CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.

The authors have nothing to disclose.

Les étapes les plus critiques pour assurer la récolte et la culture d&#39;astrocytes corticaux sont 1) le bon d&#39;exciser le cortex sans prendre le tissu situé sous le corps calleux, 2) pour enlever les méninges, 3) de dissocier complètement les cellules, et 4) pour enrichir pour GFAP + astrocytes. Bien que nous utilisions mécanique (agitation) et génétiques (restriction d&#39;une recombinaison génétique avec un vecteur d&#39;Ad5GFAPCre ou l&#39;utilisation d&#39;un transgène de transformation do...

Les astrocytomes sont tumeur la plus fréquente primaire du cerveau et le glioblastome (GBM), un astrocytome de grade IV, est le sous-type le plus commun et agressif avec une survie médiane de 1,2 mois 12-15. Invasion de astrocytomes diffus, en particulier GBM, s&#39;oppose à la résection chirurgicale complète, limite l&#39;efficacité des traitements adjuvants, et conduit inévitablement à un post-traitement récidive 3. Les patients initialement présents soit avec de novo (primaire) GBM ou l&#39;astrocytome de grade inférieur qui progresse inévitablement (secondaire) GBM 4. GBM est hétérogène sur le plan génomique et caractérisé par des mutations qui s&#39;excluent mutuellement et concomitants dans trois gènes qui régissent les voies de signalisation: la base G 1 / S (Rb) de point de contrôle du cycle cellulaire, le récepteur tyrosine kinase (RTK), et TP53 cheminements de 5-7. GBM est composée de quatre sous-types génomiques avec des profils d&#39;expression distincts qui ressemblent à différents types de cellules du cerveau, suggérant que le sous-type GBM est influtionnés par sa cellule d&#39;origine 6,8,9. Modèles d&#39;astrocytome de meilleure qualité sont nécessaires pour définir le rôle des combinaisons spécifiques de mutations dans certains types cellulaires au cours de l&#39;astrocytome pathogenèse. S&#39;appuyant sur ces modèles pour le développement préclinique de médicaments plus efficaces en fin de compte contribuer à améliorer les résultats des patients. Modèles d&#39;astrocytome actuelles comprennent des lignées cellulaires humaines, dérivées patient xénogreffes (PDX), les astrocytes humains normaux génétiquement modifiés et les cellules souches neurales (CSN) et des souris génétiquement modifiées (GEM) 10-14. Nous avons développé une solution de rechange, GEM non germinale (nGEM) modèle 15 utilisant des cellules cérébrales primaires - astrocytes corticaux et NSC - récoltées à partir de GEM abritant diverses combinaisons d&#39;allèles oncogènes Floxé. L&#39;objectif était de produire des modèles d&#39;astrocytome avec des cellules génétiquement définies que l&#39;on pourrait qualifier phénotypique à la fois in vitro et in vivo et potentiellement utilisés pour le développement préclinique de médicaments en isouris mmune-compétentes. Lignées établies de cellules humaines sont le modèle le plus couramment utilisé de l&#39;astrocytome pathogenèse et la réponse aux médicaments in vitro et in vivo. Ils sont techniquement simple, largement disponibles, et ont défini la cinétique et la tumorigénicité sur la xénogreffe orthotopique chez des souris immunodéficientes 10,11,16-18 . Leurs inconvénients comprennent l&#39;incapacité à générer des lignées cellulaires établies à partir des astrocytomes de bas grade, ce qui limite l&#39;étude que pour les astrocytomes de haut grade; l&#39;absence d&#39;une cellule définie d&#39;origine; la présence d&#39;anomalies génomiques complexes, souvent avec des profils génomiques qui diffèrent nettement de l&#39;échantillon du patient d&#39;origine; et la susceptibilité à phénotypique et génotypique dérive pendant une série de culture dans le sérum 11,17,19-22. Les conséquences phénotypiques de mutations oncogéniques individuels dans des lignées cellulaires de glioblastome humaines établies peuvent être masquées par la multitude des anomalies qui sont effectivement présents, ce qui empêche souvent elucdation des conséquences directes génotype-phénotype. PDX sont générés par le passage sous-cutanée de cellules d&#39;astrocytome patient isolé dans des souris immunodéficientes ou par leur culture sous forme de sphéroïdes non-adhérentes dans un milieu défini sans sérum avant l&#39;injection orthotopique dans le cerveau de souris immunodéficientes 12,23. PDX conserver avec plus de précision le paysage génomique d&#39;astrocytomes humains, mais proche de lignées établies de cellules humaines, l&#39;effet phénotypique de mutations oncogéniques individuels peuvent être masquées en raison de leur complexité génomique 19,24. Pour définir les conséquences phénotypiques de mutations oncogéniques spécifiques, en particulier en réponse à de nouvelles thérapies, les panneaux de lignées cellulaires humaines établies ou PDX sont fréquemment utilisées pour établir des corrélations génotype-phénotype, montrer généralisation, et minimiser la probabilité d&#39;effets spécifiques aux lignes cellulaires. Alors que PDX récapituler avec précision les caractéristiques histopathologiques de l&#39;astrocytome humain, incLUDING invasion, xénogreffes orthotopiques de lignées établies de cellules humaines font généralement pas 21,23,25. Par