Nature Protocols最新文献

{"title":"Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids.","authors":"Eva Blondeel, Sam Ernst, Felix De Vuyst, Ákos Diósdi, Cláudio Pinheiro, Diogo Estêvão, Pekka Rappu, Robin Boiy, Sándor Dedeyne, Ligia Craciun, Vera Goossens, Jonas Dehairs, Tânia Cruz, Dominique Audenaert, Wim Ceelen, Michael Linnebacher, Tom Boterberg, Jo Vandesompele, Pieter Mestdagh, Johan Swinnen, Jyrki Heino, Peter Horvath, Maria José Oliveira, An Hendrix, Pieter De Metter, Olivier De Wever","doi":"10.1038/s41596-025-01150-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01150-y","url":null,"abstract":"<p><p>Spheroids are reaggregated multicellular three-dimensional structures generated from cells or cell cultures of healthy as well as pathological tissue. Basic and translational spheroid application across academia and industry have led to the development of multiple setups and analysis methods, which mostly lack the modularity to maximally phenotype spheroids. Here we present the self-assembly of single-cell suspensions into spheroids by the liquid overlay method, followed by a modular framework for a multifaceted phenotyping of spheroids. Cell seeding, supernatant handling and compound administration are elaborated by both manual and automated procedures. The phenotyping modules contain a suite of orthogonal assays to analyze spheroids longitudinally and/or at an endpoint. Longitudinal analyses include morphometry with or without spheroid or cell state specific information and supernatant evaluation (nutrient consumption and metabolite/cytokine production). Spheroids can also be used as a starting point to monitor single and collective cell migration and invasion. At an endpoint, spheroids are lysed, fixed or dissociated into single cells. Endpoint analyses allow the investigation of molecular content, single-cell composition and state and architecture with spatial cell and subcellular specific information. Each module addresses time requirements and quality control indicators to support reproducibility. The presented complementary techniques can be readily adopted by researchers experienced in cell culture and basic molecular biology. We anticipate that this modular protocol will advance the application of three-dimensional biology by providing scalable and complementary methods.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human induced pluripotent stem cell-derived cardiomyocytes and their use in a cardiac organ-on-a-chip to assay electrophysiology, calcium and contractility.","authors":"M Iveth Garcia, Keri Dame, Verena Charwat, Brian A Siemons, Henrik Finsberg, Bhavya Bhardwaj, Ryosuke Yokosawa, Ishan Goswami, Dylan Bruckner, Samuel T Wall, Kevin A Ford, Kevin E Healy, Alexandre J S Ribeiro","doi":"10.1038/s41596-025-01166-4","DOIUrl":"https://doi.org/10.1038/s41596-025-01166-4","url":null,"abstract":"<p><p>Cardiac organs-on-a-chip (OoCs) or microphysiological systems have the potential to predict cardiac effects of new drug candidates, including unanticipated cardiac outcomes, which are among the main causes for drug attrition. This protocol describes how to prepare and use a cardiac OoC containing cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS cells). The use of cells derived from hiPS cells as reliable sources of human cells from diverse genetic backgrounds also holds great potential, especially when cultured in OoCs that are physiologically relevant culture platforms. To promote the broad adoption of hiPS cell-derived cardiac OoCs in the drug development field, there is a need to first ensure reproducibility in their preparation and use. This protocol aims to provide key information on how to reduce sources of variability during hiPS cell maintenance, differentiation, loading and maturation in OoCs. Variability in these procedures can lead to inconsistent purity after differentiation and variable function between batches of microtissues formed in OoCs. This protocol also focuses on describing the handling and functional assessment of cardiac microtissues using live-cell microscopy approaches to quantify parameters of cellular electrophysiology, calcium transients and contractility. The protocol consists of five stages: (1) thaw and maintain hiPS cells, (2) differentiate hiPS cell cardiomyocytes, (3) load differentiated cells into OoCs, (4) maintain and characterize loaded cells, and (5) evaluate and utilize cardiac OoCs. Execution of the entire protocol takes ~40 days. The required skills to carry out the protocol are experience with sterile techniques, mammalian cell culture and maintaining hiPS cells in a pluripotent state.