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Mimicking the neurotrophic factor profile of embryonic spinal cord controls the differentiation potential of spinal progenitors into neuronal cells.
PUBLISHED: 03-09-2011
Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively.
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.
19 Related JoVE Articles!
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Chicken Embryo Spinal Cord Slice Culture Protocol
Authors: Kristina C. Tubby, Dee Norval, Stephen R. Price.
Institutions: University College London.
Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.
Developmental Biology, Issue 73, Neurobiology, Neuroscience, Medicine, Cellular Biology, Molecular Biology, Anatomy, Physiology, Biomedical Engineering, Genetics, Surgery, Cells, Animal Structures, Embryonic Structures, Nervous System, spinal cord, embryo, development, Slice-Culture, motor neuron, neurons, immunostaining, chick, imaging, animal model
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Derivation of Glial Restricted Precursors from E13 mice
Authors: André W. Phillips, Sina Falahati, Roshi DeSilva, Irina Shats, Joel Marx, Edwin Arauz, Douglas A. Kerr, Jeffrey D. Rothstein, Michael V. Johnston, Ali Fatemi.
Institutions: Johns Hopkins University, Johns Hopkins School of Medicine, University of Maryland , Biogen Idec, Johns Hopkins School of Medicine, Johns Hopkins School of Medicine.
This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.
Neuroscience, Issue 64, Physiology, Medicine, periventricular leukomalacia, oligodendrocytes, glial restricted precursors, spinal cord, cell culture
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An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
Authors: Starlyn L. M. Okada, Nicole S. Stivers, Peter K. Stys, David P. Stirling.
Institutions: University of Louisville, University of Calgary.
Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.
Neuroscience, Issue 93, spinal cord injury, axon, myelin, two-photon excitation microscopy, Nile Red, axonal degeneration, axonal dieback, axonal retraction
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Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals
Authors: Shankar Ramachandran, Simon Alford.
Institutions: University of Illinois at Chicago.
Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+ into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+ currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+ and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+ entry has been characterized using imaging techniques and macroscopic current measurements – techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.
Neuroscience, Issue 92, reticulospinal synapse, reticulospinal axons, presynaptic terminal, presynaptic calcium, voltage-gated calcium channels, vesicle fusion, synaptic transmission, neurotransmitter release, spinal cord, lamprey, synaptic vesicles, acute dissociation
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Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury
Authors: Teresa A. Evans, Deborah S. Barkauskas, Jay T. Myers, Alex Y. Huang.
Institutions: Case Western Reserve University, Case Western Reserve University, Case Western Reserve University.
Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.
Cellular Biology, Issue 93, Intravital, spinal cord crush injury, chimera, microglia, macrophages, dorsal column crush, axonal dieback
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Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors
Authors: Peng Jiang, Vimal Selvaraj, Wenbin Deng.
Institutions: School of Medicine, University of California, Davis.
Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (>80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.
Neurobiology, Issue 39, pluripotent stem cell, oligodendrocyte precursor cells, differentiation, myelin, neuroscience, brain
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Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons
Authors: Chia-Yen Wu, Dosh Whye, Robert W. Mason, Wenlan Wang.
Institutions: Alfred I. duPont Hospital for Children.
Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51±0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.
Stem Cell Biology, Issue 64, Molecular Biology, Developmental Biology, Mouse embryonic stem cells, motor neurons, spinal cord, Hb9, neurosciences, retinoic acid, sonic hedgehog, Islet-1, choline acetyltransferase
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Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons
Authors: Rebecca Conrad, Sibylle Jablonka, Teresa Sczepan, Michael Sendtner, Stefan Wiese, Alice Klausmeyer.
Institutions: Ruhr-University Bochum, University of Wuerzburg.
Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.
Neuroscience, Issue 55, p75NTR, spinal cord, lectin, axon, dendrite
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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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Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
Authors: Ryan W. O'Meara, Scott D. Ryan, Holly Colognato, Rashmi Kothary.
Institutions: Ottawa Hospital Research Institute, University of Ottawa , Stony Brook University, University of Ottawa .
Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy & de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.
Neuroscience, Issue 54, Oligodendrocyte, myelination, in vitro, dorsal root ganglion neuron, co-culture, primary cells, mouse, neuroscience
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Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture
Authors: Ron Bouchard, Thomas Chong, Subbiah Pugazhenthi.
Institutions: Denver VA Medical Center, University of Colorado Denver School of Medicine.
Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.
Neuroscience, Issue 79, Neurobiology, Cellular Biology, Cells, Cultured, Neurons, Central Nervous System, Neurodegenerative Diseases, Human neuroprogenitor cells, neuronal differentiation, neuronal markers, astrocytes, laser capture microdissection, PEN membrane slides, cell culture
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Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain
Authors: Maria Chiara G. Monaco, Dragan Maric, Alexandra Bandeian, Emily Leibovitch, Wan Yang, Eugene O. Major.
Institutions: National Institute of Neurological Disorders and Stroke, National Institutes of Health, National Institute of Neurological Disorders and Stroke, National Institutes of Health.
Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation. Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.
Neuroscience, Issue 70, Developmental Biology, Medicine, Stem Cell Biology, Molecular Biology, Cellular Biology, Physiology, lineage characterization, neural progenitors, differentiation, cell culture model
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Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays
Authors: Kristen N. Fantetti, Donna M. Fekete.
Institutions: Purdue University.
The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6. Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.
Neuroscience, Issue 58, chicken, dissection, morphogen, NT-3, neurite outgrowth, spinal cord, statoacoustic ganglion, Wnt5a
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The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells
Authors: Erin M. Boisvert, Kyle Denton, Ling Lei, Xue-Jun Li.
Institutions: The University of Connecticut Health Center, The University of Connecticut Health Center, The University of Connecticut Health Center.
Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.
Stem Cell Biology, Issue 74, Neuroscience, Neurobiology, Developmental Biology, Cellular Biology, Molecular Biology, Stem Cells, Embryonic Stem Cells, ESCs, Pluripotent Stem Cells, Induced Pluripotent Stem Cells, iPSC, neural differentiation, forebrain, glutamatergic neuron, neural patterning, development, neurons
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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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A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
Authors: Brandon C. Shelley, Geneviève Gowing, Clive N. Svendsen.
Institutions: Cedars-Sinai Medical Center.
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
Neuroscience, Issue 88, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
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Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Authors: Matteo Donegà, Elena Giusto, Chiara Cossetti, Julia Schaeffer, Stefano Pluchino.
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases. These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS). This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo. Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
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Propagation of Human Embryonic Stem (ES) Cells
Authors: Laurence Daheron.
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 1, ES, embryonic stem cells, tissue culture
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Ole Isacson: Development of New Therapies for Parkinson's Disease
Authors: Ole Isacson.
Institutions: Harvard Medical School.
Medicine, Issue 3, Parkinson' disease, Neuroscience, dopamine, neuron, L-DOPA, stem cell, transplantation
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