实验方案 生物化学 生物信息学与计算生物学 生物物理学 癌症生物学 细胞生物学 发育生物学 免疫学 微生物学 分子生物学 神经科学 植物科学 干细胞 系统生物学 文章和过刊查询 现期刊物 全部刊物 Articles In Press 实验方案合集 Articles In Press 作者指南 提交流程 稿件书写指南 提交稿件 稿件评审 评审标准 AI使用指南 出版伦理 利益冲突 文章处理费 关于 关于我们 目标与定位 顾问 编辑委员会 评审委员会 领导与管理团队 开放获取政策 内容可用性和索引 期刊合作伙伴 专业组织会员 联系我们 Isolation of Human Umbilical Cord Blood Hematopoietic Stem Cells and Directed Differentiation Into Megakaryocytes 人脐带血造血干细胞的分离及向巨核细胞定向分化 Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China Department of Transfusion Medicine, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University, Chongqing, China Platelets originate from megakaryocytes, whose generation involves a series of biological processes including directed differentiation, proliferation, polyploidization, and maturation of hematopoietic stem cells. Abnormalities in megakaryocyte development and maturation can lead to quantitative and functional defects in platelets, thereby contributing to hemostatic or thrombotic disorders as well as the development of malignancies. Investigating megakaryocyte development and maturation and platelet production can provide important theoretical foundations for the diagnosis and treatment of thrombocytopenia, thrombotic diseases, and myeloproliferative neoplasms. Currently, there are three main clinical sources of hematopoietic stem cells (HSCs): bone marrow (BM), peripheral blood (PBSC), and umbilical cord blood (UCB). Among these, umbilical cord blood (UCB)-derived HSCs, due to their higher differentiation efficiency and stronger proliferative capacity, are the preferred starting cell source for studying megakaryocyte (MK) development and maturation and the mechanisms of platelet production. This article describes a detailed protocol covering all necessary steps for isolating CD34 + hematopoietic stem cells from umbilical cord blood, followed by in vitro induction culture with stem cell factor (SCF) and thrombopoietin (TPO) to generate mature megakaryocytes that highly express early megakaryocyte markers (CD41a, CD61) and late maturation markers (CD42a, CD42b). This protocol provides an effective tool for studying megakaryocyte development and platelet production and holds potential value for application in research on megakaryocyte-related diseases. Protocol for Mouse Embryonic Aorta–Gonad–Mesonephros (AGM) Region Frozen Sectioning and Immunofluorescence 小鼠胚胎主动脉-性腺-中肾（AGM）区域冰冻切片及免疫荧光染色方法 School of Life Science and Technology, Shandong Second Medical University, Weifang, China School of Life Science and Technology, Shandong Second Medical University, Weifang, China School of Life Science and Technology, Shandong Second Medical University, Weifang, China The aorta–gonad–mesonephros (AGM) region is the site where hematopoietic stem cells (HSCs) first emerge during development, and is therefore widely used to study in vivo hematopoiesis and to discover novel regulatory mechanisms. The endothelial-to-hematopoietic transition (EHT) process can be directly observed via immunofluorescence on frozen sections of the AGM region. However, the mouse AGM region is extremely delicate and lies deep within the embryo, between the notochord and the somatic mesoderm. Here, we present a step-by-step protocol covering embryo collection, fixation, dehydration, and embedding with a defined orientation, followed by frozen sectioning, immunofluorescence staining, and confocal imaging. The protocol is highly reproducible and easy to follow and provides clear instructions on orienting the embryo and anatomically locating AGM. By filling a technical gap, the protocol can enable researchers to reliably study HSC emergence and EHT in the mouse embryonic AGM. Probing the Luminal Compartment of 3D Organoids via Particle Tracking Microrheology 利用粒子追踪微流变技术探测三维类器官腔内环境 Katrina N. Lyon Department of Neurology, University of Colorado Anschutz Medical Campus, Aurora, CO, USADepartment of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USA Chemical and Biological Engineering Department, Montana State University, Bozeman, MT, USACenter for Biofilm Engineering, Montana State University, Bozeman, MT, USA Department of Mathematical Sciences, Montana State University, Bozeman, MT, USA Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USA Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USACenter for Biofilm Engineering, Montana State University, Bozeman, MT, USA The mucus layer lining the human stomach is a critical barrier that protects the underlying epithelium from gastric acid and harmful pathogens such as Helicobacter pylori . The efficacy of this barrier relies on the structural integrity of the mucus, which is determined by various biochemical and biophysical features. Human gastric organoids—3D cellular models that resemble the stomach—contain mucus and have been used to investigate gastric disease. The luminal compartment of three-dimensional epithelial organoids represents a physiologically relevant but experimentally inaccessible microenvironment. In gastric organoids, luminal accumulation of mucus creates a confined viscoelastic hydrogel that mimics native gastric mucus. However, the small volume and topological confinement of organoids preclude conventional bulk rheometry. Here, we describe a particle tracking microrheology (PTM) protocol to measure the viscoelastic