RESUMO
Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in-vitro-differentiated Th17 cells and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65, Plzp, Toso, and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Análise de Sequência de RNA , Análise de Célula Única , Células Th17/metabolismo , Células Th17/patologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Perfilação da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Linfonodos/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Imunológicos/metabolismo , Receptores Depuradores , Células Th17/imunologiaRESUMO
High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.
Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade/genética , Comunicação Parácrina , Animais , Antígenos Virais/farmacologia , Sequência de Bases , Comunicação Celular , Células Dendríticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Interferon beta/genética , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Componente Principal , RNA Mensageiro/química , RNA Mensageiro/genética , Análise de Célula ÚnicaRESUMO
CMOS microelectrode arrays (MEAs) can record electrophysiological activities of a large number of neurons in parallel but only extracellularly with low signal-to-noise ratio. Patch clamp electrodes can perform intracellular recording with high signal-to-noise ratio but only from a few neurons in parallel. Recently we have developed and reported a neuroelectronic interface that combines the parallelism of the CMOS MEA and the intracellular sensitivity of the patch clamp. Here, we report the design and characterization of the CMOS integrated circuit (IC), a critical component of the neuroelectronic interface. Fabricated in 0.18-µm technology, the IC features an array of 4,096 platinum black (PtB) nanoelectrodes spaced at a 20 µm pitch on its surface and contains 4,096 active pixel circuits. Each active pixel circuit, consisting of a new switched-capacitor current injector--capable of injecting from ±15 pA to ±0.7 µA with a 5 pA resolution--and an operational amplifier, is highly configurable. When configured into current-clamp mode, the pixel intracellularly records membrane potentials including subthreshold activities with â¼23 µVrms input referred noise while injecting a current for simultaneous stimulation. When configured into voltage-clamp mode, the pixel becomes a switched-capacitor transimpedance amplifier with â¼1 pArms input referred noise, and intracellularly records ion channel currents while applying a voltage for simultaneous stimulation. Such voltage/current-clamp intracellular recording/stimulation is a feat only previously possible with the patch clamp method. At the same time, as an array, the IC overcomes the lack of parallelism of the patch clamp method, measuring thousands of mammalian neurons in parallel, with full-frame intracellular recording/stimulation at 9.4 kHz.
RESUMO
Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output, with important functional consequences. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs or proteins simultaneously, because genomic profiling methods could not be applied to single cells until very recently. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.
Assuntos
Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Splicing de RNA/imunologia , Análise de Célula Única , Transcriptoma/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Hibridização in Situ Fluorescente , Fator Regulador 7 de Interferon , Interferons/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Fator de Transcrição STAT2 , Análise de Sequência de RNA , Vírus/imunologiaRESUMO
A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell's membrane and subsequently release surface-bound molecules directly into the cell's cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology's potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanofios/química , Silício/química , Animais , Sequência de Bases , Células Cultivadas , Células HeLa , Humanos , Proteínas Luminescentes/genética , Microscopia Eletrônica de Varredura , Nanofios/ultraestrutura , Plasmídeos/administração & dosagem , Plasmídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , TransfecçãoRESUMO
A circuit level understanding of immune cells and hematological cancers has been severely impeded by a lack of techniques that enable intracellular perturbation without significantly altering cell viability and function. Here, we demonstrate that vertical silicon nanowires (NWs) enable gene-specific manipulation of diverse murine and human immune cells with negligible toxicity. To illustrate the power of the technique, we then apply NW-mediated gene silencing to investigate the role of the Wnt signaling pathway in chronic lymphocytic leukemia (CLL). Remarkably, CLL-B cells from different patients exhibit tremendous heterogeneity in their response to the knockdown of a single gene, LEF1. This functional heterogeneity defines three distinct patient groups not discernible by conventional CLL cytogenetic markers and provides a prognostic indicator for patients' time to first therapy. Analyses of gene expression signatures associated with these functional patient subgroups reveal unique insights into the underlying molecular basis for disease heterogeneity. Overall, our findings suggest a functional classification that can potentially guide the selection of patient-specific therapies in CLL and highlight the opportunities for nanotechnology to drive biological inquiry.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Nanofios/química , RNA Interferente Pequeno/administração & dosagem , Silício/química , Animais , Linfócitos B/metabolismo , Células Cultivadas , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Nanofios/toxicidade , Interferência de RNA , RNA Interferente Pequeno/genética , Silício/toxicidadeRESUMO
Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 µm spatial resolution while a miniaturized data interface allows 8× parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platform's capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.
RESUMO
High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform's potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Ensaios de Triagem em Larga Escala/métodos , Diagnóstico por Imagem , Impedância Elétrica , EletrodosRESUMO
Electrode-based impedance and electrochemical measurements can provide cell-biology information that is difficult to obtain using optical-microscopy techniques. Such electrical methods are non-invasive, label-free, and continuous, eliminating the need for fluorescence reporters and overcoming optical imaging's throughput/temporal resolution limitations. Nonetheless, electrode-based techniques have not been heavily employed because devices typically contain few electrodes per well, resulting in noisy aggregate readouts. Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) have sometimes been used for electrophysiological measurements with thousands of electrodes per well at sub-cellular pitches, but only basic impedance mappings of cell attachment have been performed outside of electrophysiology. Here, we report on new field-based impedance mapping and electrochemical mapping/patterning techniques to expand CMOS-MEA cell-biology applications. The methods enable accurate measurement of cell attachment, growth/wound healing, cell-cell adhesion, metabolic state, and redox properties with single-cell spatial resolution (20 µm electrode pitch). These measurements allow the quantification of adhesion and metabolic differences of cells expressing oncogenes versus wild-type controls. The multi-parametric, cell-population statistics captured by the chip-scale integrated device opens up new avenues for fully electronic high-throughput live-cell assays for phenotypic screening and drug discovery applications.
