Ce travail a été financé (en partie) par le Helmholtz Alliance ICEMED - imagerie et le traitement des maladies métaboliques de l&#39;environnement, à travers le Fonds de l&#39;Initiative et le Réseau de l&#39;Association Helmholtz, une subvention de la Fondation Else Kroener-Fresenius, et une subvention de la Deutsche Forschungsgemeinschaft ( SFB 655: Les cellules dans les tissus).

Le travail adhère à la Déclaration de Helsinski et l&#39;état des animaux ARVO. Les animaux ont été utilisés pour la collecte des tissus et toutes les méthodes pertinentes ont été suivies selon les instructions de l&#39;animalerie de l&#39;Université de Dresde. Les animaux ont été manipulés et logés selon les lignes directrices fédérales allemandes pour l&#39;utilisation et l&#39;entretien des animaux de laboratoire, et l&#39;étude a été approuvé par le Landesdirektion Dresde. S&#39;il vous plaît consulter la politique vétérinaire de votre établissement (IACUC ou autre conseil) en ce qui concerne la méthodologie de l&#39;euthanasie approprié. Concentrations de travail de réactifs actions spécifiques et peuvent être trouvés dans les tableaux accompagnant ce protocole réactif. Une. Culture vaisselle Préparation <ol><li> Manteau de plats avec un volume suffisant de 75 pg / ml de poly-L-ornithine (soit 2 ml / puits d&#39;une plaque à 6 puits) pendant une nuit dans l&#39;incubateur de culture de cellules 2 jours avant la date prévue de dissection. </li><li&gt; Aspirer le poly-L-ornithine le jour et laver chaque puits 2x avec 3 ml de PBS pendant 5 min suivant. </li><li> Retirez le lavage final, puis ajouter la fibronectine (dilué 1:250, 4 ug / ml dans PBS) à la plaque et placez les plats dans l&#39;incubateur pendant la nuit. </li><li> Aspirer la fibronectine, le jour de la dissection et de lavage des plats 2x avec du PBS. Remettre les plaques contenant encore la finale PBS laver à l&#39;incubateur jusqu&#39;à ce que le tissu dissocié est prêt pour la culture. À ce moment, retirer la PBS avant l&#39;étalement du tissu. </li><li> plaques de magasins recouvertes de poly-L-ornithinein l&#39;incubateur pendant jusqu&#39;à 3 semaines. Des plaques recouvertes de fibronectine peuvent être stockées pendant 1 semaine. revêtement de fibronectine peut être fait en aussi peu que 2 heures si urgent. La fibronectine est sensible à la dénaturation, ne pas agiter la solution ou laissez sécher la vaisselle. </li></ol> 2. Dissection / placage <ol><li> Ce protocole s&#39;applique à l&#39;utilisation de trois rats adultes (3-6 mois d&#39;âge). </li><li> Chill le cerveau sectionnement bloc sur la glace avant l&#39;euthanasie chez le rat. </li><li> Placer un tube Falcon de 15 ml contenant 5 ml de milieu N2 avec bFGF ajouté sur de la glace. </li><li> Euthanasier les rats conformément à la politique vétérinaire de votre institution. </li><li> Retirer le cerveau du crâne en faisant une incision de la ligne médiane vers le bas de la tête. Utilisez une pince à décoller de l&#39;os permettant au cerveau pour être soigneusement extrait. </li><li> Placez le cerveau dans une boîte de 10 cm de Pétri contenant glacée PBS. Cela permettra d&#39;éliminer tous les poils résiduelle et le sang. (Figure 1A) </li><li> Placer le cerveau dans le bloc de sectionnement réfrigérés. (Figure 1B) </li><li> Insérez une nouvelle lame de rasoir propre dans le bloc comme illustré à la figure 1C. </li><li> Insérer un second mm caudal rasoir propre pale 3 à la première, comme illustré sur la figure 1D. </li><li> Soulevez délicatement les lames du bloc, emportant avec eux la section d&#39;intérêt. </li><li> Faire flotter le section de la lame en l&#39;immergeant dans du PBS. (Figure 1E) </li><li> tissu de récolte de la zone d&#39;intérêt (dans cet