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1.
Science ; 373(6550): 111-117, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210887

RESUMO

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.


Assuntos
Padronização Corporal/genética , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Transcriptoma , Animais , Atlas como Assunto , Encéfalo/embriologia , Movimento Celular , Camundongos , Neurogênese/genética , Neurônios/citologia
2.
Nat Commun ; 12(1): 4197, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234139

RESUMO

Single cell RNA sequencing (scRNA-seq) is a powerful tool in detailing the cellular landscape within complex tissues. Large-scale single cell transcriptomics provide both opportunities and challenges for identifying rare cells playing crucial roles in development and disease. Here, we develop GapClust, a light-weight algorithm to detect rare cell types from ultra-large scRNA-seq datasets with state-of-the-art speed and memory efficiency. Benchmarking on diverse experimental datasets demonstrates the superior performance of GapClust compared to other recently proposed methods. When applying our algorithm to an intestine and 68 k PBMC datasets, GapClust identifies the tuft cells and a previously unrecognised subtype of monocyte, respectively.


Assuntos
Algoritmos , RNA-Seq/métodos , Análise de Célula Única/métodos , Conjuntos de Dados como Assunto , Células HEK293 , Humanos , Mucosa Intestinal/citologia , Células Jurkat , Software
3.
Nat Commun ; 12(1): 3796, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145278

RESUMO

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.


Assuntos
Proteínas CLOCK/metabolismo , Relógios Circadianos/fisiologia , Criptocromos/metabolismo , Proteínas Circadianas Period/metabolismo , Análise de Célula Única/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Ritmo Circadiano/fisiologia , Criptocromos/genética , Técnicas de Introdução de Genes/métodos , Genes Reporter/genética , Células HCT116 , Humanos , Proteínas Circadianas Period/genética
4.
Methods Mol Biol ; 2277: 299-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080159

RESUMO

In light of accumulating evidence suggestive of cell type-specific vulnerabilities as a result of normal aging processes that adversely affect the brain, as well as age-related neurodegenerative disorders such as Parkinson's disease (PD), the current chapter highlights how we study mitochondrial DNA (mtDNA) changes at a single-cell level. In particular, we comment on increasing questioning of the narrow neurocentric view of such pathologies, where microglia and astrocytes have traditionally been considered bystanders rather than players in related pathological processes. Here we review the contribution made by single-cell mtDNA alterations towards neuronal vulnerability seen in neurodegenerative disorders, focusing on PD as a prominent example. In addition, we give an overview of methodologies that support such experimental investigations. In considering the significant advances that have been made in recent times for developing mitochondria-specific therapies, investigations to account for cell type-specific mitochondrial patterns and how these are altered by disease hold promise for delivering more effective disease-modifying therapeutics.


Assuntos
Encéfalo/patologia , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Doenças Neurodegenerativas/patologia , Análise de Célula Única/métodos , Envelhecimento/genética , Humanos , Doenças Neurodegenerativas/genética , Doença de Parkinson/genética , Reação em Cadeia da Polimerase/métodos
5.
Methods Mol Biol ; 2268: 207-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085271

RESUMO

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gßγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Opsinas/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
6.
Methods Mol Biol ; 2268: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085272

RESUMO

Split-TEV assay enables the identification of protein-protein interaction in mammalian cells. This method is based on the split of tobacco etch virus (TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. However, some studies have detected unspecific interaction between membrane proteins due to its higher tendency to aggregate. Here we describe a variation of the Split-TEV method developed with the aim to increase the specificity in the study of G protein-coupled receptor (GPCR) interacting proteins. This approach for monitoring interactions between GPCRs is an easy and robust assay and offers good perspectives in drug discovery.


