Ce travail a été soutenu financièrement par la Fondation Overstreet.

1. Configuration de base Avant de procéder à la dissection: <ol><li> Volume approprié de milieu complet NSC est préparée en mélangeant NeuroCult NSC moyen basale et NeuroCult NSC Suppléments prolifération à un ratio 9:1, respectivement. </li><li> Le milieu est chauffé dans un bain d&#39;eau à 37 ° C. </li><li> Froid tamponné HEPES milieu essentiel minimum (HEM) à forte concentration d&#39;antibiotiques (10%) est préparé pour la dissection et l&#39;objet à laver. Alternativement, le NSC milieu basal avec une supplémentation en antibiotiques peuvent également être utilisés à cette fin. </li><li> 25-30 ml de SEM le rhume contenant des antibiotiques est distribué dans des tubes stériles 50 ml pour la collecte des embryons. </li><li> Plusieurs 10cm en plastique des boîtes de Petri sont nécessaires pour maintenir les embryons et le cerveau lors de la dissection et aussi pour tenir les tissus disséqués. </li><li> Les outils chirurgicaux, nécessaire pour éliminer les embryons (gros ciseaux, petits ciseaux pointus, pinces grandes, petites pinces courbées) ou pour la dissection du cerveau embryonnaire (petite pince, pinces fines courbes, coudée à 45 ° pinces fines, et de petits ciseaux) sont stérilisés à l&#39;aide billes de verre stérilisateur à 250 ° C ou d&#39;autres méthodes disponibles autoclave. </li><li> Microscope à dissection est lavée à l&#39;alcool 70% et mis en place dans une hotte à flux laminaire ou PC2. </li></ol> 2. Récolte E14 cerveau de souris et de micro-dissection: <ol><li> Un temps accouplés souris enceinte est anesthésié au 14 ème jour de gestation en fonction de son protocole d&#39;animaux institutionnelle approuvée. </li><li> La dislocation cervicale est effectuée pour s&#39;assurer que l&#39;animal ne souffre pas la douleur et la détresse. </li><li> La souris anesthésiée est posé sur le dos sur un document papier absorbant, et l&#39;abdomen est rincé à l&#39;éthanol à 70% pour stériliser la région. </li><li> La peau de l&#39;abdomen est saisie en utilisant une grande pince, puis la peau et le fascia sous-jacent est coupé avec de grands ciseaux pour exposer la cavité abdominale et les cornes utérines. </li><li> Les utérus sont retirés avec une pince et des ciseaux et de petits sont transférés dans un tube contenant 50 ml conique HEM froid. </li><li> Les tissus utérins sont transférés à la hotte, puis rincée une fois ou deux fois avec un volume suffisant de produits frais HEM froide stérile pour éliminer les contaminants possibles comme le sang et les cheveux. </li><li> Les tissus utérins sont ensuite transférés dans une boîte de Petri contenant 10cm HEM froid. </li><li> Les cornes utérines sont ouverts à l&#39;aide de petites pinces et ciseaux courbes et les embryons sont transférés dans un plat de 10cm de nouvelles, qui contient HEM froid. </li><li> Les chefs des embryons sont séparés au niveau de la moelle épinière cervicale et transféré à une autre boîte de Pétri contenant HEM froid. </li><li> La boîte de Pétri est transféré sous un microscope à dissection pour enlever le cerveau du crâne. </li><li> Les têtes sont organisées avec une pince fine incurvée du côté caudal afin que la face dorsale des têtes sont vers le haut. En utilisant des micro-ciseaux, d&#39;abord une coupe horizontale est faite au-dessus des yeux et a ensuite continué dans la ligne médiane du front vers l&#39;arrière de la tête. Assurez-vous de couper à travers la peau et le crâne et de ne pas endommager le cerveau sous-jacent. </li><li> Le cerveau est enlevé du crâne en poussant les bords de la section coupée dans un mouvement vers l&#39;arrière à l&#39;aide de la pince courbe. Cette procédure est poursuivie jusqu&#39;à ce que tous les cerveaux sont retirés de ces crânes. </li><li> Le cerveau est resté stable en utilisant la pince incurvée de sorte que la face dorsale est tournée vers le haut, puis en utilisant microciseaux une coupe est réalisée à travers le cortex de chaque hémisphère s&#39;étend du bulbe olfactif à l&#39;arrière de l&#39;hémisphère pour exposer les éminences ganglionnaires. </li><li> Utilisation de la pince courbée dans une main et la pince coudée à 45 ° dans l&#39;autre, les volets coupe de cortex sont répartis et les éminences ganglionnaires sont disséquées. </li><li> Le tissu disséqué est placé dans une boîte de Pétri stériles 10cm, et cette procédure est répétée jusqu&#39;à ce que tous les cerveaux ont été micro-disséqués. </li><li> Morceaux de tissus sont recueillies à l&#39;aide de 1 mL de milieu NSC dans un bout de 1 mL pipette et transférée dans un tube à centrifuger de 15 ml. </li><li> Tissu est ensuite dissocié soigneusement, mais doucement en appuyant sur la pointe de la pipette au fond du tube et la suspension de pipetage de haut en bas. De cette façon, les mottes sont divisés en cellules individuelles. Le pipetage est réalisé de telle sorte que 3-4 fois une suspension