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143803882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized identification and characterization of small RNAs with PANDORA-seq.","authors":"Junchao Shi, Yunfang Zhang, Yun Li, Liwen Zhang, Xudong Zhang, Menghong Yan, Qi Chen, Ying Zhang","doi":"10.1038/s41596-025-01158-4","DOIUrl":"10.1038/s41596-025-01158-4","url":null,"abstract":"<p><p>Small noncoding RNAs (sncRNAs) are a diverse group of RNAs including small interfering RNAs, microRNAs, PIWI-interacting RNAs and RNAs derived from structured RNAs such as transfer RNAs, ribosomal RNAs and others. These sncRNAs have varied termini and RNA modifications, which can interfere with adaptor ligation and reverse transcription during cDNA library construction, hindering detection of many types of sncRNA by standard small RNA sequencing methods. To address this limitation, PANDORA sequencing introduces a refined methodology. The procedure includes sequential enzymatic treatments of size-selected RNAs with T4PNK and AlkB, which effectively circumvent the challenges presented by the ligation-blocking termini and reverse transcription-blocking RNA modifications, followed by tailored small RNA library construction protocols and deep sequencing. The obtained datasets are analyzed with the SPORTS pipeline, which can comprehensively analyze various types of sncRNA beyond the traditionally studied classes, to include those derived from various parental RNAs (for example, from transfer RNA and ribosomal RNA), as well as output the locations on the parental RNA from which these sncRNAs are derived. The entire protocol takes ~7 d, depending on the sample size and sequencing turnaround time. PANDORA sequencing provides a transformative tool to further our understanding of the expanding small RNA universe and to explore the uncharted functions of sncRNAs.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative molecular imaging using deep magnetic resonance fingerprinting.","authors":"Nikita Vladimirov, Ouri Cohen, Hye-Young Heo, Moritz Zaiss, Christian T Farrar, Or Perlman","doi":"10.1038/s41596-025-01152-w","DOIUrl":"https://doi.org/10.1038/s41596-025-01152-w","url":null,"abstract":"<p><p>Deep learning-based saturation transfer magnetic resonance fingerprinting (MRF) is an emerging approach for noninvasive in vivo imaging of proteins, metabolites and pH. It involves a series of steps, including sample/participant preparation, image acquisition schedule design, biophysical model formulation and artificial intelligence and computational model training, followed by image acquisition, deep reconstruction and analysis. Saturation transfer-based molecular MRI has been slow to reach clinical maturity and adoption for clinical practice due to its technical complexity, semi-quantitative contrast-weighted nature and long scan times needed for the extraction of quantitative molecular biomarkers. Deep MRF provides solutions to these challenges by providing a quantitative and rapid framework for extracting biologically and clinically meaningful molecular information. Here we define a complete protocol for quantitative molecular MRI using deep MRF. We describe in vitro sample preparation and animal and human scan considerations, and provide intuition behind the acquisition protocol design and optimization of chemical exchange saturation transfer (CEST) and semi-solid magnetization transfer (MT) quantitative imaging. We then extensively describe the building blocks for several artificial intelligence models and demonstrate their performance for different applications, including cancer monitoring, brain myelin imaging and pH quantification. Finally, we provide guidelines to further modify and expand the pipeline for imaging a variety of other pathologies (such as neurodegeneration, stroke and cardiac disease), accompanied by the related open-source code and sample data. The procedure takes between 48 min (for two proton pools or in vitro imaging) and 57 h (for complex multi-proton pool in vivo imaging) to complete and is suitable for graduate student-level users.