Assuntos
Bioensaio/métodos , Endopeptidases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Potyvirus/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Genes Reporter , Humanos , Imagem Molecular/métodos , Potyvirus/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Análise de Célula Única/métodos
7.
Methods Mol Biol ; 2268: 233-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085273

RESUMO

Cytosolic ß-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or ß-arrestin recruitment makes ß-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor ß-arrestin2 recruitment to the human histamine H1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H1 receptor.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Luciferases/metabolismo , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/metabolismo , Análise de Célula Única/métodos , beta-Arrestina 2/metabolismo , Células HEK293 , Humanos , Ligantes , Imagem Molecular/métodos , Ligação Proteica , Transdução de Sinais
8.
Methods Mol Biol ; 2268: 249-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085274

RESUMO

An understanding of the kinetic contributions to G protein-coupled receptor pharmacology and signaling is increasingly important in compound profiling. Nonequilibrium conditions are commonly present in vivo, for example, as the drug competes with dynamic changes in hormone or neurotransmitter concentration for the receptor. Under such conditions individual binding kinetic properties of the ligands can influence duration of action, local ligand concentration, and functional properties such as the degree of insurmountable inhibition. Mapping the kinetic patterns of GPCR signaling events elicited by agonists, rather than a peak response at a single timepoint, is often key to predicting their functional impact. This is also a path to a better understanding of the origins of ligand bias, and whether such ligands demonstrate their effects through selection of distinct GPCR conformations, or via their kinetic properties. Recent developments in complementation approaches, based on a small bright shrimp luciferase Nanoluc, provide a new route to kinetic analysis of GPCR signaling in living cells that is amenable to the throughput required for compound profiling. In the NanoBiT luciferase complementation system, GPCRs and effector proteins are tagged with Nanoluc fragments optimized for their low interacting affinity and stability. The interactions brought about by GPCR recruitment of the effector are reproduced by a rapid and reversible increase in NanoBiT luminescence, in the presence of its substrate furimazine. Here we discuss the methods for optimizing and validating the GPCR NanoBiT assays, and protocols for their application to study endpoint and kinetic aspects of agonist and antagonist pharmacology. We also describe how timecourse families of agonist concentration response curves, derived from a single NanoBiT assay experiment, can be used to evaluate the kinetic components in operational model derived parameters of ligand bias.


Assuntos
Bioensaio/métodos , Luciferases/metabolismo , Imagem Molecular/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Células HEK293 , Humanos , Cinética , Ligantes , Luminescência , Transdução de Sinais
9.
Nat Commun ; 12(1): 3942, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168133

RESUMO

We present dyngen, a multi-modal simulation engine for studying dynamic cellular processes at single-cell resolution. dyngen is more flexible than current single-cell simulation engines, and allows better method development and benchmarking, thereby stimulating development and testing of computational methods. We demonstrate its potential for spearheading computational methods on three applications: aligning cell developmental trajectories, cell-specific regulatory network inference and estimation of RNA velocity.


Assuntos
Simulação por Computador , Redes Reguladoras de Genes , Análise de Célula Única/métodos , Algoritmos , Benchmarking , Biologia Computacional/métodos , Perfilação da Expressão Gênica
10.
Molecules ; 26(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066773

RESUMO

Besides human red blood cells (RBC), a standard model used in AFM-single cell force spectroscopy (SCFS), little is known about apparent Young's modulus (Ea) or adhesion of animal RBCs displaying distinct cellular features. To close this knowledge gap, we probed chicken, horse, camel, and human fetal RBCs and compared data with human adults serving as a repository for future studies. Additionally, we assessed how measurements are affected under physiological conditions (species-specific temperature in autologous plasma vs. 25 °C in aqueous NaCl solution). In all RBC types, Ea decreased with increasing temperature irrespective of the suspension medium. In mammalian RBCs, adhesion increased with elevated temperatures and scaled with reported membrane sialic acid concentrations. In chicken only adhesion decreased with higher temperature, which we attribute to the lower AE-1 concentration allowing more membrane undulations. Ea decreased further in plasma at every test temperature, and adhesion was completely abolished, pointing to functional cell enlargement by adsorption of plasma components. This halo elevated RBC size by several hundreds of nanometers, blunted the thermal input, and will affect the coupling of RBCs with the flowing plasma. The study evidences the presence of a RBC surface layer and discusses the tremendous effects when RBCs are probed at physiological conditions.


Assuntos
Camelus/sangue , Adesão Celular/fisiologia , Galinhas/sangue , Eritrócitos/citologia , Cavalos/sangue , Microscopia de Força Atômica/métodos , Análise de Célula Única/métodos , Temperatura , Adulto , Animais , Membrana Celular/metabolismo , Humanos
11.
Nat Commun ; 12(1): 3679, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140473

RESUMO

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.