properties of the mucus within intact organoid lumina following microinjection of fluorescent microspheres. High-speed fluorescence imaging and particle trajectory analysis enable the quantification of viscous and elastic properties of the mucus through calculation of mean squared displacement (MSD), diffusive scaling exponent (alpha), and frequency-dependent storage (G’) and loss (G’’) moduli. This method enables rheological measurements in nanoliter-scale compartments without disrupting organoid architecture. We further discuss the impact of mucus heterogeneity and microstructure on scale-dependent mechanical behavior. This protocol is broadly applicable to other organoid systems and can be adapted to Transwell or organ-on-chip platforms for in situ luminal measurements. Generation of Budoids: 3D Multilineage Limb Models From Mouse Embryonic Stem Cells 肢芽类器官的构建：利用小鼠胚胎干细胞建立三维多谱系肢体模型 Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany Limb development requires the coordination of multiple cell types, including the limb bud mesoderm and surface ectoderm, by the apical ectodermal ridge (AER), a specialized signaling center secreting numerous morphogens. Characterizing these cell–cell interactions is crucial for understanding limb morphogenesis, but they are challenging to study in vivo. Furthermore, existing in vitro models do not capture the multilineage complexity of the limb. We recently developed a robust 7-day differentiation protocol using mouse embryonic stem cells (mESCs) to generate heterogeneous cultures containing cells with characteristics of the limb bud mesoderm, surface ectoderm, and AER. Dissociating and reaggregating these cultures in low attachment 96-well plates forms budoids, organoids that display certain limb bud–like features. Budoids undergo chondrogenesis-mediated symmetry breaking and elongation within 5 days of culture. Altogether, our protocols have enabled the study of cell–cell interactions in limb development and provide an easily scalable model adaptable for various applications, including drug testing and congenital disorder modeling. Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron–Myotube Co-cultures 人 iPSC 来源运动神经元—肌管分区共培养体系中轴突变性与 TDP-43 聚集的定量分析 Anand Ganapathy Subramaniam Gray Faculty of Medical & Health Sciences, Department of Neuroscience and Brain Disorders, Tel Aviv University, Tel Aviv, Israel Lucas Keniger de Andrade Gensas Gray Faculty of Medical & Health Sciences, Department of Neuroscience and Brain Disorders, Tel Aviv University, Tel Aviv, IsraelSagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Tal Gradus-Pery Gray Faculty of Medical & Health Sciences, Department of Neuroscience and Brain Disorders, Tel Aviv University, Tel Aviv, Israel Gray Faculty of Medical & Health Sciences, Department of Neuroscience and Brain Disorders, Tel Aviv University, Tel Aviv, IsraelSagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders. Engineering Decellularized Extracellular Matrix-Incorporated Apical-Out Airway Organoids 构建整合脱细胞细胞外基质的顶端向外气道类器官 Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA Chika S. Ikpechukwu Department of Medicine, University of Vermont, Burlington, VT, USA Dhruv Bhattaram Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USAPrecision Medicine Center for CF, Carver College of Medicine, University of Iowa, Iowa City, IA, USA Daniel J. Weiss Department of Medicine, University of Vermont, Burlington, VT, USA Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial–ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization. Generation of 3D Hemogenic Gastruloids From Mouse Embryonic Stem Cells 小鼠胚胎干细胞来源的三维造血类原肠胚构建 Sanquin Research, Landsteiner Laboratory, Amsterdam, The NetherlandsDepartment of Biosciences, College of Health, Medicine and Life Sciences, Brunel University of London, Uxbridge, United Kingdom Sanquin Research, Landsteiner Laboratory, Amsterdam, The NetherlandsCancer Center Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands

Department of Biosciences, College of Health, Medicine and Life Sciences, Brunel University of London, Uxbridge, United KingdomCentre for Genome Engineering and Maintenance, College of Health, Medicine and Life Sciences, Brunel University of London, Uxbridge, United Kingdom Embryonic blood formation encompasses the independent generation of different cell types in distinct cellular and anatomical environments, reflecting highly coordinated specific hierarchies of interacting tissues. Despite widespread use of embryonic stem cells (ESC) and induced pluripotent stem cell (iPSC)-based models to attempt to capture blood development in vitro and generate hematopoietic stem cells (HSC), a system that fully captures the spatial and temporal complexity of embryonic hematopoiesis is still lacking. In recent years, gastruloid models have emerged as powerful representations of early development, demonstrating self-organizing behaviors such as symmetry breaking, elongation, multi-axis formation, somitogenesis, and early organogenesis, with striking parallels to embryonic processes. Here, we present a protocol to generate hemogenic gastruloids (haemGx) from mouse ESC (mESC) that closely recapitulates the multi-stage, multi-niche process of blood formation and generates developmentally accurate hematopoietic