Ce travail a été soutenu par le financement de la Fondation Overstreet.

<ol>
	<li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457 (2011).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods. Mol. Biol. 750, 61-77 (2011).</li><li>Scheffler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+characterization+of+adult+brain+neuropoiesis.">Phenotypic and functional characterization of adult brain neuropoiesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 9353-9358 (2005).</li><li>Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+immature+neuronal+cells+from+neural+stem+cell+progeny.">Purification of immature neuronal cells from neural stem cell progeny.</a> PLoS One. 6, e20941-e20941 (2011).</li><li>Amariglio, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-Derived+Brain+Tumor+Following+Neural+Stem+Cell+Transplantation+in+an+Ataxia+Telangiectasia+Patient.">Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient.</a> Plos Medicine. 6, 221-231 (2009).</li></ol>

Les auteurs n&#39;ont rien à révéler.

La capacité d&#39;isoler des populations définies de cellules neurales (neurones à savoir, astrocytes et oligodendrocytes) pourrait résoudre certains des obstacles associés à l&#39;application clinique des cellules souches neurales (CSN). Tout d&#39;abord, il nous permet de caractériser complètement la population de départ de cellules du donneur. Deuxièmement, il nous permet d&#39;utiliser un type cellulaire particulier ou une combinaison de différents types de cellules avec un rapport spécifique, en fonctio...

<table border="1"><tr><td> Nom du réactif </td><td> Type </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> NeuroCult NSC moyen basal </td><td> Moyen </td><td> Technologies des cellules souches </td><td> 05700 </td><td></td></tr><tr><td> NeuroCult NSC suppléments prolifération </td><td> Supplément moyen </td><td> Technologies des cellules souches </td><td> 05701 </td><td></td></tr><tr><td> 0,05% de trypsine-EDTA </td><td> Réactif </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> Inhibiteur de trypsine de soja </td><td> Réactif </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Réactif </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> * MEM </td><td> Réactif </td><td> Gibco </td><td> 41500-018 </td><td> Composante HEM </td></tr><tr><td> * Les HEPES </td><td> Réactif </td><td> Sigma </td><td> H4034 </td><td> Composante HEM </td></tr><tr><td> * L&#39;eau distillée </td><td> Réactif </td><td> Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> Tamis cellulaire </td><td> Tamis </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 flacon </td><td> Culture de la céramique </td><td> Nalge Nunc internationale </td><td> 136196 </td><td></td></tr><tr><td> T80 flacon </td><td> Culture de la céramique </td><td> Nalge Nunc internationale </td><td> 178905 </td><td></td></tr><tr><td> Tubes de 15 ml </td><td> Culture de la céramique </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> Tubes de 50 ml </td><td> Culture de la céramique </td><td> BD Falcon </td><td> 352070 </td><td&gt; </td></tr><tr><td> EGF </td><td> Facteur de croissance </td><td> R &amp; D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Facteur de croissance </td><td> R &amp; D </td><td> 3139-FB </td><td></td></tr><tr><td> Héparine </td><td> Facteur de croissance </td><td> Sigma </td><td> H4784 </td><td> Reconstitué dans du PBS </td></tr><tr><td> Anti-PSA-NCAM-PE </td><td> Anticorps </td><td> Miltenyi Biotec </td><td> 130-093-274 </td><td></td></tr><tr><td> IgG1 souris PE-conjugué </td><td> Anticorps de contrôle isotypique </td><td> BD Pharminogen </td><td> 556029 </td><td></td></tr><tr><td> Héparine </td><td> Facteur de croissance </td><td> Sigma </td><td> H4784 </td><td> Reconstitué dans du PBS </td></tr><tr><td> PMOrh-4 </td><td> Facteur de croissance </td><td> R &amp; D </td><td> 314-BP-010 </td><td></td></tr><tr><td> Poly-L-Ornithine </td><td> Réactif </td><td> Sigma </td><td> P4957 </td><td></td></tr><tr><td> Sérum de veau foetal </td><td> Supplément moyenne </td><td> Gibco </td><td> 10099-141 </td><td></td></tr><tr><td> GFAP </td><td> Anticorps </td><td> DAKO </td><td> Z0334 </td><td></td></tr><tr><td> B-III tubuline </td><td> Anticorps </td><td> Promega </td><td> G7121 </td><td></td></tr></table> * Pour faire HEM, mélange 1 sachet x L 10 du MEM et 160 ml de HEPES 1M et porter le volume à 8,75 L avec de l&#39;eau distillée. Réglez le pH final à 7,4 et le conserver à 4 ° C.

the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry

Les cellules souches neurales (CSN) peuvent être isolés et élargi à grande échelle, en utilisant le test neurosphères et différenciés dans les trois principaux types de cellules du système nerveux central (SNC); à savoir, les astrocytes, oligodendrocytes et des neurones. Ces caractéristiques font de souches neurales et les cellules progénitrices une source inestimable renouvelable de cellules pour des études in vitro, tels que le dépistage des drogues, neurotoxicologie et l&#39;électrophysiologie et aussi pour la thérapie de remplacement cellulaire dans de nombreuses maladies neurologiques. En pratique, cependant, l&#39;hétérogénéité de la descendance NSC, la faible production de neurones et des oligodendrocytes, les astrocytes et la prédominance de la suite de différenciation limiter leurs applications cliniques. Ici, nous décrivons une nouvelle méthodologie pour la purification de production et subséquente des neurones immatures de NSC descendance murin utilisant la fluorescence tri cellulaire (FACS) de la technologie. En utilisant cette méthodologie, une population hautement enrichi de cellules souches neuronales peuvent être atteints d&#39;espritHout toute astrocytes notable et de bonne foi la contamination NSC. La procédure comprend la différenciation des produits de filiation du NSC isolé et agrandi à partir de E14 éminences ganglionnaires de souris en utilisant le test neurosphères, suivie par l&#39;isolement et l&#39;enrichissement de cellules immatures neuronaux en fonction de leurs propriétés physiques (taille et la complexité interne) et fluorescente utilisant la technologie de cytométrie en flux. Dans l&#39;ensemble, cela prend 5-7 jours pour générer neurosphères et 6-8 jours pour différencier descendance NSC et d&#39;isoler hautement purifié des cellules neuronales immatures.

Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Ce protocole vidéo démontre une nouvelle méthode pour la génération et la purification ultérieure des cellules progénitrices neuronales à partir d&#39;une source renouvelable de cellules souches neurales (CSN) en fonction de leur physique (taille et granularité interne) et les propriétés fluorescentes en utilisant la technologie de cytométrie en flux.

Le test neuroblaste: Un test pour la génération et l&#39;enrichissement des cellules souches neuronales à partir de différenciation des cellules souches neurales Progeny Par cytométrie en flux

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-neuroblast-assay-an-assay-for-generation-enrichment-neuronal

Research

JoVE Journal

Neuroscience

15.4K vues.  The University of Florida. Ce protocole vidéo démontre une nouvelle méthode pour la génération et la purification ultérieure des cellules progénitrices neuronales à partir d'une source renouvelable de cellules souches neurales (CSN) en fonction de leur physique (taille et granularité interne) et les propriétés fluorescentes en utilisant la technologie de cytométrie en flux.

Vidéo: The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

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.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

L&#39;isolement et expansion des cellules souches neurales de souris adultes utilisant le test Neurosphère

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 ...

Recherche

Neurosciences

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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&#8208;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.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Établir embryonnaires de la souris culture de cellules souches neurales utilisant le test Neurosphère

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 ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

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 &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 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 &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-Formant Colonie dosage cellulaire: Un test pour distinguer Bona Fide Cellules souches neuronales à partir de cellules progénitrices neurales

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). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Imagerie des cellules vivantes des cellules sensorielles de précurseurs d&#39;organes en Intact Drosophile Les chrysalides

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

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.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Dérivation sans chargeur-de cellules progénitrices de la crête neurale souches pluripotentes humaines cellules

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Cytométrie en flux pour les protocoles surface et intracellulaire Antigen analyses des types cellulaires de neurones

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Tri cellulaire des neurones cellules souches et progénitrices de la souris adulte-ventriculaire Zone et Live-imagerie de la dynamique de leur cycle cellulaire

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Énumération des cellules souches neurales utilisant clonales Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identification des marqueurs de surface cellulaire des cellules neurales primaires de tige et de progéniture par l'étiquetage métabolique du sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...