Les recherches menant aux présents résultats ont bénéficié du financement du septième programme-cadre de l&#39;Union européenne (FP7/2007-2013) sous convention de subvention n ° 323267, d&#39;Électricité de France (EDF) et de l&#39;Agence Nationale de la Recherche - Santé-Environnement et Santé-Travail (ANR-SEST, Neurorad).

1. Procédures d&#39;animaux Ce protocole a été conçu en conformité avec la directive du Conseil des Communautés européennes du 24 Novembre 1986 (86/609/CEE) et a été approuvé par notre comité institutionnel sur la protection des animaux (Cetea-CEA DSV IdF). <ol><li> Injections avec Edu / BrdU et l&#39;irradiation des souris gravides de E14.5 (figure 2A). <ol><li> Préparer une solution de EdU à 1 mg / ml et de BrdU à 5 mg / ml dans du PBS. Effectuez une injection intra-péritonéale (IP) de 100 pi de solution edu à l&#39;aide d&#39;une aiguille 25 G dans des souris enceintes. 1,5 heures plus tard, les souris ont irradier (irradiation corporelle totale) à 0 Gy ou 2 Gy (0,6 Gy / min). Immédiatement après, injecter (IP) 200 pi de solution de BrdU aide d&#39;une aiguille G 25 dans les souris enceintes. </li></ol></li><li> Préparation de coupes coronales de cerveau embryonnaire. <ol><li> À 1 h ou 4 h après l&#39;irradiation, euthanasier les souris enceintes par le dioxyde de carbone. </li><li> Ouvrez la cavi abdominaleté sur trois centimètres à l&#39;aide de ciseaux. Séparer les deux cornes utérines du reste de l&#39;utérus en sectionnant les deux extrémités des cornes. Transférer les cornes utérines dans une boîte de Pétri contenant du PBS 0,6% de glucose. Ouvrir les cornes utérines avec la pince afin d&#39;isoler les embryons. Retirer le sac amniotique de chaque embryon en utilisant une pince. </li><li> Disséquer têtes embryonnaires avec une pince et les fixer par immersion dans du paraformaldéhyde 4% (PFA, pH = 7,4) O / N à 4 ° C. </li><li> Rincer têtes embryonnaires dans du PBS pendant au moins une nuit à 4 ° C. Les têtes peuvent être conservés dans du PBS pendant plusieurs jours. </li><li> Têtes Intégrer dans la paraffine en utilisant un processeur d&#39;infiltration sous vide comme indiqué dans le tableau 1. Chefs d&#39;enrobage peuvent également être effectuées sous une hotte chimique pour l&#39;éthanol et bains Xylen, puis dans une étuve à 65 ° C pour les bains de paraffine. </li><li> Préparer 5 um coupes coronales d&#39;épaisseur avec un microtome. </li><li> Monter sur des lames de microscope polylysine enduit. &lt;/ Li&gt; <li> Diapositives peuvent être conservés à température ambiante (TA) pendant plusieurs mois. </li></ol></li></ol> 2. Edu / BrdU coloration <ol><li> Déparaffinage et démasquage antigénique <ol><li> Déparaffiner les coupes de cerveau paraffine par immersion des lames dans 3 bains de toluène pendant 5 min. </li><li> Réhydrater pendant 3 min dans 2 bains de chacune des solutions de la diminution de la concentration en éthanol de la manière suivante: 100% de l&#39;éthanol, de l&#39;éthanol à 95%, éthanol à 70%, et 5 min dans H 2 O. Diapositives peuvent être conservés dans de l&#39;eau déminéralisée pour plusieurs heures. </li><li> Faire bouillir les tranches pendant 10 minutes dans une solution de citrate (10 mM, pH 6), puis incuber dans de l&#39;eau désionisée pendant 5 min. </li></ol></li><li> EdU coloration <ol><li> Effectuer une détection EdU selon le protocole du fabricant. Perméabiliser les cellules avec 0,5% de triton X-100 dans du PBS pendant 10 min. </li><li> Préparer additif tampon 1X EdU par dilution de la solution 10X dans de l&#39;eau désionisée. Cette solution doit être fraîchement préparé et utilisé sur la même day. </li><li> Préparer EdU réaction cocktail, y compris EdU Alexa Fluor 488 azoture. Il est important d&#39;ajouter les ingrédients dans l&#39;ordre indiqué dans le tableau 2, sinon, la réaction ne sera pas procéder de manière optimale. Utilisez la réaction cocktail EdU dans les 15 minutes de préparation. </li><li> Retirez le tampon de perméabilisation (étape 2.1.1), puis laver deux fois avec PBS. </li><li> Ajouter 150 pi de EdU réaction cocktail, mettre une lamelle et incuber pendant 30 minutes dans l&#39;obscurité à la température ambiante. </li><li> Retirer la réaction cocktail EdU, puis laver une fois avec PBS. </li></ol></li><li> BrdU coloration <ol><li> Préparer du tampon de saturation avec 7,5% de sérum de chèvre et 7,5% de sérum fœtal bovin dans du PBS. Ajouter 150 ul de tampon de saturation sur des tranches de cerveau, mettre une lamelle couvre-objet, et incuber pendant 1 heure à température ambiante pour bloquer la liaison d&#39;anticorps non spécifique. </li><li> Retirer la lamelle. Ajouter 150 pi de souris anti-BrdU anticorps primaire dilué à 1/300 dans le tampon de saturation, mettre une lamelle et incuber O /N à 4 ° C. </li><li> Retirer la lamelle, puis laver 3 fois en PBS. </li><li> Ajouter 150 ul d&#39;anticorps de chèvre anti-souris-Alexa594 anticorps secondaire dilué au 1/400 dans du tampon de saturation, placer une lamelle couvre-objet, et incuber pendant 1 heure à température ambiante. </li><li> Retirer la lamelle, puis lavez une fois dans du PBS. </li><li> Coloration nucléaire: ajouter 150 ul de 4 &#39;,6-diamidino-2-phénylindole (DAPI) à 1 pg / ml dans du PBS, mettre une lamelle et incuber pendant 2 minutes à température ambiante. </li><li> Monter les lames dans un milieu de montage. </li></ol></li></ol> 3. Analyse <ol><li> Examiner des coupes de cerveau sous un microscope à fluorescence avec un objectif 20X ou un microscope confocal et acquérir des images à trois voies (488 nm, 594 nm et UV) comme sépare fichiers. </li><li> Empilez les images avec le logiciel Photoshop. </li><li> Analyser des coupes de cerveau dans un secteur standard de la paroi cérébrale dorsomédial. Ce secteur est de 100 um dans sa dimension médiane-latérale et est divisée en 18 bacs (ou plus) de 10 μm de hauteur dans sa dimension radiale à l&#39;aide d&#39;une grille superposée sur les images (figure 1) comme précédemment décrit 31. <ol><li> Aligner la grille tel que le premier bac est à la surface ventriculaire, avec son grand axe parallèle à la frontière ventriculaire. Numéroter la EdU marqué, BrdU et / ou noyaux pycnotiques (correspondant à noyaux apoptotiques) dans chaque bac. Compter un noyau situé sur la limite de deux bacs dans le bac qui contient sa plus grande partie, ou dans le bac inférieur, lorsque le noyau occupe des aires égales dans les deux bacs. </li></ol></li><li> L&#39;analyse statistique. </li></ol> Analyser au moins deux tranches de cortex par embryon. Répétez expériences sur au moins 3 embryons de différentes portées. Les résultats doivent être données en tant que moyenne ± erreur type de la moyenne (SEM). Les analyses statistiques sont effectuées avec Graphpad Prism utilisant ANOVA à deux voies et de multiples tests de comparaison POSTHOC Bonferroni.

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Les auteurs n&#39;ont pas de conflit d&#39;intérêt.

Le dispositif expérimental décrit ici sur la base de l&#39;incorporation de EdU 1,5 heure avant l&#39;irradiation et l&#39;incorporation de BrdU immédiatement après irradiation a permis la démonstration que NSPCs sont capables d&#39;activer S et G2 / M points de contrôle, mais pas au point de contrôle G1 / S au cours de la 1 ère heure après un stress génotoxique dans le cerveau de fœtus de souris. Nous avons effectué d&#39;autres expériences dans lesquelles Edu a été injecté à différents tem...

Au cours du développement embryonnaire du cerveau, les neurones de projection du cortex cérébral sont générés dans la zone ventriculaire, un épithélium pseudo composée de différents types de cellules neuronales souches et de cellules progénitrices (NSPC) qui tapisse les ventricules latéraux. Parmi NSPC, les cellules radiales gliales (RGC), qui servent de cellules souches neurales, subissent une migration nucléaire interkinetic (INM): ils effectuent la mitose à la surface du ventricule et de la phase S à la limite de base de la zone ventriculaire (VZ) 1, 2,3. Ils peuvent se diviser de manière symétrique à générer deux RGC ou asymétrique pour générer un RGC et un neurone ou une cellule intermédiaire progénitrices (IPC) 4, 5. IPC migrent à une couche sus-jacente en prolifération dite zone sous-ventriculaire (SVZ), où, après une dernière division symétrique ils génèrent deux neurones de projection immatures 8.6. Contrairement à RGC, l&#39;IPC ne subissent pas l&#39;INM (revue dans 9). Neurones nouvellement générés émigrésmangé radialement le long des fibres radiales dans la zone intermédiaire (IZ) pour atteindre leur destination finale dans la plaque corticale (CP) 10, 8. Le timing parfait de tous ces événements est essentielle pour un développement cortical correct. Par exemple, le passage de la prolifération de la différenciation des populations de cellules progénitrices neurales est contrôlée par la durée de la phase G1 11, 12. L&#39;allongement de la phase G1 corrèle donc avec la différenciation cellulaire. Stress génotoxique, tels que les rayonnements ionisants, le développement du cerveau sérieusement affaiblir (revue dans 13). Nous et d&#39;autres avons montré que CNPS sont très sujettes à l&#39;apoptose induite par le rayonnement 14, 15,16. Les rayonnements ionisants induisent cassures double brin qui sont les plus graves dommages aux cellules proliférantes. Une composante essentielle de la réponse de l&#39;ADN de dommages (DDR) dans les cellules de cyclisme est l&#39;activation des points de contrôle du cycle cellulaire au G1 / S ou G2 / M transitions ou pendant Sphase (intra-S checkpoint) 17-21. Ils bloquent la progression du cycle cellulaire pour fournir des temps de réparation de l&#39;ADN ou de l&#39;élimination des cellules trop endommagés. Par conséquent, la mort cellulaire ainsi que la progression du cycle cellulaire retardée peuvent altérer le développement du cerveau en réponse à l&#39;exposition au rayonnement ionisant de 22 à 24. Il était donc intéressant de développer une méthode pour évaluer l&#39;activation des points de contrôle du cycle cellulaire dans NSPC chez la souris irradiée cerveau embryonnaire. La progression du cycle cellulaire est habituellement suivie en utilisant l&#39;incorporation d&#39;un analogue de la thymidine, la 5-bromo-2&#39;-désoxyuridine (BrdU). BrdU est incorporé au cours de la phase S du cycle cellulaire, lorsque l&#39;ADN se réplique. L&#39;utilisation d&#39;un anticorps contre BrdU permet par la suite de la détection de cellules qui étaient en phase S pendant l&#39;impulsion de BrdU. Un nouvel analogue de la thymidine, la 5-éthynyl-2&#39;-désoxyuridine (EDU) est détectée par un azoture fluorescent. Les différentes façons de détecter EdU et B<html> RDU ne cross-réagit pas 25, ce qui permet la détection simultanée de deux des analogues de la thymidine, qui est utile pour l&#39;étude de la progression du cycle cellulaire. Habituellement cellules sont d&#39;abord marquées avec Edu puis puisées avec BrdU, où le temps entre les deux incorporations dure deux heures 25, 26. L&#39;addition de BrdU dans des milieux de culture contenant EdU entraîne préférentiellement incorporation de BrdU dans l&#39;ADN, à l&#39;exclusion de EdU, alors que l&#39;addition simultanée de 25 EdU avec BrdU équimolaire ou équimolaire par rapport à la moitié des résultats de médias dans seulement l&#39;incorporation de BrdU 27. Cela simplifie le protocole de marquage double en supprimant les étapes de lavage qui sont normalement requises pour éliminer la première étiquette à partir du milieu de culture avant l&#39;addition de la deuxième étiquette. C&#39;est également un intérêt particulier pour l&#39;étude in vivo, où les étapes de lavage ne ​​sont pas possibles, afin de déterminer le moment précis de l&#39;entrée ou de sortie de la population de cellules en phase S. t &quot;&gt; Récemment, un procédé de marquage à double impulsion dans le cerveau de souris embryonnaire en utilisant EdU et BrdU 23, 24,22 a été développé pour analyser la progression du cycle cellulaire et l&#39;INM de NSPC après l&#39;irradiation in utero. En outre, il a été démontré que, lors de phase S, les cellules de souris reproduire d&#39;abord les régions euchromatine et l&#39;hétérochromatine alors péricentrique 28,29,30. intéressant, hétérochromatine péricentrique de chromosomes différents clusters dans les noyaux interphasiques pour former des foyers d&#39;hétérochromatine aussi connu comme chromocentres et facilement détectable par coloration DAPI comme foyers lumineux. Par conséquent, les différentiels Edu et BrdU colorations de l&#39;euchromatine et chromocentres nous ont aidés à analyser plus précisément la progression de la phase S du CNPS. Cette méthode a permis la démonstration de l&#39;absence apparente de G1 / S checkpoint dans NSCP 22-24, ce qui est assez surprenant, car ce point de contrôle est censé être critique pour la stabilité du génome. Plusieurs expconceptions de erimental basés sur diverses combinaisons d&#39;impulsions edu et BrdU ont été utilisées pour analyser la progression du cycle cellulaire dans le télencéphale ventral et dorsal. Ici, nous donnons un exemple de protocoles permettant l&#39;étude des dommages de l&#39;ADN aiguë de NSPC dans le 4 premières heures suivant l&#39;irradiation in utero d&#39;embryons de souris E14.5.

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			<td height="40" style="height:40px;width:228px;">EdU</td>
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			<td style="width:125px;">A10044</td>
			<td style="width:163px;">1 mg/ml</td>
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			<td height="37" style="height:37px;width:228px;">BrdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">B5002</td>
			<td style="width:163px;">5 mg/ml</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:228px;">IBL 637</td>
			<td style="width:212px;">CIS BIO International</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
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		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Tissu Tek VIP</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Microtome</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;">RM 2125 RT</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Triton X-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td align="right" style="width:125px;">93443</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:228px;">Click-iT EdU Alexa 488 imaging kit</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">C10083</td>
			<td style="width:163px;"></td>
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		<tr height="47">
			<td height="47" style="height:47px;width:228px;">Anti-BrdU</td>
			<td style="width:212px;">GE Healthcare</td>
			<td style="width:125px;">RPN202</td>
			<td style="width:163px;">1/300</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:228px;">Goat anti mouse-Alex594</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A11001</td>
			<td style="width:163px;">1/400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Fluoromount</td>
			<td style="width:212px;">SouthernBiotech</td>
			<td style="width:125px;">0100-01</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Polysine slide</td>
			<td style="width:212px;">Thermo scientific</td>
			<td style="width:125px;">J2800AMNZ</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Paraformaldehyde</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">P6148</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:228px;">PBS</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">20012-068</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:228px;">DAPI</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">D9542</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Microscope BX51</td>
			<td style="width:212px;">Olympus</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Confocal microscope SPE</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Prism software</td>
			<td style="width:212px;">Graphpad</td>
			<td style="width:125px;"></td>
			<td style="width:163px;">Version 5.0c</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Photoshop software</td>
			<td style="width:212px;">Adobe</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
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assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

Les neurones du cortex cérébral sont générées lors du développement du cerveau à partir de différents types de cellules souches et progénitrices neurales (CNPS), qui forment un épithélium pseudo tapisse les ventricules latéraux du cerveau embryonnaire. Stress génotoxiques, tels que les rayonnements ionisants, ont des effets très néfastes sur le développement du cerveau liée à la grande sensibilité de la NSPC. L&#39;élucidation des mécanismes cellulaires et moléculaires impliqués dépend de la caractérisation de la réponse aux dommages de l&#39;ADN de ces types particuliers de cellules, ce qui nécessite une méthode précise pour déterminer la progression NSPC à travers le cycle cellulaire dans les tissus endommagés. Ici on trouve une méthode basée sur des injections intrapéritonéales successives de EdU et BrdU chez les souris enceintes et en outre la détection de ces deux analogues de la thymidine en coupes coronales du cerveau embryonnaire. EdU BrdU et sont tous les deux incorporés dans l&#39;ADN pendant la replication des cellules en phase S et sont détectés par deux techniques différentes (azide ouun anticorps spécifique, respectivement), ce qui facilite leur détection simultanée. EdU BrdU et la coloration sont ensuite déterminés pour chaque noyau NSPC en fonction de sa distance à partir de la marge ventriculaire dans une région standard du télencéphale dorsal. Ainsi, cette technique de double marquage permet de distinguer les cellules qui ont progressé à travers le cycle cellulaire à partir de ceux qui ont activé un point de contrôle du cycle cellulaire, conduisant à un arrêt du cycle cellulaire en réponse à des dommages à l&#39;ADN. Un exemple d&#39;expérience est présentée, dans laquelle Edu a été injectée avant l&#39;irradiation et BrdU immédiatement après et les analyses effectuées dans le 4 h après l&#39;irradiation. Ce protocole fournit une analyse précise de la réponse aiguë de lésions de l&#39;ADN de NSPC en fonction de la phase du cycle cellulaire à laquelle ils ont été irradiées. Ce procédé est aisément transposable à d&#39;autres systèmes en vue de déterminer l&#39;incidence d&#39;un traitement particulier sur la progression du cycle cellulaire dans les tissus vivants.

Dans l&#39;expérience décrite dans la figure 2, on a administré EdU 1,5 h avant l&#39;irradiation et la BrdU juste après l&#39;irradiation. Quatre types de cellules ont ensuite été distingués dans les tranches de cortex préparés en 1 ou 4 heures après l&#39;irradiation, selon la constitution de l&#39;une ou EdU BrdU, les deux ou aucun (Figures 2A et 2B). Surtout, ni EdU ni l&#39;incorporation de BrdU changé le niveau d&#39;apoptose radio-induite (données non...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress | Neuroscience | Video

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

L&#39;administration de deux analogues de la thymidine, et EdU BrdU, dans des souris gravides permet l&#39;analyse de la progression du cycle cellulaire dans les neurones et les cellules progénitrices dans le cerveau de souris embryonnaire. Cette méthode est utile pour déterminer les effets du stress génotoxique, y compris les rayonnements ionisants, au cours du développement du cerveau.

Évaluation de la progression du cycle cellulaire des cellules souches neurales et progénitrices dans le développement du cerveau de souris après un stress génotoxique

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

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15.2K vues.  CEA DSV iRCM SCSR.  L'administration de deux analogues de la thymidine, et EdU BrdU, dans des souris gravides permet l'analyse de la progression du cycle cellulaire dans les neurones et les cellules progénitrices dans le cerveau de souris embryonnaire. Cette méthode est utile pour déterminer les effets du stress génotoxique, y compris les rayonnements ionisants, au cours du développement du cerveau. 

Vidéo: Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Imagerie time-lapse en direct de relation clonale cellules progénitrices neurales dans le cerveau antérieur développement Zebrafish

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

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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 ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagerie en direct de la mitose dans les développement Cortex souris embryonnaires

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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 ...

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. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Modélisation d’infection par le Virus de Zika du développement cerveau humain In Vitro à l’aide de cellules souches dérivées cérébrale organoïdes

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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 expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.