progenitors. The haemGx model has been proven valuable in understanding embryonic hematopoiesis, as well as an in vitro model of forms of infant leukemia with an embryonic, in utero origin. Protocol for Measuring Drug–Target Engagement in Mouse Colorectal Cancer Organoids Using NanoBRET Assay 利用NanoBRET检测小鼠结直肠癌类器官中药物与靶标结合的实验方法 Hammed A. Badmos School of Cancer Sciences, Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow, UK School of Cancer Sciences, Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow, UK School of Cancer Sciences, Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow, UK Organoids as a drug discovery platform represent an emerging field that continues to refine its tools. NanoBRET (bioluminescence resonance energy transfer) has emerged as a proximity-based and highly sensitive assay to measure protein–protein and protein–ligand interactions. NanoBRET assays were developed and are currently used for 2D cell line experiments. Here, we present the development of the first organoid-compatible Nanoluciferase (Nluc) for 3D model systems. We utilise the Nluc for NanoBRET assays to test drug–target engagement. We describe steps for seeding, transfecting, and replating of mouse colorectal cancer organoids. In addition, we provide detailed procedures for the NanoBRET assay. Various lines of evidence have shown significant difference in drug response between 2D human cell lines and 3D model systems, including patient-derived organoids. Our protocol provides a template for measuring this difference in the context of drug–target engagement. Satellite Cell Isolation, Culture, and Infection After Retroviral Preparation 逆转录病毒制备后卫星细胞的分离培养与感染 Department of Biochemistry and Molecular Biology, University of Iowa, Iowa City, IA, USA Department of Biochemistry and Molecular Biology, University of Iowa, Iowa City, IA, USA Elizabeth H. Chen Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USADepartment of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Satellite cells are adult skeletal muscle stem cells that play essential roles in muscle regeneration. Understanding their behavior is critical for elucidating the mechanisms of muscle repair and advancing muscle regenerative therapies. This requires efficient methods for genetic manipulation in these cells. Retroviral-mediated gene delivery is commonly used for stable transgene expression in immortalized cell lines. However, existing approaches are not optimized for primary satellite cells, often resulting in variable efficiency and inconsistent outcomes. Here, we describe an optimized protocol for satellite cell isolation and culture, as well as retroviral production and infection of primary satellite cells that achieves high transduction efficiency. The satellite cell isolation procedure enriches for myofiber fragments prior to satellite cell release, thereby reducing contamination by non-myogenic cells and improving cell purity. Another key feature of this protocol is the concentration of retroviral particles and their resuspension in satellite cell growth medium prior to infection, which minimizes satellite cell exposure to packaging cell-conditioned medium. Compared to standard approaches, this protocol improves both infection efficiency and reproducibility. It is readily adaptable to a wide range of downstream applications, including microscopies, biochemical assays, and molecular biology analyses. Histological Processing of Organoids for Immunostaining 类器官免疫染色的组织学处理方法 Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Victor Perreaux Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France Guillaume Lamirault Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France Nantes Université, CHU Nantes, CNRS, Inserm, BioCore, US16, SFR Bonamy, Nantes, France Nathalie Gaborit Nantes Université, CHU Nantes, CNRS, INSERM, l’institut du thorax, Nantes, France Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, FranceCenter for Stem Cell and Organoid Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Organoids are three-dimensional cell structures derived from stem cells that recapitulate the architecture and function of native tissues. Histological analysis of organoids is essential for assessing their structure, cellular composition, and responses to experimental conditions. However, their small size and fragility make standard paraffin embedding workflows difficult. Here, we describe a robust and reproducible protocol for the fixation, paraffin embedding, and sectioning of human organoids, enabling high-quality histological and immunostaining analysis. The method involves direct fixation within the culture matrix and inclusion in HistoGel to prevent organoid loss during processing. The protocol is compatible with hematoxylin–eosin (H&E) staining and multiplex immunofluorescence. Critical steps, troubleshooting, and adaptations for intestinal and cardiac organoids are discussed. This cost-effective and accessible method supports long-term preservation and detailed structural analysis of organoid models. Stepwise Differentiation of Mouse Embryonic Stem Cells Into Murine Blood Vessel Organoids With Endothelial Lineage Tracing for Quality Control 将小鼠胚胎干细胞逐步分化为小鼠血管类器官并通过内皮细胞谱系追踪进行质量控制 Laboratory of Tumor Microenvironment and Therapeutic Resistance, VIB-KU