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Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease.
Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.
Authors: Asma Yaghi, Aisha Zaman, Myrna Dolovich.
Published: 03-26-2010
Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and ≤1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37°C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.
20 Related JoVE Articles!
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Imaging Centrosomes in Fly Testes
Authors: Marcus L. Basiri, Stephanie Blachon, Yiu-Cheung Frederick Chim, Tomer Avidor-Reiss.
Institutions: University of Toledo.
Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.
Developmental Biology, Issue 79, biology (general), genetics (animal and plant), animal biology, animal models, Life Sciences (General), Centrosome, Spermatogenesis, Spermiogenesis, Drosophila, Centriole, Cilium, Mitosis, Meiosis
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
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Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis
Authors: Denise Wernike, Chloe van Oostende, Alisa Piekny.
Institutions: Concordia University.
This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.
Neuroscience, Issue 85, C. elegans, morphogenesis, cytokinesis, neuroblasts, anillin, microscopy, cell division
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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Authors: Daniel T. Claiborne, Jessica L. Prince, Eric Hunter.
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
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In vitro Cell Culture Model for Toxic Inhaled Chemical Testing
Authors: Shama Ahmad, Aftab Ahmad, Keith B. Neeves, Tara Hendry-Hofer, Joan E. Loader, Carl W. White, Livia Veress.
Institutions: University of Colorado, Colorado School of Mines.
Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.
Bioengineering, Issue 87, air-liquid interface, chlorine exposure, toxic inhaled chemicals, Transepithelial Electrical Resistance,Immunocytochemistry
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Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Authors: Kristen K. McCampbell, Kristin N. Springer, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
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From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Authors: Wen-Ting Tsai, Ahmed Hassan, Purbasha Sarkar, Joaquin Correa, Zoltan Metlagel, Danielle M. Jorgens, Manfred Auer.
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
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Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy
Authors: Johnny L. Carson.
Institutions: The University of North Carolina at Chapel Hill.
Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.
Biophysics, Issue 91, Freeze-fracture; Freeze-etch; Membranes; Intercellular junctions; Materials science; Nanotechnology; Electron microscopy
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Live Imaging of Drosophila Larval Neuroblasts
Authors: Dorothy A. Lerit, Karen M. Plevock, Nasser M. Rusan.
Institutions: National Institutes of Health.
Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.
Neuroscience, Issue 89, live imaging, Drosophila, neuroblast, stem cell, asymmetric division, centrosome, brain, cell cycle, mitosis
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Using Coculture to Detect Chemically Mediated Interspecies Interactions
Authors: Elizabeth Anne Shank.
Institutions: University of North Carolina at Chapel Hill .
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
Microbiology, Issue 80, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
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An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection
Authors: Marloes Vissers, Marrit N. Habets, Inge M. L. Ahout, Jop Jans, Marien I. de Jonge, Dimitri A. Diavatopoulos, Gerben Ferwerda.
Institutions: Radboud university medical center.
Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.
Immunology, Issue 82, Blood Cells, Respiratory Syncytial Virus, Human, Respiratory Tract Infections, Paramyxoviridae Infections, Models, Immunological, Immunity, HRSV culture, purification, quantification, PBMC isolation, stimulation, inflammatory pathways
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Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface
Authors: Hilaire C. Lam, Augustine M.K. Choi, Stefan W. Ryter.
Institutions: Harvard Medical School, University of Pittsburgh.
Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.
Medicine, Issue 48, Air-Liquid Interface, Cell isolation, Cigarette smoke, Epithelial cells
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Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells
Authors: Susan Fotheringham, Keren Levanon, Ronny Drapkin.
Institutions: Dana-Farber Cancer Institute, Boston, MA, Chaim Sheba Medical Center, Brigham and Women's Hospital.
Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.
Cellular Biology, Issue 51, Primary human epithelial cells, ovarian cancer, serous, ex-vivo, cell biology, fallopian tube, fimbria
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Generation of Organotypic Raft Cultures from Primary Human Keratinocytes
Authors: Daniel Anacker, Cary Moody.
Institutions: University of North Carolina-Chapel Hill, University of North Carolina-Chapel Hill.
The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.
Immunology, Issue 60, Epithelium, organotypic raft culture, virus, keratinocytes, papillomavirus
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Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Authors: Xiaofeng Zheng, Guang Hu.
Institutions: National Institute of Environmental Health Sciences.
Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2). To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8. Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.
Stem Cell Biology, Issue 63, Molecular Biology, Genetics, Embryonic stem cell, ESC, self-renewal, differentiation, Oct4, GFP, reporter assay, RNAi
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Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium
Authors: Karolina Piotrowska-Nitsche, Tamara Caspary.
Institutions: Emory University School of Medicine, IGAB Polish Academy of Sciences.
We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.
Neuroscience, Issue 74, Genetics, Neurobiology, Cellular Biology, Molecular Biology, Developmental Biology, ex vivo live imaging, cell division, imaging neuroepithelium, primary cilia, Shh, time-lapse confocal imaging, microscopy, immunofluorescence, cell culture, mouse, embryo, animal model
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Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle
Authors: Guangliang Wang, H. Joseph Yost, Jeffrey D. Amack.
Institutions: Upstate Medical University, University of Utah .
Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-β superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow. In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs—in combination with optical transparency and rapid development of the zebrafish embryo—make zebrafish KV an excellent model system to study LR ciliated cells. Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time. Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.
Developmental Biology, Issue 73, Genetics, Cellular Biology, Neurobiology, Neuroscience, Molecular Biology, Bioengineering, Biophysics, Anatomy, Physiology, Cilia, Zebrafish, Danio rerio, Gene Knockdown Techniques, Left-right asymmetry, cilia, Kupffer's Vesicle, morpholinos, microinjection, animal model
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Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia
Authors: Richard Francis, Cecilia Lo.
Institutions: University of Pittsburgh.
An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.
Biomedical Engineering, Issue 78, Developmental Biology, Cellular Biology, Molecular Biology, Anatomy, Physiology, Respiratory Mucosa, Trachea, Ciliary Motility Disorders, Animal Experimentation, Microscopy, Fluorescence, Interference, Polarization, Video, Airway, mucociliary clearance, microscopy, animal model
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Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface
Authors: Loretta Müller, Luisa E. Brighton, Johnny L. Carson, William A. Fischer II, Ilona Jaspers.
Institutions: The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill.
In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.
Cellular Biology, Issue 80, Epithelium, Cell culture models, ciliated, air pollution, co-culture models, nasal epithelium
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Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
Authors: Robert M. Hoffman, Lingna Li.
Institutions: AntiCancer, Inc..
There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.
Cellular Biology, Issue 13, Springer Protocols, hair follicles, liposomes, adenovirus, genes, stem cells
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