Name: isolation and culture of embryonic mouse neural stem cells
Uploaded: 2018-11-11T14:30:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Embryonic Mouse Neural Stem Cells at JoVE.com

Ce travail a été soutenu par l’Institut de Royan.

Toutes les chirurgies et les procédures sur les animaux ont été approuvées par le Comité de l’éthique animale de l’Institut de Royan, Téhéran, Iran.
1. préparation des Instruments chirurgicaux, lavage tampon (HEPES-MEM), milieux de Culture cellulaire et plaques de Culture cellulaire
<ol><li>Stériliser les instruments chirurgicaux (ciseaux, poignée de lame de bistouri Swann-Morton et forceps) par autoclavage en fonction sur les lignes directrices de stérilisation systématique...

<ol>
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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thrombin+Receptor+Restricts+Subventricular+Zone+Neural+Stem+Cell+Expansion+and+Differentiation.">The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation.</a> Scientific Reports. 8 (1), 9360 (2018).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+novel+rat+neural+progenitor+clone+that+expresses+Dlx+family+transcription+factors+and+gives+rise+to+functional+GABAergic+neurons+in+culture.">Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture.</a> Developmental Neurobiology. 72 (6), 805-820 (2012).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+phenotypically+diverse+neural+culture+from+hESC+by+combining+adherent+and+dissociation+methods.">Derivation of phenotypically diverse neural culture from hESC by combining adherent and dissociation methods.</a> Journal of Neuroscience Methods. , (2018).</li><li>Dhara, S. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Chromosomal+Analysis+of+Cultured+Mouse+Neural+Stem+Cell+Lines.">Systematic Chromosomal Analysis of Cultured Mouse Neural Stem Cell Lines.</a> Stem Cells and Development. 20 (8), 1411-1423 (2011).</li><li>Hemmer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Neural+Stem+Cells+Achieve+Long-Term+Survival+and+Functional+Integration+in+the+Adult+Mouse+Brain.">Induced Neural Stem Cells Achieve Long-Term Survival and Functional Integration in the Adult Mouse Brain.</a> Stem Cell Reports. 3 (3), 423-431 (2014).</li><li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry+Protocols+for+Surface+and+Intracellular+Antigen+Analyses+of+Neural+Cell+Types.">Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types.</a> Journal of Visualized Experiments. (94), 52241 (2014).</li><li>Zhang, Z., Wang, Z., Rui, W., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+lithospermate+B+promotes+proliferation+and+differentiation+of+neural+stem+cells+in+vitro+and+enhances+neurogenesis+in+vivo.">Magnesium lithospermate B promotes proliferation and differentiation of neural stem cells in vitro and enhances neurogenesis in vivo.</a> Tissue and Cell. 53, 8-14 (2018).</li><li>Zilkha-Falb, R., Gurevich, M., Hanael, E., Achiron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prickle1+as+positive+regulator+of+oligodendrocyte+differentiation.">Prickle1 as positive regulator of oligodendrocyte differentiation.</a> Neuroscience. 364, 107-121 (2017).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+differentiation+from+human+neural+stem+cells:+A+novel+role+for+c-Src.">Oligodendrocyte differentiation from human neural stem cells: A novel role for c-Src.</a> Neurochemistry International. 120, 21-32 (2018).</li><li>Palanisamy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+alters+cell+fate+selection+of+rat+neural+progenitor+cells+in+vitro.">Oxytocin alters cell fate selection of rat neural progenitor cells in vitro.</a> PLoS ONE. 13 (1), e0191160 (2018).</li><li>Llewellyn-Smith, I. 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À l’aide d’une source de cellules souches neuronales est très important pour les chercheurs en neurosciences. Des cellules souches neurales pourraient être récoltés de différentes zones du cerveau de l’embryon et qu’ils peuvent générer des types spécifiques de neurones et les cellules gliales. Il existe plusieurs méthodes pour l’induction de la différenciation des cellules souches neurales de les amener à générer des neurones matures et les cellules gliales. Il y a de nombreux rapports indiquant q...

Des cellules souches neurales (CNS) résident dans différentes régions du cerveau adulte et de l’embryon, et ils ont tendance à générer différents types de neurones et cellules gliales1. Les zones sous-ventriculaire dans le cerveau des mammifères adultes2 et éminence ganglionnaire dans le cerveau de l’embryon sont NSC riches régions3. Dans le développement du cerveau, l’éminence ganglionnaire fournit la plupart des interneurones corticaux et particulièrement GABAergique interneurones3. Il y a aussi des méthodes moins invasives pour la production de cellules souches neurales de cellules souches embryonnaires (CSE) ou utiliser des cellules souches pluripotentes induites (CISP) qui réduisent la demande de l’animal. Malgré le fait que générer des CNS d’ESCs ou CISP est possible4,5, il a ses avantages et inconvénients par rapport à l’isolement des cellules souches neurales de l’adulte ou l’embryon cerveau6,7, 8. Les protocoles pour induire la différenciation des ces et CISP vers phénotypes neurones sont toujours temps et coût de consommation et le taux de succès (70-80 % Nestin cellules positives)5 est plus faible comparativement à isolement direct des CNS le cerveau animal ( plus de 99 % Nestin cellules positives)9. En outre, les cellules souches perdent leur tendance de stabilité et de la différenciation génétique après plusieurs passages10,11. Même s’il existe d’autres nouveaux rapports sur la conversion directe de cellules somatiques en CNS, ces cellules sont génétiquement modifiées et ne sont pas facilement accessibles dans chaque laboratoire12. Par conséquent, il y a toujours une grande demande pour isoler des cellules souches neurales dans le cerveau de l’animal ; Il est possible de réduire la quantité d’utilisation de l’animale en améliorant les techniques chirurgicales. En réduisant le temps chirurgical et en améliorant les techniques, il est possible de garder les cellules des dommages et d’obtenir le taux le plus élevé des CNS de chaque animal.
Ici, nous présentons une technique simplifiée et reproductible pour l’isolement de cellules souches neuronales du cerveau de souris E13 embryons.

<table border="0" cellpadding="0" cellspacing="0" style="width:942px;" width="941">
	<tbody>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">15 mL tubes</td>
			<td dir="LTR" style="width:145px;">Falcon</td>
			<td dir="LTR" style="width:235px;">352097</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">50 mL tubes</td>
			<td dir="LTR" style="width:145px;">Falcon</td>
			<td dir="LTR">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Adson Forceps, 12 cm, Straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">14226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">B27</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">17504-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">bFGF</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">F0291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">bovine serum albumin (BSA)</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">dish 10cm&#160;</td>
			<td dir="LTR" style="width:145px;">Falcon</td>
			<td dir="LTR" style="width:235px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Dressing Forceps, 12.5 cm, Straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">15908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Dumont Tweezers, 11 cm, Straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR" style="width:235px;">500342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">EGF</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">E9644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Ethanol</td>
			<td dir="LTR" style="width:145px;">Merck</td>
			<td dir="LTR" style="width:235px;">100983&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Glutamate</td>
			<td dir="LTR" style="width:145px;">Sigma&#160;</td>
			<td dir="LTR" style="width:235px;">G3291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Glutamax</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">goat anti rabbit FITC conjugated secondary antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">AP307F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">goat serum</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Heparin&#160;&#160;</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">h3149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">HEPES</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">83264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">HEPES</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR">90909C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">insulinsyringe with 25-27 gauge Needle&#160;</td>
			<td dir="LTR" style="width:145px;">SUPA medical</td>
			<td dir="LTR">A1SNL127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">laminin</td>
			<td dir="LTR" style="width:145px;">sigma&#160;&#160;&#160;</td>
			<td dir="LTR" style="width:235px;">L2020</td>
			<td></td>
		</tr>
		<tr height="26">
			<td dir="LTR" height="26" style="height:26px;width:389px;">MEM</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">M2279</td>
			<td></td>
		</tr>
		<tr height="26">
			<td dir="LTR" height="26" style="height:26px;width:389px;">N2 supplement</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">NB medium</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">21103-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Non-essential amino acid (NEAA)</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">PBS without Ca and Mg</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR">20012050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Penicilin/ Streptomycin</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">5140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Poly-L-ornithine</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">&#160;P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">rabbit anti mouse beta tubulin-III antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">T2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">rabbit anti mouse GFAP antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">G4546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">rabbit anti mouse Nestin antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">N5413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Scalpel Handle #3</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">500236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Scissors curve</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR" style="width:235px;">14396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Scissors sharp straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">14192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Soybean trypsin inhibitor</td>
			<td dir="LTR" style="width:145px;">Roche</td>
			<td dir="LTR" style="width:235px;">10109886001&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Tissue culture flasks, T25</td>
			<td dir="LTR" style="width:145px;">BD</td>
			<td dir="LTR" style="width:235px;">353014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Tissue culture flasks, T75</td>
			<td dir="LTR" style="width:145px;">BD</td>
			<td dir="LTR" style="width:235px;">353024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Tween 20</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Vannas Scissors,&#160; 8 cm, Straight&#160;</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td>14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">&#946;-mercaptoethanol</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR">M6250</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of embryonic mouse neural stem cells

Les cellules souches neurales (CNS) sont multipotentes et peut donner lieu à trois types cellulaires majeurs du système nerveux central (CNS). La culture in vitro et l’expansion des CNS fournissent une source appropriée de cellules pour les chercheurs en neurosciences étudier la fonction des neurones et des cellules gliales ainsi que leurs interactions. Il existe plusieurs techniques signalés pour isoler des cellules souches neurales du cerveau mammifère adulte ou embryonnaire. Pendant l’opération microchirurgie pour isoler des CNS de différentes régions du SNC embryonnaire, il est très important réduire les dommages au cerveau des cellules pour obtenir le taux élevé de cellules souches vivantes et extensibles. Une technique possible pour la réduction du stress au cours de l’isolement de ces cellules du cerveau d’embryon de souris est la réduction du temps chirurgical. Ici, nous démontrons une technique mise au point pour l’isolement rapide de ces cellules de l’éminence E13 souris embryon ganglionnaire. Procédures chirurgicales incluent récolte d’embryons de souris13 E de l’utérus, coupant la fontanelle frontale de l’embryon avec une pointe d’aiguille tordue, extraire le cerveau du crâne, microdissection du cerveau isolé de récolter l’éminence ganglionnaire, dissociation du tissu récolté dans le milieu du NSC pour obtenir une suspension de cellules du même et enfin électrodéposition de cellules en suspension pour générer des neurospheres.

Micro-Dissection, l’isolement de cellules et Culture Neurosphère. Ici, nous avons présenté une méthode rapide et efficace pour la souris E13 cerveau microchirurgie et isolement des cellules souches neurales. Cet article montre qu’il est possible d’enlever tout le cerveau d’un crâne d’embryon de souris E13 à travers une rupture fine à fontanelle frontale. Avec cette méthode, le cerveau subit moins de dégâts et il est possible d’i...

Watch this Scientific Journal Video about Isolation and Culture of Embryonic Mouse Neural Stem Cells at JoVE.com

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Ici, nous présentons une nouvelle technique microchirurgicale pour isoler des cellules souches neurales de l’éminence E13 souris embryon ganglionnaire.

Isolement et Culture des embryons de souris des cellules souches neurales

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

L’objectif de ce protocole vidéo est d’établir une source et un protocole efficace pour l’isolement et la culture des cellules souches neurales de souris embryonnaires. Bonjour, je suis Maryam Sadeghi-Zadeh. Je suis étudiant à la maîtrise en biologie cellulaire et moléculaire à l’Institut Royan.Aujourd’hui, mon collègue et moi voulons vous montrer comment récolter les cellules souches neurales à partir de 13 éminence ganglionnaires de souris embryonnaires. Bonjour, je suis Bahareh Alizadeh-Shoorjestan. Je suis étudiant à la maîtrise en biologie cellulaire et moléculaire au département de cellules souches de l’Institut Royan pour la biotechnologie.Bonjour, je suis Reza Dehghani-Varnamkhasti. J’étudie aussi la biologie cellulaire et moléculaire à l’Institut Royan. Les interventions chirurgicales comprennent la récolte de chaque 13 embryons de souris de l’utérus.Couper l’avant du fontanel de l’embryon avec une pointe d’aiguille pliée extrayant le cerveau du crâne. Micro-dissection du cerveau isolé pour récolter l’éminence ganglionnaire. Dissociation du tissu récolté dans un milieu de cellules souches neurales pour obtenir une suspension cellulaire unique.Et enfin, placage des cellules dans une culture de suspension pour générer des cellules nerveuses. Nettoyez et désinfectez la peau de l’abdomen en pulvérisant à 70 % d’éthanol. Contrôlez la peau vers le haut de l’abdomen, puis coupez la peau et soulignez le visage avec des ciseaux pour découvrir la cavité abdominale et les cornes utérine.Retirer les cornes utérinnes contenant les embryons par ciseaux et les transférer dans un tube conique de 50 ml contenant 25 ml de tampon HEPES MEM froid. Placez le tube conique sous le capot d’écoulement laminal. Ouvrez le bouchon sous le capot.Sortez les cornes utérinnes du tube et rincez trois fois avec suffisamment de volume de la tampon fraîchement froide HEPES MEM pour éliminer les débris et le sang. Transférer le tissu utérin dans une boîte de Pétri de 10 cm contenant de 7 à 10 ml de tampon HEPES MEM froid. Ouvrez les cornes utérinnes à l’aide de ciseaux fins et transférez les embryons dans un nouveau plat contenant de 7 à 10 ml de tampon HEPES MEM froid.Maintenant, mettez le plat de 10 cm avec des embryons sous le microscope de dissection pour l’opération de micro-dissection. Pliez le bout d’une aiguille de seringue en poussant sa pointe sur une poignée de lame de scalpel en acier. Pliez le bout de l’aiguille jusqu’à ce que la pointe soit pliée à un angle de 70 à 90 degrés.Vérifiez le bout de l’aiguille sous le microscope disséquant pour voir la courbure à l’extrémité de l’aiguille. Naturellement, les embryons ont une position latérale dans ce. Sans changer leur position, a tenu le cou et le corps de l’embryon avec des forceps fins, puis insérer la partie pliée de l’aiguille profondément dans l’avant de la fontanel de la tête de l’embryon.Il y aurait un petit saignement ou un caillot de sang là-bas. Déplacez la pointe de l’aiguille superficiellement pendant que la partie pliée est à l’intérieur du renflement vers le front, puis vers l’arrière de la tête. Assurez-vous de couper à travers la peau et le crâne, et de ne pas endommager le cerveau sous-jacent.Poussez la peau avec la pointe de l’aiguille latéralement pour découvrir le cerveau. Insérez l’aiguille du côté latéral de la peau au dessous du cadre du côté latéral de la peau à la zone sous le cerveau. Et puis enlever le cerveau de ce cuir chevelu en le poussant à sortir du crâne disséqué.L’éminence ganglionnaire est clairement montrée en vidéo avec ses parties médials et latérales. A maintenu le cerveau stable en utilisant les forceps de soins, puis en utilisant des microscisseurs pour couper le cortex de chaque hémisphère pour exposer l’éminence ganglionnaire. A tenu le cerveau et place l’éminence ganglionnaire disséquée dans le tube de centrifugeuse de 15 ml contenant le média neural de cellules souches.Répétez cette procédure jusqu’à ce que tout le cerveau ait été micro-disséqué. Coupez l’éminence ganglionnaire disséquée en plaçant la pointe pipette contre le fond du tube et pipette la suspension de haut en bas pendant 2 5 fois pour briser le tissu pour obtenir une suspension à cellule unique. Centrifuger la suspension à 110 g pendant 5 minutes.Retirer le supernatant et résuspendre les cellules dans 1 ml de tube de 0,05% dans EDTA. Incuber la suspension cellulaire dans un 37 degrés Celsius pendant 10 minutes. Et puis ajouter un volume équivalent d’inhibiteur de la trypsine de soja pour arrêter l’activité de la trypsine.Pipette la suspension cellulaire de haut en bas pour s’assurer que la trypsine a été totalement inactivée. Centrifuger la suspension cellulaire à 110 g pendant 5 minutes. Enlevez le supernatant et resuspendez les cellules dans 1 ml de milieu complet de cellules souches neurales et comptez le nombre de cellules.Plaquez les cellules au milieu des cellules souches neurales dans le flacon de culture tissulaire de taille appropriée. Transférer 5 ml de 2T25, 20 ml au T75 et 40 ml dans des flacons T175. Les neurosphères apparaîtront après 3 jours de culture à 37 degrés Celsius dans un incubateur humidifié avec 5% de CO2.Pour aspirer la culture des neurosphères, transférer le milieu avec des neurosphères suspendues à la centrifugeuse à la centrifugeuse à 110 g pendant 5 minutes, puis jeter le supernatant. À 1 ml de 0,05% trypsine EDTA et incuber à 37 degrés Celsius pendant 10 minutes. Puis inactivez la trypsine à l’aide de l’inhibiteur de la tyrpsine de soyben et pipette les neurosphères doucement de haut en bas pour en faire des cellules simples.Centrifuger la suspension cellulaire à 110 g pendant 5 minutes. Jetez le supernatant et resuspendez les cellules dans 1 ml de cellules souches neurales moyennes. Ces cellules sont prêtes pour la culture ou la culture de prochaine étape sur la surface stratifié et entièrement enduite pour des expériences avancées.En cultivant un million de cellules souches neurales primaires après sept jours, le nombre de cellules passera à trois à quatre millions. Après six à sept jours, les sphères doivent mesurer entre 150 et 200 micromètres de diamètre et seront prêtes pour la sous-culture sur les plats enrobés de poly ornithine stratifié en l’absence de bFGF et d’EGF pour la différenciation neuronale. Les résultats de l’analyse de cytométrie de flux montrent que près de 95% des cellules constituant les neurosphères étaient nestin positives qui est un marqueur connu pour les cellules souches neurales.Les analyses utilisant des anticorps bêta tubulin III et ganglio fibrous de protéine révèlent qu’en cultivant des cellules souches neurales sur la surface enduite environ 94% ont montré le phénotype neuronal et environ 5% montrent le phénotype glial. Dans cette courte vidéo, une technique de laboratoire pour l’isolement rapide des cellules souches neurales de 13 embryons de souris éminence ganglionnaire. J’espère que vous réussirez dans votre culture de cellules souches neurales.

isolation-and-culture-of-embryonic-mouse-neural-stem-cells

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JoVE Journal

Neuroscience

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 ...

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 ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolement et culture des cellules souches neurales Crête des follicules pileux humains

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

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.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectine à base d&#39;isolement et de la culture des motoneurones embryonnaires de la souris

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolement et culture de cellules embryonnaires de rat neurones: un protocole rapide

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Expansion du embryonnaires et cellules souches neurales adultes par<em&gt; In Utero</em&gt; Électroporation ou virale par injection stéréotaxique

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolement et culture de cellules endothéliales de la embryonnaire du cerveau antérieur

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Une souris, deux cultures: Isolement et culture des cellules souches adultes de neurones à partir des deux Neurogenic Zones de souris individuelle

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...