exemple, nous avons utilisé la commissure antérieure, (antérieure) ACA, figure 1F) en utilisant # 5 forceps et recueillir dans un tube conique de 15 ml contenant 1 ml de milieu N2 contenant bFGF. </li><li> Déplacer le tube dans une hotte de culture de cellules à flux laminaire, en continuant le reste du protocole en conditions stériles. </li><li> Mécaniquement dissocier le tissu à l&#39;aide d&#39;une pipette de 1 ml attaché à une pipette P1000. Introduire à la pipette de haut en bas plusieurs fois (environ 20 fois à raison de 1 cycle de pipetage par seconde) tout en appuyant sur ​​l&#39;ouverture de la pointe de la pipette contre le fond du tube conique (figures 1G 1-3). Arrêtez trituration quand la solution devient homogène. </li><li> Laisser reposer la solution pendant 2 minutes pour permettre à tout le tissu non dissociée plus grande à se déposer au fond. (Figure 1G 4) </li><li> Recueillir le homogeneous surnageant et le placer dans un tube conique contenant 8,5 ml de milieu N2 plus 20 ng ml de bFGF /, 500 ng ml Dll4 /, 500 ng / ml Ang2, et 200 nMJAK inhibiteur. </li><li> Plate-les dans 3 puits d&#39;une plaque à 6 puits à l&#39;aide de 3 ml de solution cellulaire diluée / puits. </li><li> Placer la plaque dans un incubateur humidifié de cellules à 37 ° C, 5% de CO 2, 5% d&#39;O 2. </li></ol> 3. Entretien quotidien <ol><li> Remplacez le support sur les boîtes de culture avec N2 milieu contenant bFGF, Ang2, Dll4, et JAK inhibiteur 24 heures après placage. </li><li> Ajouter un bolus de bFGF, Dll4, Ang2, et l&#39;inhibiteur de JAK le jour suivant. </li><li> Continuez ce processus alternatif de changements des médias et des ajouts de facteurs pour un total de 10-14 jours. </li></ol> 4. L&#39;induction de la différenciation <ol><li> Retirer bFGF, Dll4, Ang2, et JAK inhibiteur contenant du milieu et de le remplacer par un milieu N2 qui ne contient pas de facteurs supplémentaires. </li><li> Changez le milieu tous les deux jours. </li><li> Fixer les cellules après 10 jours et d&#39;effectuer immunocytochimie. </li></ol> 5. Détection immunocytochimique <ol><li> Aspirer le milieu et fixer des plaques de culture de cellules avec 2 ml de paraformaldéhyde à 4% pendant 20 min. </li><li> Laver 2 fois avec 3 ml de PBS pendant 5 min chacun. </li><li> Aspirer le PBS, puis bloquer et perméabiliser les cellules par addition de 2 ml de PBS contenant 5% de sérum normal d&#39;âne (NDS) et 0,1% de Triton X-100 pendant 20 min. </li><li> Préparer le mélange d&#39;anticorps primaire dans du PBS contenant 5% de NDS. Anticorps anti-nestine peut être utilisé pour identifier SC, tandis que Tuj1 (classe III β-tubuline), anticorps GFAP, et CNPase peut être utilisé pour un triple coloration de marqueurs de différenciation. </li><li> Aspirer la solution de blocage et ajouter 1,5 ml du mélange d&#39;anticorps primaire à chaque puits. Incuber à température ambiante pendant 90 min. </li><li> Retirez les anticorps primaires et laver 2 fois avec 3 ml de PBS, et 1x avec 3 ml de PBS contenant 5% de NDS pour 5 min chacun. </li><li> Prepare les anticorps secondaire dans du PBS contenant 5% de NDS. </li><li> Aspirer le lavage final et ajouter 1,5 ml de solution d&#39;anticorps secondaire dans chaque puits. Incuber dans l&#39;obscurité pendant 40 min à température ambiante. </li><li> Retirer la solution d&#39;anticorps secondaire et ajouter 2 ml d&#39;une tache DAPI fraîchement préparé (500 ng / ml dans du PBS). Incuber pendant 3 min. </li><li> Aspirer la solution DAPI puis laver 3 fois avec 3 ml de PBS pendant 5 min chacun. Les cellules peuvent maintenant être visualisées avec un microscope à fluorescence. </li></ol>

<ol>
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Nous n&#39;avons rien à communiquer.

Un grand nombre des étapes critiques de l&#39;isolement, de l&#39;expansion et la différenciation des cellules souches neuronales du cerveau adulte sont partagées en commun par des techniques de culture tissulaire standard. L&#39;objectif de garder à l&#39;esprit est que les CNS isolés du cerveau adulte devrait maintenir le plus grand nombre de leurs caractéristiques in vivo que possible (Figure 2J). Souvent un équilibre entre la prudence nécessaire et la vitesse appropriée doit être ...

Le dogme central de la neurobiologie, prévue par les observations fondamentales de la cytoarchitecture de cerveau fait par Ramón Y. Cajal il ya plus d&#39;un siècle, a estimé que la neurogenèse est peu probable après l&#39;adolescence étant donné la complexité des réseaux de neurones présents dans le système nerveux central 1. Malgré les travaux de Altman dans les années 1960, et plus tard Kaplan, ce qui démontre que la 3H-thymidine pourrait être trouvée dans les neurones matures indiquant que, en fait, les neurones ont été générés dans des zones distinctes du cerveau adulte, le dogme a continué à tenir deux, trois. La preuve a continué à monter avec la recherche de Nottebohm décrivant les changements saisonniers dans le nombre de neurones présents dans le cerveau des oiseaux chanteurs 4. Ce n&#39;était pas jusqu&#39;en 1999, quand Gould et al travail. Publié sur la génération de neurones dans l&#39;hippocampe augmente avec l&#39;exécution des tâches d&#39;apprentissage associatif chez le rat, ainsi que les observations de Kornack et Rakic ​​démonstrationTing a continué la neurogenèse chez le macaque adulte que le concept d&#39;un cerveau moins rigide, plus plastique, a été reconnu 5, 6. La recherche de la source cellulaire de ces neurones générés de novo conduit à la découverte d&#39;une population distincte de cellules souches (CS) qui résident dans les zones du cerveau appelée niches 7. La zone sous-ventriculaire et la zone granulaire sous de l&#39;hippocampe sont considérés comme les deux principales régions neurogènes 8,9. Cellules isolées à partir de ces endroits présentent la caractéristique classique de CS dérivés embryonnaires ou fœtales, l&#39;auto-renouvellement et potentiel de différenciation. Dans le cas des cellules souches neurales (NSC), ils peuvent être différenciées en neurones, astrocytes et oligodendrocytes,. En outre, ces SCs tache positive pour les marqueurs du CNS tels que la foetales intermédiaire nestine de protéine de filament 10. Des travaux plus récents souligne que les conseils sectoriels pourraient ne pas se limiter à ces Two domaines, et sont en fait localisée dans le cerveau comme une population en grande partie de repos de cellules étroitement associés à la vascularisation 11. Les observations que les CS sont mobilisés en réponse à une blessure suggère la possibilité d&#39;être en mesure d&#39;utiliser ces cellules à des fins de régénération pour aider à la récupération de maladie neurodégénérative et course 12, 13. Cela n&#39;est pas sans rappeler le rôle que les cellules souches mésenchymateuses (CSM) jouent dans la cicatrisation des tissus conjonctifs, qui sont présents sous forme de cellules périvasculaires qui ont le potentiel de devenir des ostéoblastes, les chondrocytes, les cellules adipeuses et 14. Cependant, les CNS ne peuvent être récoltées de la même manière que les cellules souches mésenchymateuses à partir de la moelle osseuse par aspiration de routine et les techniques de centrifugation à gradient de densité et par la suite utilisées dans les thérapies à base de cellules autologues. En conséquence, d&#39;autres sources de cellules, telles que l&#39;utilisation de NNC foetales ou des précurseurs neuronaux issus de embcellules souches ryonic ont été largement explorée dans la maladie de modèles animaux et de blessures avec différents degrés de succès 15. Technologies de cellules souches pluripotentes induites utilisant des sources de cellules somatiques offrent un autre moyen potentiel pour produire des thérapies à base de cellules thérapeutiquement utiles pour un large éventail d&#39;applications, en surmontant la disponibilité limitée et des préoccupations éthiques concernant l&#39;utilisation des cellules embryonnaires et les tissus fœtaux 16. Cependant, la traduction clinique de ces résultats s&#39;est avéré être une tâche difficile, comme l&#39;a démontré dans les luttes de traitement de divers troubles neurologiques avec thérapeutiques à base SC approches 17,18, ainsi que d&#39;un chemin tortueux à l&#39;autorisation réglementaire. Comme une approche alternative, l&#39;introduction de traitements pharmacologiques spécifiques peut moduler le nombre de CSN et de faciliter la récupération dans les modèles de la maladie de Parkinson et les AVC 19. Quelle que soit la stratégie pourrait être, de comprendre comment manipuler efficacement ces cellulesexige un système in vitro accessible. Cultures du CNS peuvent être réalisés soit sous forme de cultures globales, aussi appelées neurosphères, ou comme une monocouche 8,20. Les deux techniques ont été largement utilisés, ce qui permet l&#39;établissement de conditions de culture définies, à savoir l&#39;utilisation de facteur de croissance épidermique (EGF) ou le facteur de croissance basique des fibroblastes (bFGF) en tant que source de mitogène, qui assurent l&#39;expansion de précurseurs multipotentes. Bien que les cultures de neurosphères peuvent être mieux adaptés à l&#39;étude de la capacité de propagation clonale d&#39;un type de cellule isolée, le système a été montré pour produire une population mixte de cellules lors de la détente 21. En outre, la structure fermée de neurosphères rend la manipulation pharmacologique des cellules peu pratique, et l&#39;interprétation de l&#39;influence de ces facteurs peuvent avoir pourrait être confondue en raison du microenvironnement établi dans la neurosphère lui-même. Des cultures monocouches, d&#39;autre part, peuvent êtreutilisé dans les écrans à haut débit de banques de petites molécules, en fournissant un outil puissant pour explorer les mécanismes de transduction du signal qui régulent la croissance et la différenciation SC et ouvre la possibilité de découvrir de nouveaux composés qui ciblent spécifiquement cette population de cellules. En conséquence, la capacité de générer de manière reproductible des cultures de NNC adultes provenant de différentes régions d&#39;intérêt dans le cerveau peut être utilisée dans un large éventail d&#39;applications de recherche, allant d&#39;études sur le développement du système nerveux central (SNC) à explorer de nouvelles approches de la médecine régénérative . Le protocole présenté ici montre comment disséquer et évaluer le potentiel de différenciation de SNC Centres isolés du cerveau de rongeur adulte.

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well tissue culture dishes</td>
			<td style="width:95px;">BD Biosciences</td>
			<td style="width:119px;">353934</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="190" rowspan="1" style="height:190px;width:216px;">Poly-L-ornithine</td>
			<td rowspan="1" style="width:95px;">Sigma-Aldrich</td>
			<td rowspan="1" style="width:119px;">P-3655</td>
			<td style="width:167px;">
			5 mg/ml stock solution prepared in double distilled water (Stable for several months at -20 &#176;C).
			Working concentration 0.5 mg/ml in water (Stable for 1 month at 4 &#176;C).
			</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fibronectin</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">1030-Fn</td>
			<td style="width:167px;">Do not agitate stock solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Filtration Apparatus</td>
			<td style="width:95px;">Corning Life Sciences</td>
			<td style="width:119px;">430769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">DMEM/F-12</td>
			<td style="width:95px;">Mediatech</td>
			<td style="width:119px;">10-090-CV</td>
			<td style="width:167px;">See note below for complete N2 media preparation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Apo-transferrin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-2036</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Insulin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-0516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:216px;">Putrescine</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-5780</td>
			<td style="width:167px;">1 M stock solution in ddH2O (Stable at 20 &#176;C for 6 months)</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:216px;">Sodium Selenite</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">S-5261</td>
			<td style="width:167px;">500 &#181;M stock solution in ddH2O (Stable at -20 &#176;C for 6 months)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Progesterone</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8783</td>
			<td style="width:167px;">100 &#181;M stock solution in ethanol (Stable at -20&#176;C for 6 months)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:95px;">Mediatech</td>
			<td style="width:119px;">21-040-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Basic Fibroblast Growth Factor (bFGF)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB</td>
			<td style="width:167px;">Working concentration 20 ng/ml&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Delta4 (Dll4)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">1389-D4</td>
			<td style="width:167px;">Working concentration&#160; 500 ng/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Angiopoetin 2 (Ang2)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">623-AN</td>
			<td style="width:167px;">Working concentration&#160; 500 ng/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">JAK Inhibitor</td>
			<td style="width:95px;">Calbiochem</td>
			<td style="width:119px;">420099</td>
			<td style="width:167px;">Working concentration 200 nM&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">A-2058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">15- and 50-ml Conical tubes</td>
			<td style="width:95px;">Corning Life Sciences</td>
			<td style="width:119px;">430053, 430829</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="243">
			<td colspan="4" height="243" style="height:243px;width:596px;">Other necessary items include: General dissection instruments, including razor blade and forceps, Adult rat (3-6 months old; Sprague-Dawley or Long Evans), CO2 intoxication chamber, Laminar flow hood for cell culture and incubator Incubator (humidified, 37 &#176;C, 5% CO2, 5% O2). Note: For complete N2 media preparation, to one bottle of DMEM/F-12 (500 ml) add 0.05 g of apotransferin, 0.0125 g of insulin (freshly predissolved in 1 ml of 10 mM NaOH), 50 &#956;l of putrescine, 30 &#956;l of sodium selenite, 100 &#956;l of progesterone stocks, and 5 ml of penicillin/streptomycin solution.&#160; Adjust pH to 7.2, if needed. Filter-sterilize and store at 4 &#176;C for up to 3 weeks and protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>Immunofluorescence Reagents Table</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Paraformaldehyde</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15719</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Normal Donkey Serum (NDS)</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">D-9663</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Triton X-100</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">4,6-Diamidino-2-phenylindole (DAPI)</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">D-8417</td>
			<td style="width:167px;">5 mg/ml stock solution in methanol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Primary Antibody Table</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Nestin</td>
			<td rowspan="1" style="width:95px;">Chemicon</td>
			<td rowspan="1" style="width:119px;">MAB353</td>
			<td style="width:167px;">
			Dilution Factor: 1:400
			Species: Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Hes3</td>
			<td rowspan="1" style="width:95px;">Santa Cruz</td>
			<td rowspan="1" style="width:119px;">sc-25393</td>
			<td style="width:167px;">
			Dilution Factor: 1:100
			Species: Rabbit IgG
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Sox2</td>
			<td rowspan="1" style="width:95px;">R&#38;D Systems</td>
			<td rowspan="1" style="width:119px;">MAB2018&#160;</td>
			<td style="width:167px;">
			Dilution Factor: 1:100
			Species: Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">CNPase</td>
			<td rowspan="1" style="width:95px;">Chemicon</td>
			<td rowspan="1" style="width:119px;">MAB326</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">&#946;-tubulin III (TUJ1)</td>
			<td rowspan="1" style="width:95px;">R&#38;D Systems</td>
			<td rowspan="1" style="width:119px;">MAB1195</td>
			<td style="width:167px;">
			Dilution Factor: 1:500
			Species: Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Glial Fibrillary Acidic Protein (GFAP)</td>
			<td rowspan="1" style="width:95px;">Dako North America</td>
			<td rowspan="1" style="width:119px;">Z0334</td>
			<td style="width:167px;">
			Dilution Factor 1:500
			Species: Rabbit
			</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Secondary Antibody Table</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Alexa&#160;&#160; 568</td>
			<td rowspan="1" style="width:95px;">Invitrogen</td>
			<td rowspan="1" style="width:119px;">A-21124</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Alexa 488</td>
			<td rowspan="1" style="width:95px;">Invitrogen</td>
			<td rowspan="1" style="width:119px;">A-21131</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Cy5</td>
			<td rowspan="1" style="width:95px;">Jackson ImmunoResearch</td>
			<td rowspan="1" style="width:119px;">59883</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Rabbit
			</td>
		</tr>
	</tbody>
</table>

growing neural stem cells from conventional and nonconventional regions of the adult rodent brain

Des travaux récents montrent que le système nerveux central (SNC), la régénération et la tumorigenèse implique des populations de cellules souches (SC) résidant dans le cerveau adulte. Cependant, les mécanismes de ces cellules normalement quiescentes emploient pour assurer le bon fonctionnement des réseaux de neurones, ainsi que leur rôle dans la récupération de blessures et d&#39;atténuation des processus neurodégénératifs sont peu compris. Ces cellules se trouvent dans les régions appelées «niches» qui offrent un environnement maintien impliquant des signaux modulateurs à la fois le système vasculaire et le système immunitaire. L&#39;isolement, l&#39;entretien et la différenciation des CS CNS dans des conditions de culture définies qui excluent facteurs inconnus, qui les rend accessibles à un traitement par des moyens pharmacologiques ou génétiques, fournissant ainsi un aperçu de leur comportement in vivo. Nous vous proposons ici des informations détaillées sur les méthodes pour générer des cultures de Centres de SNC régions distinctes du cerveau adulte et approches pour évaluer leur dpotentiel de ifferentiation dans les neurones, les astrocytes, les oligodendrocytes et in vitro. Cette technique donne une population cellulaire homogène d&#39;une culture en monocouche qui peut être visualisée à étudier SC individuel et leur descendance. En outre, il peut être appliqué sur différents systèmes de modèles animaux et des échantillons cliniques, utilisé précédemment pour prédire des réactions de régénération dans le système nerveux adulte endommagé.

L&#39;identification de la région d&#39;intérêt à partir de laquelle la récolte de cellules souches neurales est la première étape critique et définir la quantité de temps qu&#39;il faut pour obtenir une plaque de cellules confluentes. Par exemple, la SVZ est une région neurogène classique et a une proportion relativement plus élevée de cellules souches neurales donc. Cependant, la technique présentée ici peut être utilisé avec d&#39;autres régions pas souvent reconnus comme ayant un potentiel neurogè...

Watch this Scientific Journal Video about Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain at JoVE.com

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Les cellules souches neurales récoltés dans le cerveau adulte sont de plus en plus utilisés dans des applications allant de la recherche fondamentale du développement du système nerveux à l&#39;exploration des applications cliniques potentielles en médecine régénérative. Cela rend le contrôle rigoureux des conditions d&#39;isolement et de culture utilisés pour cultiver ces cellules essentielles au son des résultats expérimentaux.

Grandir cellules souches neurales de régions classiques et non conventionnelles du cerveau adulte rongeurs

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

growing-neural-stem-cells-from-conventional-nonconventional-regions

Research

JoVE Journal

Neuroscience

12.1K vues.  University of Dresden.  Les cellules souches neurales récoltés dans le cerveau adulte sont de plus en plus utilisés dans des applications allant de la recherche fondamentale du développement du système nerveux à l'exploration des applications cliniques potentielles en médecine régénérative. Cela rend le contrôle rigoureux des conditions d'isolement et de culture utilisés pour cultiver ces cellules essentielles au son des résultats expérimentaux. 

Vidéo: Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Croissance et différenciation des cellules adultes hippocampe Arctic écureuil souches neurales au sol

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

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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 ...

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 ...

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 ...

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. ...

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

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

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

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

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

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Utilisation de Zinc synaptique histochimie pour révéler les différentes régions et les limbes dans le cerveau en développement et Adult

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

Collection de régions de cerveau de rongeur congelés pour des analyses en aval

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Livraison intra-artérielle de cellules souches neuronales au cerveau du rat et de la souris : application à l’ischémie cérébrale

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...