RESUMO
Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype.
Assuntos
Senilidade Prematura/metabolismo , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Progéria/metabolismo , Senilidade Prematura/genética , Linhagem Celular , Sobrevivência Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Progéria/genética , RNA Interferente Pequeno , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Genomes are arranged non-randomly in the 3D space of the cell nucleus. Here, we have developed HIPMap, a high-precision, high-throughput, automated fluorescent in situ hybridization imaging pipeline, for mapping of the spatial location of genome regions at large scale. High-throughput imaging position mapping (HIPMap) enabled an unbiased siRNA screen for factors involved in genome organization in human cells. We identify 50 cellular factors required for proper positioning of a set of functionally diverse genomic loci. Positioning factors include chromatin remodelers, histone modifiers, and nuclear envelope and pore proteins. Components of the replication and post-replication chromatin re-assembly machinery are prominently represented among positioning factors, and timely progression of cells through replication, but not mitosis, is required for correct gene positioning. Our results establish a method for the large-scale mapping of genome locations and have led to the identification of a compendium of cellular factors involved in spatial genome organization.
Assuntos
Núcleo Celular/genética , Genes , Técnicas Genéticas , Linhagem Celular , Replicação do DNA , Humanos , Processamento de Imagem Assistida por Computador/métodos , Hibridização in Situ Fluorescente/métodos , Análise de Célula Única/métodosRESUMO
The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.
Assuntos
Montagem e Desmontagem da Cromatina , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Vírus do Tumor Mamário do Camundongo , Camundongos , Modelos Biológicos , Método de Monte Carlo , Nucleossomos/metabolismo , Receptores de Estrogênio/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Células HeLa , Chaperonas de Histonas , Humanos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
BACKGROUND: Image segmentation pipelines are commonly used in microscopy to identify cellular compartments like nucleus and cytoplasm, but there are few standards for comparing segmentation accuracy across pipelines. The process of selecting a segmentation assessment pipeline can seem daunting to researchers due to the number and variety of metrics available for evaluating segmentation quality. RESULTS: Here we present automated pipelines to obtain a comprehensive set of 69 metrics to evaluate segmented data and propose a selection methodology for models based on quantitative analysis, dimension reduction or unsupervised classification techniques and informed selection criteria. CONCLUSION: We show that the metrics used here can often be reduced to a small number of metrics that give a more complete understanding of segmentation accuracy, with different groups of metrics providing sensitivity to different types of segmentation error. These tools are delivered as easy to use python libraries, command line tools, Common Workflow Language Tools, and as Web Image Processing Pipeline interactive plugins to ensure a wide range of users can access and use them. We also present how our evaluation methods can be used to observe the changes in segmentations across modern machine learning/deep learning workflows and use cases.
Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Microscopia , Aprendizado de Máquina , CitoplasmaRESUMO
The interaction of regulatory proteins with the complex nucleoprotein structures that are found in mammalian cells involves chromatin reorganization at multiple levels. Mechanisms that support these transitions are complex on many timescales, which range from milliseconds to minutes or hours. In this Review, we discuss emerging concepts regarding the function of regulatory elements in living cells. We also explore the involvement of these dynamic and stochastic processes in the evolution of fluctuating transcriptional activity states that are now commonly reported in eukaryotic systems.
Assuntos
Cromatina/genética , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Humanos , Modelos Genéticos , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação ProteicaRESUMO
Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Dano ao DNA/genética , Translocação Genética/genética , Animais , Linfócitos B/citologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Posicionamento Cromossômico , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Genes myc/genética , Genoma/genética , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Proteína de Replicação A/metabolismoRESUMO
3D organotypic culture models such as organoids and multicellular tumor spheroids (MCTS) are becoming more widely used for drug discovery and toxicology screening. As a result, 3D culture technologies adapted for high-throughput screening formats are prevalent. While a multitude of assays have been reported and validated for high-throughput imaging (HTI) and high-content screening (HCS) for novel drug discovery and toxicology, limited HTI/HCS with large compound libraries have been reported. Nonetheless, 3D HTI instrumentation technology is advancing and this technology is now on the verge of allowing for 3D HCS of thousands of samples. This review focuses on the state-of-the-art high-throughput imaging systems, including hardware and software, and recent literature examples of 3D organotypic culture models employing this technology for drug discovery and toxicology screening.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Hepatócitos/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imageamento Tridimensional/métodos , Esferoides Celulares/ultraestrutura , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Imageamento Tridimensional/instrumentação , Bibliotecas de Moléculas Pequenas/farmacologia , Software , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Higher-order genome organization shows tissue-specific patterns. However, functional relevance and the mechanisms shaping the genome architecture are poorly understood. Here we report a profound shift from promiscuous to highly selective genome organization that accompanies the effector lineage choice of differentiating T cells. As multipotent naive cells receive antigenic signals and commit to a T helper (Th) pathway, the genome-wide contacts of a lineage-specific cytokine locus are preferentially enriched for functionally relevant genes. Despite the establishment of divergent interactomes and global reprogramming of transcription in Th1 versus Th2, the overall expression status of the contact genes is surprisingly similar between the two lineages. Importantly, during differentiation, the genomic contacts are retained and strengthened precisely at DNA binding sites of the specific lineage-determining STAT transcription factor. In cells from the specific STAT knock-out mouse, the signature cytokine locus is unable to shed the promiscuous contacts established in the naive T cells, indicating the importance of genomic STAT binding. Altogether, the global aggregation of STAT binding loci from genic and nongenic regions highlights a new role for differentiation-promoting transcription factors in direct specification of higher-order nuclear architecture through interacting with regulatory regions. Such subnuclear environments have significant implications for efficient functioning of the mature effector lymphocytes.