laiteuse comme est atteint. Ensuite, la suspension est autorisé à s&#39;installer pendant 1-2 minutes afin que les touffes non dissociée précipités. </li><li> Près de la suspension cellulaire entière est transférée à l&#39;autre tube, puis un autre 1 mL de milieu NSC est ajouté à la mottes restantes et dissocié de cellule unique, comme décrit. </li><li> Le contenu des tubes sont regroupées et centrifugé pendant 5 minutes à température ambiante, à 700rpm (110g). </li><li> Le surnageant est aspiré vers le hors réelles de granulés, et les cellules sont remises en suspension dans 1 ml de milieu complet NSC. </li><li> Le milieu est doucement introduit à la pipette haut et bas pouravoir une suspension cellulaire homogène et unique. </li><li> 10 ul de la suspension cellulaire est mélangé avec 90 ul de bleu trypan pour effectuer un comptage des cellules. </li><li> Enfin, les cellules sont étalées à la densité de 2x10 5 cellules / mL dans NSC milieu complet supplémenté avec 20ng / ml de facteur de croissance épidermique (EGF). 5 ml de milieu est utilisé pour T25, T75 pour les 20 ml et 40 ml pour T175 flacons. </li><li> Neurosphères sont formés dans les 5-7 jours lors d&#39;une incubation à 37 ° C dans un incubateur humidifié avec 5% de CO 2. Dans 6-7 jours, les sphères doit mesurer entre 150-200 microns et sera prêt à la sous-culture (de passage). </li></ol> 3. Le repiquage et l&#39;expansion du CNS embryonnaires: <ol><li> Lorsque les neurosphères sont prêts pour la sous-culture (150-200 um de diamètre), le milieu avec des sphères suspendues est retiré de la flacons, placé dans une taille de stériles tube approprié la culture de tissus, et centrifugé à 700 rpm (110 g) pendant 5 min à la température ambiante. </li><li> Le surnageant est éliminé et les sphères sont remis en suspension dans 1 ml de 0,05% de trypsine-EDTA. </li><li> La suspension cellulaire est ensuite incubée dans un bain d&#39;eau à 37 ° C pendant 2-3 min, puis un volume égal d&#39;inhibiteur de trypsine du soja est utilisé pour arrêter l&#39;activité de la trypsine. </li><li> La suspension cellulaire est doucement introduit à la pipette de haut en bas pour s&#39;assurer que la trypsine a été complètement inactivé. </li><li> La suspension cellulaire est centrifugée à 700 rpm (110g) pendant 5 min. Ensuite, le surnageant est éliminé et les cellules sont resuspendues dans 1 ml de milieu complet NSC. </li><li> 10 ul de la suspension cellulaire est mélangé avec 90 ul de bleu trypan pour effectuer un comptage des cellules. </li><li> Les cellules sont étalées à une concentration de 5x10 4 cellules / mL dans le NSC milieu complet supplémenté avec 20ng / ml EGF dans une fiole de culture de la taille des tissus appropriés. 5 ml de milieu est utilisé pour T25, T75 pour les 20 ml et 40 ml pour T175 flacons. </li><li> Neurosphères secondaires sont formées dans les 5-7 jours lors d&#39;une incubation à 37 ° C dans un incubateur humidifié avec 5% de CO 2. </li></ol> 4. Les résultats représentatifs: En premier embryon NSC culture, la majorité des cellules deviennent hypertrophiques et attacher à la culture de tissus plats sur placage. Alors que la majorité des cellules meurent ou se différencier, après 2-3 jours, les cellules prolifératives font de petits amas de cellules qui se détachent du substrat (figure 1). Formation de grands agrégats sphéroïdaux dans les 48 premières heures de la culture ne doit pas être confondu avec les sphères primaires. La formation d&#39;agrégats dépend principalement de la quantité de débris et de mottes de tissu non dissociée de la culture. Neurosphères sont vrais phase brillante et devenir plus sphérique que la taille augmente (figure 2). Microspikes Petite apparaît sur la surface extérieure des sphères viable et saine (figure 3). Après 5-7 jours, les sphères doivent être rondes mais pas compacté et doit mesurer entre 150 et 200 um de diamètre. Si neurosphères sont autorisés à devenir trop volumineux (après 9-10 jours de culture), ils pourraient former des agrégats ou à devenir de couleur sombre à cause de la mort cellulaire au centre de la sphère (voir vidéo). Grand neurosphères pourrait éventuellement commencer à se différencier in situ (fixation sur le substrat et la migration vers la périphérie). Il est également difficile de dissocier neurosphères grandes et les sous-culture. <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="Figure 1" /> Figure 1. Primaire E14 NSC culture 3 jours après l&#39;étalement. Les flèches indiquent les clusters prolifération des CNS. Le grossissement est de; 20x <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="Figure 2" /> Figure 2. Primaire E14 NSC culture de 7 jours après l&#39;étalement. Le grossissement est de; 10x <img src="/files/ftp_upload/2457/2457fig3.jpg" alt="Figure 3" /> Figure 3. Seul passage E14 neurosphères 5 jours après placage. Notez le micro-pointes (flèches) à la périphérie des sphères. Grossissement original; 20x

