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p38 MAPK-Mediated Bmi-1 down-regulation and defective proliferation in ATM-deficient neural stem cells can be restored by Akt activation.
PUBLISHED: 01-06-2011
A-T (ataxia telangiectasia) is a genetic disease caused by a mutation in the Atm (A-T mutated) gene that leads to neurodegeneration. Despite an increase in the numbers of studies in this area in recent years, the mechanisms underlying neurodegeneration in human A-T are still poorly understood. Previous studies demonstrated that neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm(-/-) mouse brains show defective self-renewal and proliferation, which is accompanied by activation of chronic p38 mitogen-activated protein kinase (MAPK) and a lower level of the polycomb protein Bmi-1. However, the mechanism underlying Bmi-1 down-regulation and its relevance to defective proliferation in Atm(-/-) NSCs remained unclear. Here, we show that over-expression of Bmi-1 increases self-renewal and proliferation of Atm(-/-) NSCs to normal, indicating that defective proliferation in Atm(-/-) NSCs is a consequence of down-regulation of Bmi-1. We also demonstrate that epidermal growth factor (EGF)-induced Akt phosphorylation renders Bmi-1 resistant to the proteasomal degradation, leading to its stabilization and accumulation in the nucleus. However, inhibition of the Akt-dependent Bmi-1 stabilizing process by p38 MAPK signaling reduces the levels of Bmi-1. Treatment of the Atm(-/-) NSCs with a specific p38 MAPK inhibitor SB203580 extended Bmi-1 posttranscriptional turnover and H2A ubiquitination in Atm(-/-) NSCs. Our observations demonstrate the molecular basis underlying the impairment of self-renewal and proliferation in Atm(-/-) NSCs through the p38 MAPK-Akt-Bmi-1-p21 signaling pathway.
Authors: Steven W. Poser, Andreas Androutsellis-Theotokis.
Published: 11-18-2013
Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as "niches" that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.
21 Related JoVE Articles!
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Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice
Authors: Rasika Jinadasa, Gabriel Balmus, Lee Gerwitz, Jamie Roden, Robert Weiss, Gerald Duhamel.
Institutions: Cornell University.
Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.
Immunology, Issue 50, mouse, thymic lymphoma, Atm, p53, T-cell lines
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Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Authors: Hilary C. Rees, Voichita Ianas, Patricia McCracken, Shannon Smith, Anca Georgescu, Tirdad Zangeneh, Jane Mohler, Stephen A. Klotz.
Institutions: University of Arizona, University of Arizona.
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age. HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal. The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
Medicine, Issue 77, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
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Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay
Authors: Hassan Azari, Maryam Rahman, Sharareh Sharififar, Brent A. Reynolds.
Institutions: Shiraz University of Medical Sciences, Shiraz, Iran , University of Florida.
Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes. This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.
Neuroscience, Issue 45, Adult Neural Stem Cells, Neurosphere Assay, Isolation, Expansion
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Neonatal Subventricular Zone Electroporation
Authors: David M. Feliciano, Carlos A. Lafourcade, Angélique Bordey.
Institutions: Yale University School of Medicine .
Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates. Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.
Neuroscience, Issue 72, Developmental Biology, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Anatomy, Biomedical Engineering, Stem Cell Biology, Genetics, Neurogenesis, Growth and Development, Surgery, Subventricular Zone, Electroporation, Neural Stem Cells, NSC, subventricular zone, brain, DNA, injection, genetic engineering, neonatal pups, animal model
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Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue
Authors: Jie Lu, Laurent C. Delli-Bovi, Jonathan Hecht, Rebecca Folkerth, Volney L. Sheen.
Institutions: Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Beth Israel Deaconess Medical Center, Brigham and Women's Hospital.
Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.
Neuroscience, Issue 51, neural stem cell, human, brain, cortex, fetus, cell culture
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Strategies for Study of Neuroprotection from Cold-preconditioning
Authors: Heidi M. Mitchell, David M. White, Richard P. Kraig.
Institutions: The University of Chicago Medical Center.
Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident. Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
Neuroscience, Issue 43, innate immunity, hormesis, microglia, hippocampus, slice culture, immunohistochemistry, neural-immune, gene expression, real-time PCR
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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Authors: Kevin Wilhelmsen, Katherine Farrar, Judith Hellman.
Institutions: University of California, San Francisco, University of California, San Francisco.
The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.
Immunology, Issue 78, Cellular Biology, Infection, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Endothelium, Vascular, Neutrophils, Inflammation, Inflammation Mediators, Neutrophil, Leukocyte Adhesion, Endothelial cells, assay
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Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
Authors: Alexandra Amaro-Ortiz, Jillian C. Vanover, Timothy L. Scott, John A. D'Orazio.
Institutions: University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine.
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Medicine, Issue 79, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
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Simultaneous Scalp Electroencephalography (EEG), Electromyography (EMG), and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding
Authors: Thomas C. Bulea, Atilla Kilicarslan, Recep Ozdemir, William H. Paloski, Jose L. Contreras-Vidal.
Institutions: National Institutes of Health, University of Houston, University of Houston, University of Houston, University of Houston.