Leuven Center for Cancer Biology, Leuven, BelgiumDepartment of Oncology, KU Leuven, Leuven, Belgium Laboratory of Tumor Microenvironment and Therapeutic Resistance, VIB-KU Leuven Center for Cancer Biology, Leuven, BelgiumDepartment of Oncology, KU Leuven, Leuven, Belgium Gabriele Bergers Laboratory of Tumor Microenvironment and Therapeutic Resistance, VIB-KU Leuven Center for Cancer Biology, Leuven, BelgiumDepartment of Oncology, KU Leuven, Leuven, Belgium In vitro vascular models are most informative when they recapitulate endothelial assembly within a 3D microenvironment. Blood vessel organoids (BVOs) enable the study of vascular heterogeneity, function, and organ-instructive cues in development, homeostasis, and disease. Here, we present a robust stepwise method to generate murine blood vessel organoids (mBVOs) from feeder-dependent mouse embryonic stem cells (mESCs) of common genetic backgrounds. Embryoid bodies (EBs) are formed using strain-specific seeding densities (day 0–3), followed by mesoderm induction (day 3–6) and vascular induction (day 6–8). Induced EBs are embedded in collagen I with Geltrex to drive sprouting and network formation (day 8–13). Vascular networks are microdissected and grown in suspension to yield mature mBVOs (day 21–30). The inclusion of a Cre-inducible VE-cadherin-GFP reporter line enables a quantitative quality control, reducing variability by excluding poorly differentiated organoids. The protocol reliably produces ~100 mBVOs per differentiation and is compatible with engineered mouse strains for gain- and loss-of-function studies, functional assays of vascular plasticity, and syngeneic grafting to assess perfusion. Thus, mBVOs provide a scalable and traceable 3D platform that bridges endothelial assays, mouse models, and human organoid systems. Generation of Functional Patient-Specific Thymus Organoids From Human Pluripotent Stem Cells (hPSCs) Using Air–Liquid Interface Culture 利用气液界面培养从人多能干细胞（hPSCs）构建患者特异性功能性胸腺类器官 Stephan A. Ramos Department of Developmental Biology, Stanford School of Medicine; Stanford, CA, USA Department of Pathology and Therapeutics, University of Florida, Gainesville, FL, USADiabetes Institute, University of Florida, Gainesville, FL, USA The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system, but it involutes with age, resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However, these approaches require access to primary human tissues or murine bone marrow stromal cells, are allogeneic, and do not support negative selection. Recently, we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here, we combined hPSC-derived TEPs, hematopoietic progenitor cells (HPCs), and mesenchymal cells, differentiated from the same hPSC line, and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic, patient-specific thymic organoids in vitro. Isolation, Culture, and Differentiation of Bovine Muscle Resident Stem Cells 牛肌肉驻留干细胞的分离、培养与分化方法 Department of Animal Science, College of Agricultural and Environmental Sciences, University of California, Davis, CA, USADepartment of Neurobiology, Physiology, & Behavior, College of Biological Sciences, University of California, Davis, CA, USA Madison W. Stewart Department of Neurobiology, Physiology, & Behavior, College of Biological Sciences, University of California, Davis, CA, USA Department of Animal Science, College of Agricultural and Environmental Sciences, University of California, Davis, CA, USA Department of Neurobiology, Physiology, & Behavior, College of Biological Sciences, University of California, Davis, CA, USA Rachel Espinoza Department of Neurobiology, Physiology, & Behavior, College of Biological Sciences, University of California, Davis, CA, USA Department of Animal Science, College of Agricultural and Environmental Sciences, University of California, Davis, CA, USA Department of Neurobiology, Physiology, & Behavior, College of Biological Sciences, University of California, Davis, CA, USADepartment of Physical Medicine and Rehabilitation, School of Medicine, University of California, Davis, CA, USA Bovine muscle satellite cells (MuSC) and fibro-adipogenic progenitor cells (FAP) are muscle resident stem cells that are responsible for postnatal muscle growth, intramuscular fat deposition, and extracellular matrix generation. These cells are of increasing interest for the cultivated meat community due to their ability to generate all the major components of meat; additionally, these cells are of interest to conventional animal science research to elucidate mechanisms to improve meat quality. To use these cells for these goals, efficient and accurate cell isolation, culture, and differentiation are essential to evaluate their cell fate decisions and behaviors. In this protocol, we detail a simultaneous isolation of both MuSCs and FAPs with multiple intermediate stopping points, allowing for flexibility for day-of time constraints. We also detail improved growth conditions to maximize cell expansion and procedures to assess cell differentiation. This protocol provides a flexible isolation procedure that is compatible with sampling in modern slaughterhouses or from biopsies. Additionally, the differentiation procedures provide improved differentiation but still allow in vitro treatment and assessment.