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. d. s. <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. , (2010).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li></ol>

Le test est Neurosphère la méthode de choix pour l&#39;isolement et l&#39;expansion des cellules souches neurales 1-5 raison de sa simplicité et de reproductibilité. Ce test est un outil précieux pour production à grande échelle des cellules précurseurs du SNC indifférencié, qui pourrait être utilisé à la fois in vitro et in vivo. Il convient de souligner que neurosphères pourrait être généré à la fois du bona fide des cellules souches neurales et les progéniteurs plus re...

* Pour faire HEM, mélanger 110L paquet de MEM and160ml d&#39;HEPES 1M et porter le volume à 8,75 L à l&#39;aide d&#39;eau distillée. Réglez le pH final à 7,4 et le stocker à 4 ° C.

establishing embryonic mouse neural stem cell culture using the neurosphere assay

Dans les mammifères, les cellules souches agit comme une source de cellules indifférenciées de maintenir la genèse des cellules et de renouvellement dans les différents tissus et organes pendant la durée de vie de l&#39;animal. Ils peuvent éventuellement remplacer les cellules qui sont perdues dans le processus de vieillissement ou dans le processus de blessure et de maladie. L&#39;existence de cellules souches neurales (NSC) a d&#39;abord été décrit par Reynolds et Weiss (1992) dans le système nerveux des mammifères adultes central (SNC) en utilisant un système de culture sans sérum roman, l&#39;essai de Neurosphère (NSA). En utilisant ce dosage, il est également possible d&#39;isoler et d&#39;élargir NSCs de différentes régions du SNC embryonnaire. Ces NSCs dans élargi in vitro sont multipotentes et peuvent donner lieu à trois principaux types de cellules du SNC. Alors que la NSA semble relativement simple à réaliser, attention aux procédures démontré ici est nécessaire afin d&#39;obtenir des résultats fiables et cohérents. Cette vidéo montre pratiquement la NSA pour générer et développer NSCs du jour embryonnaire du tissu cérébral de 14 souris et fournit les détails techniques, on peut obtenir pour les cultures Neurosphère reproductibles. La procédure comprend la récolte d&#39;embryons de souris E14, microdissection cerveau pour récolter les éminences ganglionnaires, la dissociation des tissus récoltés dans le milieu de NSC pour gagner une suspension cellulaire unique, et enfin plaquage cellules en culture NSA. Après 5-7 jours de culture, les neurosphères résultant primaires sont passées à élargir le nombre des CNS pour des expériences futures.

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Ce protocole vidéo démontre l&#39;application du test Neurosphère pour l&#39;isolement et l&#39;expansion des cellules souches neurales de la éminences ganglionnaires du jour embryonnaire 14 cerveau de souris.

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 ...

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Neuroscience

24.5K vues.  Shiraz University of Medical Sciences, Shiraz, Iran. Ce protocole vidéo démontre l'application du test Neurosphère pour l'isolement et l'expansion des cellules souches neurales de la éminences ganglionnaires du jour embryonnaire 14 cerveau de souris.

Vidéo: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Neurosciences

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 ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

Localisation des protéines en 3D de la culture de cellules souches neuronales: une méthodologie de visualisation hybride

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

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). ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Transplantation chirurgicale de cellules souches neurales de souris dans la moelle épinière des souris infectées avec le virus de l&#39;hépatite murine neurotrope

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

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

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.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

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 ...

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 ...

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. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolement et culture des adultes cellules souches neurales de la Subcallosal Zone Souris

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Élaboration d’un protocole raffiné pour Trans-sclérale Transplantation sous-rétinienne des cellules épithéliales humaines pigmentaire dans les yeux de Rat