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Author Correction: Life-long functional regeneration of in vivo airway epithelium by the engraftment of airway basal stem cells.","authors":"Liang Ma, Bibek R Thapa, Jake A Le Suer, Andrew Tilston-Lünel, Michael J Herriges, Feiya Wang, Pushpinder S Bawa, Xaralabos Varelas, Finn J Hawkins, Darrell N Kotton","doi":"10.1038/s41596-025-01180-6","DOIUrl":"https://doi.org/10.1038/s41596-025-01180-6","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143753617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correlative light and electron microscopy for human brain and other biological models.","authors":"Notash Shafiei, Daniel Stӓhli, Domenic Burger, Marta Di Fabrizio, Lukas van den Heuvel, Jean Daraspe, Carolin Böing, Sarah H Shahmoradian, Wilma D J van de Berg, Christel Genoud, Henning Stahlberg, Amanda J Lewis","doi":"10.1038/s41596-025-01153-9","DOIUrl":"https://doi.org/10.1038/s41596-025-01153-9","url":null,"abstract":"<p><p>Correlative light and electron microscopy (CLEM) combines light microscopy, for identifying a target via genetic labels, dyes, antibodies and morphological features, with electron microscopy, for analyzing high-resolution subcellular ultrastructures. Here, we describe step-by-step instructions to perform a CLEM experiment, optimized for the investigation of ultrastructural features in human brain tissue. The procedure is carried out at room temperature and can be adapted to other human and animal tissue samples. The procedure requires 8 d to complete and includes the stages of sample fixation for optimal ultrastructural preservation, immunofluorescence staining, image acquisition and multimodal image correlation and is executable within standard electron microscopy laboratories. Serving as a critical tool for characterizing human tissue and disease models, room-temperature CLEM facilitates the identification and quantification of subcellular morphological features across brain regions.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143753622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tutorial: guidelines for quality filtering of whole-exome and whole-genome sequencing data for population-scale association analyses.","authors":"Julia M Sealock, Franjo Ivankovic, Calwing Liao, Siwei Chen, Claire Churchhouse, Konrad J Karczewski, Daniel P Howrigan, Benjamin M Neale","doi":"10.1038/s41596-025-01169-1","DOIUrl":"https://doi.org/10.1038/s41596-025-01169-1","url":null,"abstract":"<p><p>Genetic sequencing technologies are powerful tools for identifying rare variants and genes associated with Mendelian and complex traits; indeed, whole-exome and whole-genome sequencing are increasingly popular methods for population-scale genetic studies. However, careful quality control steps should be taken to ensure study accuracy and reproducibility, and sequencing data require extensive quality filtering to delineate true variants from technical artifacts. Although processing standards are harmonized across pipelines to call variants from sequencing reads, there currently exists no standardized pipeline for conducting quality filtering on variant-level datasets for the purpose of population-scale association analysis. In this Tutorial, we discuss key quality control parameters, provide guidelines for conducting quality filtering of samples and variants, and compare commonly used software programs for quality control of samples, variants and genotypes from sequencing data. As sequencing data continue to gain popularity in genetic research, establishing standardized quality control practices is crucial to ensure consistent, reliable and reproducible results across studies.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742928","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cyborg organoids integrated with stretchable nanoelectronics can be functionally mapped during development.","authors":"Zuwan Lin, Wenbo Wang, Ren Liu, Qiang Li, Jaeyong Lee, Charles Hirschler, Jia Liu","doi":"10.1038/s41596-025-01147-7","DOIUrl":"10.1038/s41596-025-01147-7","url":null,"abstract":"<p><p>Organoids are in vitro miniaturized cellular models of organs that offer opportunities for studying organ development, disease mechanisms and drug screening. Understanding the complex processes governing organoid development and function requires methods suitable for the continuous, long-term monitoring of cell activities (for example, electrophysiological and mechanical activity) at single-cell resolution throughout the entire three-dimensional (3D) structure. Cyborg organoid technology addresses this need by seamlessly integrating stretchable mesh nanoelectronics with tissue-like properties, such as tissue-level flexibility, subcellular feature size and mesh-like networks, into 3D organoids through a 2D-to-3D tissue reconfiguration process during organogenesis. This approach enables longitudinal, tissue-wide, single-cell functional mapping, thereby overcoming the limitations of existing techniques including recording duration, spatial coverage, and the ability to maintain stable contact with the tissue during organoid development. This protocol describes the fabrication and characterization of stretchable mesh nanoelectronics, their electrical performance, their integration with organoids and the acquisition of long-term functional organoid activity requiring multimodal data analysis techniques. Cyborg organoid technology represents a transformative tool for investigating organoid development and function, with potential for improving in vitro disease models, drug screening and personalized medicine. The procedure is suitable for users within a multidisciplinary team with expertise in stem cell biology, tissue engineering, nanoelectronics fabrication, electrophysiology and data science.