Assuntos
Implantação do Embrião/genética , Desenvolvimento Embrionário , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Análise de Célula Única/métodos , Via de Sinalização Wnt , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Linhagem da Célula , Células Cultivadas , Implantação do Embrião/fisiologia , Embrião de Mamíferos , Fatores de Crescimento de Fibroblastos/metabolismo , Gastrulação/fisiologia , Camadas Germinativas/citologia , Humanos , Processamento de Imagem Assistida por Computador , Família Multigênica , Proteína Nodal/antagonistas & inibidores , RNA-Seq , Análise Espaço-Temporal
12.
Nat Commun ; 12(1): 3727, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140517

RESUMO

Clonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution from cell clones that harbor inducible proviruses to plasma viremia is poorly understood. Here, we describe a single-cell approach to simultaneously sequence the TCR, integration sites and proviral genomes from translation-competent reservoir cells, called STIP-Seq. By applying this approach to blood samples from eight participants, we show that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5'-end of the genome, often involving the major splice donor site. TCR and integration site sequencing reveal that cell clones with predicted pathogen-specificity can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we find several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, suggesting that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Provírus/genética , Análise de Célula Única/métodos , Viremia/virologia , Linfócitos T CD4-Positivos/virologia , DNA Viral/sangue , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Ionomicina/farmacologia , Masculino , Pessoa de Meia-Idade , Filogenia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Deleção de Sequência , Carga Viral/genética
13.
Development ; 148(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34170290

RESUMO

The third 'Symposium for the Next Generation of Stem Cell Research' (SY-Stem) was held virtually on 3-5 March 2021, having been cancelled in 2020 due to the COVID-19 pandemic. As in previous years, the meeting highlighted the work of early career researchers, ranging from postgraduate students to young group leaders working in developmental and stem cell biology. Here, we summarize the excellent work presented at the Symposium, which covered topics ranging from pluripotency, species-specific aspects of development and emerging technologies, through to organoids, single-cell technology and clinical applications.


Assuntos
Congressos como Assunto/organização & administração , Invenções/tendências , Pesquisa com Células-Tronco , Animais , COVID-19/epidemiologia , Diferenciação Celular , Congressos como Assunto/história , Congressos como Assunto/tendências , História do Século XXI , Humanos , Internet , Invenções/história , Sistemas On-Line , Pandemias , Análise de Célula Única/métodos , Análise de Célula Única/tendências , Pesquisa com Células-Tronco/história , Células-Tronco/fisiologia , Técnicas de Cultura de Tecidos/métodos , Técnicas de Cultura de Tecidos/tendências
14.
Nat Commun ; 12(1): 3358, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099733

RESUMO

Early stages of embryogenesis depend on subcellular localization and transport of maternal mRNA. However, systematic analysis of these processes is hindered by a lack of spatio-temporal information in single-cell RNA sequencing. Here, we combine spatially-resolved transcriptomics and single-cell RNA labeling to perform a spatio-temporal analysis of the transcriptome during early zebrafish development. We measure spatial localization of mRNA molecules within the one-cell stage embryo, which allows us to identify a class of mRNAs that are specifically localized at an extraembryonic position, the vegetal pole. Furthermore, we establish a method for high-throughput single-cell RNA labeling in early zebrafish embryos, which enables us to follow the fate of individual maternal transcripts until gastrulation. This approach reveals that many localized transcripts are specifically transported to the primordial germ cells. Finally, we acquire spatial transcriptomes of two xenopus species and compare evolutionary conservation of localized genes as well as enriched sequence motifs.


Assuntos
Rastreamento de Células/métodos , Embrião não Mamífero/metabolismo , RNA Mensageiro/genética , Transcriptoma/genética , Peixe-Zebra/genética , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Análise de Célula Única/métodos , Análise Espaço-Temporal , Especificidade da Espécie , Xenopus/embriologia , Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Peixe-Zebra/embriologia
15.
Nat Commun ; 12(1): 3826, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158507

RESUMO

Single-cell omics is the fastest-growing type of genomics data in the literature and public genomics repositories. Leveraging the growing repository of labeled datasets and transferring labels from existing datasets to newly generated datasets will empower the exploration of single-cell omics data. However, the current label transfer methods have limited performance, largely due to the intrinsic heterogeneity among cell populations and extrinsic differences between datasets. Here, we present a robust graph artificial intelligence model, single-cell Graph Convolutional Network (scGCN), to achieve effective knowledge transfer across disparate datasets. Through benchmarking with other label transfer methods on a total of 30 single cell omics datasets, scGCN consistently demonstrates superior accuracy on leveraging cells from different tissues, platforms, and species, as well as cells profiled at different molecular layers. scGCN is implemented as an integrated workflow as a python software, which is available at https://github.com/QSong-github/scGCN .


