes how ILCs differentially integrate signals from the microbial microenvironment to generate phenotypic and functional plasticity.
Assuntos
Microbioma Gastrointestinal , Imunidade Inata/genética , Intestinos/imunologia , Intestinos/microbiologia , Linfócitos/imunologia , Linfócitos/microbiologia , Animais , Sequência de Bases , Cromatina/metabolismo , Citocinas/imunologia , Epigênese Genética , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Análise de Célula Única , Transcrição GênicaRESUMO
Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.
Assuntos
Hematopoese , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Análise de Célula Única , Transcriptoma , Animais , Transplante de Medula Óssea , Proteínas Estimuladoras de Ligação a CCAAT/genética , Técnicas de Inativação de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Fatores de Transcrição/metabolismoRESUMO
The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.
RESUMO
Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogrammed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.
Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Histonas/metabolismo , Macrófagos/metabolismo , Animais , Cromatina/metabolismo , Feminino , Código das Histonas , Macrófagos/citologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Especificidade de Órgãos , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.
Assuntos
Implantação do Embrião , Embrião de Mamíferos , Desenvolvimento Embrionário , Células-Tronco Embrionárias Humanas , Humanos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Fertilização , Gastrulação , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Células-Tronco Embrionárias Humanas/citologia , Trofoblastos/citologia , Saco Vitelino/citologia , Saco Vitelino/embriologia , Células Gigantes/citologiaRESUMO
Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.
Assuntos
Tolerância a Antígenos Próprios , Linfócitos T , Timo , Animais , Camundongos , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Tolerância a Antígenos Próprios/imunologia , Tolerância a Antígenos Próprios/fisiologia , Linfócitos T/classificação , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Tecido Parenquimatoso , Células Musculares , Células Endócrinas , Cromatina , Transcrição Gênica , GrelinaRESUMO
The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.
Assuntos
Técnicas de Cultura Embrionária , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Técnicas In Vitro , Organogênese , Animais , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Feminino , Gastrulação , Masculino , Camundongos , Fatores de Tempo , ÚteroRESUMO
Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.
Assuntos
Hipocampo/metabolismo , MicroRNAs/genética , Microglia/fisiologia , Neurônios/fisiologia , Ribonuclease III/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Reparo do DNA , Feminino , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Atividade Motora , Plasticidade Neuronal , Ribonuclease III/genéticaRESUMO
The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB γ-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in γ-neurons for their developmental regrowth into the distal γ4/5 zones, where both Dpr12 and its interacting protein, DIP-δ, are enriched. DIP-δ functions in a subset of dopaminergic neurons that wire with γ-neurons within the γ4/5 zone. During metamorphosis, these dopaminergic projections arrive to the γ4/5 zone prior to γ-axons, suggesting that γ-axons extend through a prepatterned region. Thus, Dpr12/DIP-δ transneuronal interaction is required for γ4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution.