Nature Protocols最新文献_第3页

Precise kilobase-scale genomic insertions in mammalian cells using PASTE. 使用PASTE在哺乳动物细胞中精确插入千碱基规模的基因组。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2024-12-15 DOI: 10.1038/s41596-024-01090-z

Christopher W Fell, Cian Schmitt-Ulms, Dario V Tagliaferri, Jonathan S Gootenberg, Omar O Abudayyeh

{"title":"Precise kilobase-scale genomic insertions in mammalian cells using PASTE.","authors":"Christopher W Fell, Cian Schmitt-Ulms, Dario V Tagliaferri, Jonathan S Gootenberg, Omar O Abudayyeh","doi":"10.1038/s41596-024-01090-z","DOIUrl":"10.1038/s41596-024-01090-z","url":null,"abstract":"Programmable gene integration technologies are an emerging modality with exciting applications in both basic research and therapeutic development. Programmable addition via site-specific targeting elements (PASTE) is a programmable gene integration approach for precise and efficient programmable integration of large DNA sequences into the genome. PASTE offers improved editing efficiency, purity and programmability compared with previous methods for long insertions into the mammalian genome. By combining the specificity and cargo size capabilities of site-specific integrases with the programmability of prime editing, PASTE can precisely insert cargoes of at least 36 kb with efficiencies of up to 60%. Here we outline best practices for design, execution and analysis of PASTE experiments, with protocols for integration of EGFP at the human NOLC1 and ACTB genomic loci and for readout by next generation sequencing and droplet digital PCR. We provide guidelines for designing and optimizing a custom PASTE experiment for integration of desired payloads at alternative genomic loci, as well as example applications for in-frame protein tagging and multiplexed insertions. To facilitate experimental setup, we include the necessary sequences and plasmids for the delivery of PASTE components to cells via plasmid transfection or in vitro transcribed RNA. Most experiments in this protocol can be performed in as little as 2 weeks, allowing for precise and versatile programmable gene insertion.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1546-1583"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142829368","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}

引用次数: 0

A consensus platform for antibody characterization. 抗体特征描述的共识平台。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2024-12-17 DOI: 10.1038/s41596-024-01095-8

Riham Ayoubi, Joel Ryan, Sara Gonzalez Bolivar, Charles Alende, Vera Ruiz Moleon, Maryam Fotouhi, Mona Alqazzaz, Kathleen Southern, Walaa Alshafie, Matt R Baker, Alexander R Ball, Danielle Callahan, Jeffery A Cooper, Katherine Crosby, Kevin J Harvey, Douglas W Houston, Ravindran Kumaran, Meghan Rego, Christine Schofield, Hai Wu, Michael S Biddle, Claire M Brown, Richard A Kahn, Anita Bandrowski, Harvinder S Virk, Aled M Edwards, Peter S McPherson, Carl Laflamme

{"title":"A consensus platform for antibody characterization.","authors":"Riham Ayoubi, Joel Ryan, Sara Gonzalez Bolivar, Charles Alende, Vera Ruiz Moleon, Maryam Fotouhi, Mona Alqazzaz, Kathleen Southern, Walaa Alshafie, Matt R Baker, Alexander R Ball, Danielle Callahan, Jeffery A Cooper, Katherine Crosby, Kevin J Harvey, Douglas W Houston, Ravindran Kumaran, Meghan Rego, Christine Schofield, Hai Wu, Michael S Biddle, Claire M Brown, Richard A Kahn, Anita Bandrowski, Harvinder S Virk, Aled M Edwards, Peter S McPherson, Carl Laflamme","doi":"10.1038/s41596-024-01095-8","DOIUrl":"10.1038/s41596-024-01095-8","url":null,"abstract":"Antibody-based research applications are critical for biological discovery. Yet there are no industry standards for comparing the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed and approved jointly by industry and academic researchers, that enables the systematic comparison of antibody performance in western blot, immunoprecipitation and immunofluorescence. The scalable protocols, which require minimal technological resources, consist of (1) the identification of appropriate cell lines for antibody characterization studies, (2) development/contribution of isogenic knockout controls, and (3) a series of antibody characterization procedures focused on the most common applications of antibodies in research. We provide examples of expected outcomes to guide antibody users in evaluating antibody performance. Central to our approach is advocating for transparent and open data sharing, enabling a community effort to identify specific antibodies for all human proteins. Mid-level graduate students with training in biochemistry and prior experience in cell culture and microscopy can complete the protocols for a specific protein within 1 month while working part-time on this effort. Antibody characterization is needed to meet standards for resource validation and data reproducibility, which are increasingly required by journals and funding agencies.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1509-1545"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142847106","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}

引用次数: 0

fMRI data acquisition and analysis for task-free, anesthetized rats. 无任务麻醉大鼠的fMRI数据采集与分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-01-28 DOI: 10.1038/s41596-024-01110-y

Roël M Vrooman, Monica van den Berg, Gabriel Desrosiers-Gregoire, Wessel A van Engelenburg, Marie E Galteau, Sung-Ho Lee, Andor Veltien, David A Barrière, Diana Cash, M Mallar Chakravarty, Gabriel A Devenyi, Alessandro Gozzi, Olli Gröhn, Andreas Hess, Judith R Homberg, Ileana O Jelescu, Georgios A Keliris, Tom Scheenen, Yen-Yu Ian Shih, Marleen Verhoye, Claire Wary, Marcel Zwiers, Joanes Grandjean

{"title":"fMRI data acquisition and analysis for task-free, anesthetized rats.","authors":"Roël M Vrooman, Monica van den Berg, Gabriel Desrosiers-Gregoire, Wessel A van Engelenburg, Marie E Galteau, Sung-Ho Lee, Andor Veltien, David A Barrière, Diana Cash, M Mallar Chakravarty, Gabriel A Devenyi, Alessandro Gozzi, Olli Gröhn, Andreas Hess, Judith R Homberg, Ileana O Jelescu, Georgios A Keliris, Tom Scheenen, Yen-Yu Ian Shih, Marleen Verhoye, Claire Wary, Marcel Zwiers, Joanes Grandjean","doi":"10.1038/s41596-024-01110-y","DOIUrl":"10.1038/s41596-024-01110-y","url":null,"abstract":"Templates for the acquisition of large datasets such as the Human Connectome Project guide the neuroimaging community to reproducible data acquisition and scientific rigor. By contrast, small animal neuroimaging often relies on laboratory-specific protocols, which limit cross-study comparisons. The establishment of broadly validated protocols may facilitate the acquisition of large datasets, which are essential for uncovering potentially small effects often seen in functional MRI (fMRI) studies. Here, we outline a procedure for the acquisition of fMRI datasets in rats and describe animal handling, MRI sequence parameters, data conversion, preprocessing, quality control and data analysis. The procedure is designed to be generalizable across laboratories, has been validated by using datasets across 20 research centers with different scanners and field strengths ranging from 4.7 to 17.2 T and can be used in studies in which it is useful to compare functional connectivity measures across an extensive range of datasets. The MRI procedure requires 1 h per rat to complete and can be carried out by users with limited expertise in rat handling, MRI and data processing.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1393-1412"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143059667","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}

引用次数: 0

Pair correlation microscopy of intracellular molecular transport. 细胞内分子运输的对相关显微镜。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-02-06 DOI: 10.1038/s41596-024-01097-6

Julissa Sanchez-Velasquez, Ashleigh Solano, Michelle A Digman, Enrico Gratton, Francesco Cardarelli, Elizabeth Hinde

{"title":"Pair correlation microscopy of intracellular molecular transport.","authors":"Julissa Sanchez-Velasquez, Ashleigh Solano, Michelle A Digman, Enrico Gratton, Francesco Cardarelli, Elizabeth Hinde","doi":"10.1038/s41596-024-01097-6","DOIUrl":"10.1038/s41596-024-01097-6","url":null,"abstract":"Pair correlation microscopy is a unique approach to fluorescence correlation spectroscopy that can track the long-range diffusive route of a population of fluorescent molecules in live cells with respect to intracellular architecture. This method is based on the use of a pair correlation function (pCF) that, through spatiotemporal comparison of fluctuations in fluorescence intensity recorded throughout a microscope data acquisition, enables changes in a molecule's arrival time to be spatially mapped and statistically quantified. In this protocol, we present guidelines for the measurement and analysis of line scan pair correlation microscopy data acquired on a confocal laser scanning microscope (CLSM), which will enable users to extract a fluorescent molecule's transport pattern throughout a living cell, and then quantify the molecular accessibility of intracellular barriers encountered or the mode of diffusion governing a molecular trafficking event. Finally, we demonstrate how this protocol can be extended to a two-channel line scan acquisition that, when coupled with a cross pCF calculation, enables a fluorescent molecule's transport pattern to be selectively tracked as a function of complex formation with a spectrally distinct fluorescent ligand. For a skilled user of a CLSM, the line scan data acquisition and analysis described in this protocol will take ~1-2 d, depending on the sample and the number of experiments to be processed.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1651-1677"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143365259","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}

引用次数: 0

Unraveling cell-cell communication with NicheNet by inferring active ligands from transcriptomics data. 通过从转录组学数据推断活性配体，揭示细胞与NicheNet的通信。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-03-04 DOI: 10.1038/s41596-024-01121-9

Chananchida Sang-Aram, Robin Browaeys, Ruth Seurinck, Yvan Saeys

{"title":"Unraveling cell-cell communication with NicheNet by inferring active ligands from transcriptomics data.","authors":"Chananchida Sang-Aram, Robin Browaeys, Ruth Seurinck, Yvan Saeys","doi":"10.1038/s41596-024-01121-9","DOIUrl":"10.1038/s41596-024-01121-9","url":null,"abstract":"Ligand-receptor interactions constitute a fundamental mechanism of cell-cell communication and signaling. NicheNet is a well-established computational tool that infers ligand-receptor interactions that potentially regulate gene expression changes in receiver cell populations. Whereas the original publication delves into the algorithm and validation, this paper describes a best practices workflow cultivated over four years of experience and user feedback. Starting from the input single-cell expression matrix, we describe a 'sender-agnostic' approach that considers ligands from the entire microenvironment and a 'sender-focused' approach that considers ligands only from cell populations of interest. As output, users will obtain a list of prioritized ligands and their potential target genes, along with multiple visualizations. We include further developments made in NicheNet v2, in which we have updated the data sources and implemented a downstream procedure for prioritizing cell type-specific ligand-receptor pairs. Although a standard NicheNet analysis takes <10 min to run, users often invest additional time in making decisions about the approach and parameters that best suit their biological question. This paper serves to aid in this decision-making process by describing the most appropriate workflow for common experimental designs like case-control and cell-differentiation studies. Finally, in addition to the step-by-step description of the code, we also provide wrapper functions that enable the analysis to be run in one line of code, thus tailoring the workflow to users at all levels of computational proficiency.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1439-1467"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143557451","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}

引用次数: 0

YCharOS protocol for antibody validation. YCharOS 抗体验证协议。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 DOI: 10.1038/s41596-024-01108-6

Fátima L Monteiro, Jan L A Voskuil, Cecilia Williams

引用次数: 0

Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications. 用于生化传感应用的锁频低语消失谐振器（FLOWER）。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-01-09 DOI: 10.1038/s41596-024-01096-7

Sartanee Suebka, Adley Gin, Judith Su

{"title":"Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications.","authors":"Sartanee Suebka, Adley Gin, Judith Su","doi":"10.1038/s41596-024-01096-7","DOIUrl":"10.1038/s41596-024-01096-7","url":null,"abstract":"Sensitive, rapid and label-free biochemical sensors are needed for many applications. In this protocol, we describe biochemical detection using FLOWER (frequency locked optical whispering evanescent resonator)-a technique that we have used to detect single protein molecules in aqueous solution as well as exosomes, ribosomes and low part-per-trillion concentrations of volatile organic compounds. Whispering gallery mode microtoroid resonators confine light for extended time periods (hundreds of nanoseconds). When light circulates within the resonator, a portion of the electromagnetic field extends beyond the cavity, forming an evanescent field. This field interacts with bound analytes resulting in a change in the cavity's effective refractive index, which can be tracked by monitoring shifts in the resonance wavelength. The surface of the microtoroid can be functionalized to respond specifically to an analyte or biochemical interaction of interest. The frequency-locking feature of frequency locked optical whispering evanescent resonator means that the instruments respond to perturbations in the surface by very rapidly finding the new resonant frequency. Here we describe microtoroid fabrication (4-6 h), how to couple light into these devices using tapered optical fibers (20-40 min) and procedures for coupling antibodies as well as G-protein coupled receptors to the microtoroid's surface (from 1 h to 1 d depending on the target analyte). In addition, we describe our liquid handling perfusion system as well as the use of a rotary selector valve and custom fluidic chamber to optimize sample delivery. Step-by-step details on how to perform biosensing experiments and analyze the data are described; this takes 1-2 d.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1616-1650"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142952016","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}

引用次数: 0

PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics. PEPPI-MS：凝胶样品预分离，用于深层自上而下和中向下的蛋白质组学。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-01-16 DOI: 10.1038/s41596-024-01100-0

Ayako Takemori, Philipp T Kaulich, Andreas Tholey, Nobuaki Takemori

{"title":"PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics.","authors":"Ayako Takemori, Philipp T Kaulich, Andreas Tholey, Nobuaki Takemori","doi":"10.1038/s41596-024-01100-0","DOIUrl":"10.1038/s41596-024-01100-0","url":null,"abstract":"Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS-PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS-PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS-PAGE in solution with a median efficiency of 68% within 10 min and, unlike conventional electroelution methods, requires no special equipment, contributing to a remarkably economical implementation. The entire protocol from electrophoresis to protein purification can be performed in <5 h. By combining the resulting PEPPI fraction with other protein-separation techniques, such as reversed-phase liquid chromatography and ion mobility techniques, multidimensional proteome separations for in-depth proteoform analysis can be easily achieved.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1413-1438"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008633","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}

引用次数: 0

Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers. 轨道阱分析仪多路电荷检测质谱的标准化工作流程。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-01-02 DOI: 10.1038/s41596-024-01091-y

Pei Su, John P McGee, Michael A R Hollas, Ryan T Fellers, Kenneth R Durbin, Joseph B Greer, Bryan P Early, Ping F Yip, Vlad Zabrouskov, Kristina Srzentić, Michael W Senko, Philip D Compton, Neil L Kelleher, Jared O Kafader

{"title":"Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers.","authors":"Pei Su, John P McGee, Michael A R Hollas, Ryan T Fellers, Kenneth R Durbin, Joseph B Greer, Bryan P Early, Ping F Yip, Vlad Zabrouskov, Kristina Srzentić, Michael W Senko, Philip D Compton, Neil L Kelleher, Jared O Kafader","doi":"10.1038/s41596-024-01091-y","DOIUrl":"10.1038/s41596-024-01091-y","url":null,"abstract":"Individual ion mass spectrometry (I2MS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, I2MS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. I2MS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution. A mass distribution or 'profile' can be created for any desired sample regardless of composition or heterogeneity. To assist in reducing I2MS analysis to practice, we developed this workflow for data acquisition and subsequent data analysis, which includes (i) protein sample preparation, (ii) attenuation of ion signals to obtain individual ions, (iii) the creation of a charge-calibration curve from standard proteins with known charge states and finally (iv) producing a meaningful mass spectral output from a complex or unknown sample by using the STORIboard software. This protocol is suitable for users with prior experience in mass spectrometry and bioanalytical chemistry. First, the analysis of protein standards in native and denaturing mode is presented, setting the foundation for the analysis of complex mixtures that are intractable via traditional mass spectrometry techniques. Examples of complex mixtures included here demonstrate the relevant analysis of an intact human monoclonal antibody and its intricate glycosylation patterns.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1485-1508"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12151780/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation and long-term culture of human cerebellar organoids from pluripotent stem cells. 多能干细胞生成和长期培养人小脑类器官。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2024-12-02 DOI: 10.1038/s41596-024-01093-w

Alexander Atamian, Marcella Birtele, Negar Hosseini, Giorgia Quadrato

{"title":"Generation and long-term culture of human cerebellar organoids from pluripotent stem cells.","authors":"Alexander Atamian, Marcella Birtele, Negar Hosseini, Giorgia Quadrato","doi":"10.1038/s41596-024-01093-w","DOIUrl":"10.1038/s41596-024-01093-w","url":null,"abstract":"The advancement of research on human cerebellar development and diseases has been hindered by the lack of a cell-based system that mirrors the cellular diversity and functional characteristics of the human cerebellum. Here, we describe our protocol for a human pluripotent stem cell-derived human cerebellar organoid (hCerO) model, which successfully replicates the cellular diversity of the fetal cerebellum along with some of its distinct cytoarchitectural features. Our approach involves the patterning of human pluripotent stem cells, resulting in the generation of both cerebellar excitatory and inhibitory progenitor populations-specifically, the rhombic lip and ventricular zone progenitors, respectively. This patterning strategy leads to the reproducible differentiation of the major neurons of the cerebellum such as granule cells and Purkinje cells within just one month of culture. hCerOs serve as platforms for molecular, cellular and functional assays, including single-cell transcriptomics, immunohistochemistry and investigations into calcium dynamics and electrophysiological properties. Remarkably, the cultivation of hCerOs for up to 8 months enables the healthy survival and maturation of Purkinje cells, which exhibit molecular and electrophysiological features akin to their in vivo counterparts. Overall, our protocol generates and allows for the long-term culture of all major cell types within the cerebellum. Consequently, this significant advancement provides the developmental neurobiology field with a robust platform for exploring both cerebellar development and diseases within an all-human system. This protocol can be easily implemented by a technician with cell culture experience and takes 1-2 months to complete with an option for extended maturation over the course of several months.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1584-1615"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142770598","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}

引用次数: 0