Recent studies support the involvement of supraspinal networks in control of bipedal human walking. Part of this evidence encompasses studies, including our previous work, demonstrating that gait kinematics and limb coordination during treadmill walking can be inferred from the scalp electroencephalogram (EEG) with reasonably high decoding accuracies. These results provide impetus for development of non-invasive brain-machine-interface (BMI) systems for use in restoration and/or augmentation of gait- a primary goal of rehabilitation research. To date, studies examining EEG decoding of activity during gait have been limited to treadmill walking in a controlled environment. However, to be practically viable a BMI system must be applicable for use in everyday locomotor tasks such as over ground walking and turning. Here, we present a novel protocol for non-invasive collection of brain activity (EEG), muscle activity (electromyography (EMG)), and whole-body kinematic data (head, torso, and limb trajectories) during both treadmill and over ground walking tasks. By collecting these data in the uncontrolled environment insight can be gained regarding the feasibility of decoding unconstrained gait and surface EMG from scalp EEG.
Behavior, Issue 77, Neuroscience, Neurobiology, Medicine, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Electroencephalography, EEG, Electromyography, EMG, electroencephalograph, gait, brain-computer interface, brain machine interface, neural decoding, over-ground walking, robotic gait, brain, imaging, clinical techniques
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Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay
Authors: Hassan Azari, Sharareh Sharififar, Maryam Rahman, Saeed Ansari, Brent A. Reynolds.
Institutions: Shiraz University of Medical Sciences, Shiraz, Iran , The University of Florida.
In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum‐free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.
Neuroscience, Issue 47, Embryonic Neural Stem Cells, Neurosphere Assay, Isolation, Expansion
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Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells
Authors: Hassan Azari, Sharon A. Louis, Sharareh Sharififar, Vinata Vedam-Mai, Brent A. Reynolds.
Institutions: University of Florida, Shiraz University of Medical Sciences, Inc..
The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies < 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are < 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated.
Neuroscience, Issue 49, Stem Cells, Neural Colony Forming Cell Assay, Progenitor Cells, enumeration
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Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury
Authors: Paul Lu, Lori Graham, Yaozhi Wang, Di Wu, Mark Tuszynski.
Institutions: Veterans Administration Medical Center, San Diego, University of California, San Diego.
Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.
Neuroscience, Issue 89, nervous system diseases, wounds and injuries, biological factors, therapeutics, surgical procedures, neural stem cells, transplantation, spinal cord injury, fibrin, growth factors
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The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry
Authors: Hassan Azari, Sharareh Sharififar, Jeff M. Fortin, Brent A. Reynolds.
Institutions: The University of Florida, Shiraz University of Medical Sciences, Shiraz, Iran .
Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.
Neuroscience, Issue 62, neural Stem Cell, Neuronal Progenitor Cells, Flow Cytometry, Isolation, Enrichment
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
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Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Authors: Valentina Gandin, Kristina Sikström, Tommy Alain, Masahiro Morita, Shannon McLaughlan, Ola Larsson, Ivan Topisirovic.
Institutions: McGill University, Karolinska Institutet, McGill University.
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
Biochemistry, Issue 87, Cells, Eukaryota, Nutritional and Metabolic Diseases, Neoplasms, Metabolic Phenomena, Cell Physiological Phenomena, mRNA translation, ribosomes, protein synthesis, genome-wide analysis, translatome, mTOR, eIF4E, 4E-BP1
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Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans
Authors: Rivkeh Y. Haryono, Madeline A. Sprajcer, Russell S. J. Keast.
Institutions: Deakin University.
Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.
Neuroscience, Issue 88, taste, overweight and obesity, dietary fat, fatty acid, diet, fatty food liking, detection threshold
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Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Authors: Jacob Michael Froehlich, Iban Seiliez, Jean-Charles Gabillard, Peggy R. Biga.
Institutions: University of Alabama at Birmingham, INRA UR1067, INRA UR1037.
Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.
Basic Protocol, Issue 86, myogenesis, zebrafish, myoblast, cell culture, giant danio, moustached danio, myotubes, proliferation, differentiation, Danioninae, axolotl
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
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Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Authors: Daniel A. Lee, Juan Salvatierra, Esteban Velarde, John Wong, Eric C. Ford, Seth Blackshaw.
Institutions: California Institute of Technology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, University Of Washington Medical Center, Johns Hopkins University School of Medicine.
The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.
Neuroscience, Issue 81, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
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Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Authors: Chitra Venugopal, Nicole M. McFarlane, Sara Nolte, Branavan Manoranjan, Sheila K. Singh.
Institutions: McMaster University .
Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres. To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR. The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.
Cancer Biology, Issue 67, Stem Cell Biology, Medicine, Cellular Biology, Molecular Biology, BTIC (brain tumor initiating cells), tumorspheres, self-renewal, flow cytometry, single cell RT-PCR
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Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells
Authors: Nadja Zeltner, Fabien G. Lafaille, Faranak Fattahi, Lorenz Studer.
Institutions: Sloan-Kettering Institute for Cancer Research, The Rockefeller University.
Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.
Neuroscience, Issue 87, Embryonic Stem Cells (ESCs), Pluripotent Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Neural Crest, Peripheral Nervous System (PNS), pluripotent stem cells, neural crest cells, in vitro differentiation, disease modeling, differentiation protocol, human embryonic stem cells, human pluripotent stem cells
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