Assuntos
Diferenciação Celular , Fator de Transcrição STAT4/metabolismo , Células Th1/citologia , Células Th2/citologia , Animais , Sítios de Ligação , Linhagem da Célula/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromossomos/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Loci Gênicos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Fator de Transcrição STAT4/genéticaRESUMO
The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Perfilação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , RNA/genética , RNA/metabolismo , Receptores de Glucocorticoides/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing.
Assuntos
Processamento Alternativo , Epigênese Genética , Antígenos de Histocompatibilidade/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Hipóxia Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation.
Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Proteínas/genética , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Cromatina/metabolismo , Dexametasona/farmacologia , Células Epiteliais/química , Células Epiteliais/ultraestrutura , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/metabolismo , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/genética , Transcrição GênicaRESUMO
Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7-14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Diferenciação Celular , Clonagem MolecularRESUMO
Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five- to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.
RESUMO
Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.
Assuntos
Corpos Embrioides , Células-Tronco Pluripotentes , Humanos , Corpos Embrioides/metabolismo , Reprodutibilidade dos Testes , Organoides , Diferenciação CelularRESUMO
Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.
Assuntos
Astrócitos , Células-Tronco Pluripotentes , Humanos , Astrócitos/metabolismo , Células Ependimogliais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Neurogênese , Diferenciação Celular , Proteína Glial Fibrilar Ácida/metabolismoRESUMO
Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types. Following principles of developmental biology and translational applicability, here we developed an efficient stepwise differentiation method for peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest, followed by differentiation into sensory neurons. Detailed characterization, including ultrastructural analysis, confirmed that the hPSC-derived nociceptors displayed cellular and molecular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors relevant for pain research and axonal growth/regeneration studies (e.g., TRPV1, NAV1.7, NAV1.8, TAC1, CALCA, GAP43, DPYSL2, NMNAT2). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.
Assuntos
Neurônios , Células-Tronco Pluripotentes , Humanos , Neurônios/metabolismo , Nociceptores , Diferenciação Celular , Transdução de Sinais , Gânglios Espinais/metabolismo , Células Receptoras SensoriaisRESUMO
Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Robótica , Automação , Linhagem da Célula , Células Cultivadas , Corpos Embrioides/citologia , Hepatócitos/citologia , Hepatócitos/virologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/virologia , Neurônios/citologia , RNA-Seq , Padrões de Referência , Análise de Célula Única , Infecção por Zika virus/patologiaRESUMO
Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).
RESUMO
Chromatin and associated regulatory proteins regulate gene expression in the natural environment of the intact cell nucleus. Specific combinations of DNA-binding transcription factors and recruited coregulatory proteins alter the conformation of chromatin at promoters and enhancers of target genes to stimulate or repress transcription. The dynamic nature of the regulatory proteins active in these processes allows the cell to modulate gene expression very rapidly, an important feature in many physiological processes. Live cell imaging and photobleaching studies of fluorescently-tagged proteins reveal that many transcription factors and other chromatin-associated proteins rapidly move through the nucleoplasm. Transcription factors also transiently interact with specific regulatory sequences in chromatin, suggesting that gene activation does not require the formation of stable long-lived regulatory complexes on the chromatin. In this review we discuss how dynamic interactions allow transcriptional regulatory proteins find their targets within the nucleus, alter target chromatin structure, and modulate physiological gene expression.