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143720178","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A detailed guide to assessing genome assembly based on long-read sequencing data using Inspector.","authors":"Yan Guo, Yuwei Song, Limin Jiang, Yu Chen, Michele Ceccarelli, Min Gao, Zechen Chong","doi":"10.1038/s41596-025-01149-5","DOIUrl":"10.1038/s41596-025-01149-5","url":null,"abstract":"<p><p>Long-read sequencing technologies yield extended DNA sequences capable of spanning intricate, repetitive genome regions, thereby facilitating the generation of more precise and comprehensive genome assemblies. However, assembly errors are inevitable owing to inherent genomic complexity and limitations of sequencing technology and assembly algorithms, making assembly evaluation crucial. The genome assembly evaluation tool Inspector presents several advantages over existing long-read de novo assembly evaluation tools, including (1) both reference-free and reference-guided assembly evaluation; (2) the ability to detect both small- and large-scale structural errors; (3) the option of assembly error correction, which can improve the quality value of the original assembly; and (4) the ability to perform haplotype-resolved assembly evaluation. Inspector can provide not only basic contig and alignment statistics, but also the precise locations and types of the different structural errors. These advantages provide a robust framework for long-read assembly evaluation. In this Protocol, we showcase four procedures to demonstrate the different applications of Inspector for long-read assembly evaluation. Inspector software and additional guides can be found at https://github.com/ChongLab/Inspector_protocol .</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143720173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CellPhoneDB v5: inferring cell-cell communication from single-cell multiomics data.","authors":"Kevin Troulé, Robert Petryszak, Batuhan Cakir, James Cranley, Alicia Harasty, Martin Prete, Zewen Kelvin Tuong, Sarah A Teichmann, Luz Garcia-Alonso, Roser Vento-Tormo","doi":"10.1038/s41596-024-01137-1","DOIUrl":"10.1038/s41596-024-01137-1","url":null,"abstract":"<p><p>Cell-cell communication is essential for tissue development, function and regeneration. The revolution of single-cell genomics technologies offers an unprecedented opportunity to uncover how cells communicate in vivo within their tissue niches and how disruption of these niches can lead to diseases and developmental abnormalities. CellPhoneDB is a bioinformatics toolkit designed to infer cell-cell communication by combining a curated repository of bona fide ligand-receptor interactions with methods to integrate these interactions with single-cell genomics data. Here we present a protocol for the latest version of CellPhoneDB (v5), offering several new features. First, the repository has been expanded by one-third with the addition of new interactions, including ~1,000 interactions mediated by nonpeptidic ligands such as steroidogenic hormones, neurotransmitters and small G-protein-coupled receptor (GPCR)-binding ligands. Second, we outline a new way of using the database that allows users to tailor queries to their experimental designs. Third, the update incorporates novel strategies to prioritize specific cell-cell interactions, leveraging information from other modalities such as tissue microenvironments derived from spatial transcriptomics technologies or transcription factor activities derived from a single-cell assay for transposase accessible chromatin assays. Finally, we describe the new CellPhoneDBViz module to interactively visualize and share results. Altogether, CellPhoneDB v5 enhances the precision of cell-cell communication inference, offering new insights into tissue biology in physiological microenvironments. This protocol typically takes ~15 min and requires basic knowledge of python.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143710776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}