Assuntos
Técnicas de Cultura de Células , Semicondutores , Fenômenos Eletrofisiológicos , Microeletrodos , ÓxidosRESUMO
Current electrophysiological or optical techniques cannot reliably perform simultaneous intracellular recordings from more than a few tens of neurons. Here we report a nanoelectrode array that can simultaneously obtain intracellular recordings from thousands of connected mammalian neurons in vitro. The array consists of 4,096 platinum-black electrodes with nanoscale roughness fabricated on top of a silicon chip that monolithically integrates 4,096 microscale amplifiers, configurable into pseudocurrent-clamp mode (for concurrent current injection and voltage recording) or into pseudovoltage-clamp mode (for concurrent voltage application and current recording). We used the array in pseudovoltage-clamp mode to measure the effects of drugs on ion-channel currents. In pseudocurrent-clamp mode, the array intracellularly recorded action potentials and postsynaptic potentials from thousands of neurons. In addition, we mapped over 300 excitatory and inhibitory synaptic connections from more than 1,700 neurons that were intracellularly recorded for 19 min. This high-throughput intracellular-recording technology could benefit functional connectome mapping, electrophysiological screening and other functional interrogations of neuronal networks.
Assuntos
Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Potenciais da Membrana , Nanotecnologia/instrumentação , Neurônios/fisiologia , Animais , Células Cultivadas , Estimulação Elétrica , Microeletrodos , Ratos , Sinapses/fisiologiaRESUMO
The synaptic connections between neurons are traditionally determined by correlating the action potentials (APs) of a pre-synaptic neuron and small-amplitude subthreshold potentials of a post-synaptic neuron using invasive intracellular techniques, such as patch clamping. Extracellular recording by a microelectrode array can non-invasively monitor network activities of a large number of neurons, but its reduced sensitivity usually prevents direct measurements of synaptic signals. Here, we demonstrate that a newly developed complementary metal-oxide-semiconductor (CMOS) nanoelectrode array (CNEA) is capable of extracellularly determining direct synaptic connections in dense, multi-layer cultures of dissociated rat neurons. We spatiotemporally correlate action potential signals of hundreds of active neurons, detect small (â¼1 pA after averaging) extracellular synaptic signals at the region where pre-synaptic axons and post-synaptic dendrites/somas overlap, and use those signals to map synaptic connections. We use controlled stimulation to assess stimulation-dependent synaptic strengths and to titrate a synaptic blocker (CNQX: IC50 â¼ 1 µM). The new capabilities demonstrated here significantly enhance the utilities of CNEAs in connectome mapping and drug screening applications.
Assuntos
Axônios , Neurônios , Potenciais de Ação , Animais , Óxidos , Ratos , Semicondutores , SinapsesRESUMO
Developing a new tool capable of high-precision electrophysiological recording of a large network of electrogenic cells has long been an outstanding challenge in neurobiology and cardiology. Here, we combine nanoscale intracellular electrodes with complementary metal-oxide-semiconductor (CMOS) integrated circuits to realize a high-fidelity all-electrical electrophysiological imager for parallel intracellular recording at the network level. Our CMOS nanoelectrode array has 1,024 recording/stimulation 'pixels' equipped with vertical nanoelectrodes, and can simultaneously record intracellular membrane potentials from hundreds of connected in vitro neonatal rat ventricular cardiomyocytes. We demonstrate that this network-level intracellular recording capability can be used to examine the effect of pharmaceuticals on the delicate dynamics of a cardiomyocyte network, thus opening up new opportunities in tissue-based pharmacological screening for cardiac and neuronal diseases as well as fundamental studies of electrogenic cells and their networks.
Assuntos
Diagnóstico por Imagem , Cardiopatias/metabolismo , Ventrículos do Coração/metabolismo , Potenciais da Membrana , Miócitos Cardíacos/metabolismo , Animais , Eletrodos , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Ventrículos do Coração/patologia , Miócitos Cardíacos/patologia , RatosRESUMO
Deciphering the neuronal code--the rules by which neuronal circuits store and process information--is a major scientific challenge. Currently, these efforts are impeded by a lack of experimental tools that are sensitive enough to quantify the strength of individual synaptic connections and also scalable enough to simultaneously measure and control a large number of mammalian neurons with single-cell resolution. Here, we report a scalable intracellular electrode platform based on vertical nanowires that allows parallel electrical interfacing to multiple mammalian neurons. Specifically, we show that our vertical nanowire electrode arrays can intracellularly record and stimulate neuronal activity in dissociated cultures of rat cortical neurons and can also be used to map multiple individual synaptic connections. The scalability of this platform, combined with its compatibility with silicon nanofabrication techniques, provides a clear path towards simultaneous, high-fidelity interfacing with hundreds of individual neurons.