Assuntos
Algoritmos , Inteligência Artificial , Genômica/métodos , Redes Neurais de Computação , Análise de Célula Única/métodos , Células A549 , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Internet , Rim/citologia , Rim/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Software
16.
Nat Commun ; 12(1): 3896, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162837

RESUMO

Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.


Assuntos
Neoplasias Renais/genética , Rim/metabolismo , RNA Mensageiro/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Transcriptoma , Adulto , Algoritmos , Criança , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/embriologia , Neoplasias Renais/embriologia , Neoplasias Renais/metabolismo , Modelos Genéticos , Transdução de Sinais/genética
17.
Nat Commun ; 12(1): 3876, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162856

RESUMO

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Homeostase/genética , Células-Tronco Mesenquimais/metabolismo , Regeneração/genética , Testículo/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Célula Única/métodos , Testículo/citologia
18.
Nat Commun ; 12(1): 3895, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162860

RESUMO

Brain tumors are typically immunosuppressive and refractory to immunotherapies for reasons that remain poorly understood. The unbiased profiling of immune cell types in the tumor microenvironment may reveal immunologic networks affecting therapy and course of disease. Here we identify and validate the presence of hematopoietic stem and progenitor cells (HSPCs) within glioblastoma tissues. Furthermore, we demonstrate a positive link of tumor-associated HSPCs with malignant and immunosuppressive phenotypes. Compared to the medullary hematopoietic compartment, tumor-associated HSPCs contain a higher fraction of immunophenotypically and transcriptomically immature, CD38- cells, such as hematopoietic stem cells and multipotent progenitors, express genes related to glioblastoma progression and display signatures of active cell cycle phases. When cultured ex vivo, tumor-associated HSPCs form myeloid colonies, suggesting potential in situ myelopoiesis. In experimental models, HSPCs promote tumor cell proliferation, expression of the immune checkpoint PD-L1 and secretion of tumor promoting cytokines such as IL-6, IL-8 and CCL2, indicating concomitant support of both malignancy and immunosuppression. In patients, the amount of tumor-associated HSPCs in tumor tissues is prognostic for patient survival and correlates with immunosuppressive phenotypes. These findings identify an important element in the complex landscape of glioblastoma that may serve as a target for brain tumor immunotherapies.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Progressão da Doença , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Estimativa de Kaplan-Meier , RNA-Seq/métodos , Transdução de Sinais/genética , Análise de Célula Única/métodos , Microambiente Tumoral/genética
19.
Nat Commun ; 12(1): 3913, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162888

RESUMO

Human FOXP3+ regulatory T (Treg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate Treg cells into naïve, activated, and effector stages, and resolve the HLA-DRhi, LIMS1hi, highly suppressive FOXP3hi, and highly proliferative MKI67hi effector subsets. Trajectory analysis assembles Treg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3hi and MKI67hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3hi and MKI67hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of Treg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of Treg complexity in health and disease.


Assuntos
Diferenciação Celular/genética , Fatores de Transcrição Forkhead/imunologia , Transdução de Sinais/genética , Análise de Célula Única/métodos , Linfócitos T Reguladores/imunologia , Transcriptoma/genética , Western Blotting , Ensaio de Imunoadsorção Enzimática , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , RNA-Seq/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo
20.
Nat Commun ; 12(1): 3341, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099695

RESUMO

Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.


Assuntos
Proteômica/métodos , Análise de Célula Única/métodos , Humanos , Leucemia Mieloide Aguda , Espectrometria de Massas , Células-Tronco Neoplásicas , Proteoma/metabolismo , RNA , Fluxo de Trabalho
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