ailleurs, les astrocytes humains normaux et NSC ont été modifiés par génie génétique des mutations oncogéniques définis pour modéliser astrocytome la tumorigenèse in vitro et in vivo 13,14,26. Ces cellules n&#39;ont pas la complexité du génome de lignées cellulaires humaines établies et PDX et récapitulent précision histopathologie astrocytome humain, mais nécessitent xénogreffe chez les rongeurs immunodéprimés in vivo. Parce que tous les modèles de cellules humaines nécessitent hôtes rongeurs immunodéprimés pour prévenir le rejet de xénogreffe à médiation immunitaire, ces modèles ne permettent pas de récapituler les interactions tumeur-stroma natif d&#39;un système syngénique et manquent d&#39;un système immunitaire intact, ce qui limite investigation préclinique d&#39;immuno-modulatrice du stroma ciblée et thérapies 10,11. GEM examen du permis de les conséquences phénotypiques de combinaisons prédéterminées de muta oncogènetions in vivo au cours de la tumorigenèse in situ. Considérant que GEM non conditionnelle ont des mutations dans tous les tissus au cours du développement, GEM conditionnelle ont floxé allèles oncogènes qui permettent le ciblage des mutations en limitant la recombinaison médiée par Cre à des types spécifiques de cellules grâce à l&#39;utilisation de cellules promoteurs spécifiques de type 10,11,15,18. Astrocytome GEM conditionnelle ont été utilisés pour élucider les rôles fonctionnels des mutations oncogéniques dans des types cellulaires distincts au sein d&#39;un cerveau intact 11. L&#39;utilitaire préclinique de gliomagenèse in situ en utilisant GEM conditionnelle est limitée par un certain nombre de facteurs, y compris 1) l&#39;absence d&#39;une corrélation in vitro, 2) la difficulté à générer de grandes cohortes de souris avec des génotypes complexes, 3) longue période de latence du développement tumoral in situ , 4) et stochastique progression tumorale. Parce que la tumorigenèse in situ manque un modèle in vitro correspondant, le test de médicament in vitro ne peut être réalisée avecmodèles conditionnels classiques vec GEM. Contrairement à d&#39;autres cancers, les modèles GEM conditionnelles des astrocytomes sont rarement induites par des mutations oncogéniques individuelles 11. Ainsi, les programmes de sélection complexes sont nécessaires pour produire GEM conditionnelle avec de multiples mutations oncogéniques. En outre, l&#39;astrocytome initiation se produit avec une pénétrance variable après une longue période de latence dans ces modèles, alors que la progression de l&#39;astrocytome de haut grade se produit généralement dans un non-uniforme, stochastique manière et donne finalement naissance à des tumeurs avec des paysages génomiques complexes et de la cinétique de croissance rapide 27, 28. La pénétrance variable et la nature stochastique de la progression maligne dans les modèles GEM conditionnelles exige que les souris individuelles seront examinées par imagerie radiographique pour détecter la présence et l&#39;emplacement des astrocytomes de haut grade avant leur inscription dans les essais de médicaments précliniques. Pris ensemble, ces limites entravent la production et les essais des grandes cohortes de GEM conditionnelle requis pour prdépistage des drogues eClinique. Le système de GEM RCAS-tva, qui utilise rétroviraux aviaires (CR) des vecteurs d&#39;infecter GEM modifié pour exprimer le récepteur viral (tva) sur des types spécifiques de cellules neurales, a été largement utilisé pour modéliser l&#39;astrocytome 11 tumorigenèse. Contrairement à GEM conditionnelle, ce système modèle permet l&#39;introduction de multiples mutations oncogènes des types de cellules spécifiques sans l&#39;obligation pour les programmes de sélection complexes. Toutefois, il est limité par une pénétrance variable, l&#39;exigence de cellules en division active pour réaliser l&#39;intégration virale, et l&#39;insertion aléatoire de transgènes dans le génome de l&#39;hôte 29. Non germinales GEM (nGEM) modèles, qui utilisent les cellules récoltées à partir de GEM, sont de plus en plus important car ils surmonter bon nombre des limitations d&#39;autres systèmes modèles 15. Le rôle d&#39;initiateur de type cellulaire et des mutations concomitants dans l&#39;astrocytome pathogenèse sont difficiles à dissuaderla mine en utilisant des lignées de cellules humaines de glioblastome ou PDX parce qu&#39;ils sont issus de tumeurs en phase terminale qui ont accumulé de vastes mutations génétiques dans des types cellulaires définis au cours de la progression maligne. En revanche, toutes les qualités de astrocytomes peuvent être modélisés à l&#39;aide nGEM en induisant défini mutations génétiques dans les types de cellules du cerveau purifiés spécifiques 11,30. Ainsi, l&#39;influence de mutations génétiques spécifiques et le type de cellule sur des phénotypes cellulaires et moléculaires peut être déterminée in vitro et in vivo. Similaires à des lignées de cellules de glioblastome humaines établies, le test initial de médicament in vitro à l&#39;aide nGEM peut être utilisé pour établir des priorités des médicaments pour les essais in vivo en utilisant les mêmes cellules. Tumorigenèse in vivo peut être déterminée par allogreffe de cellules nGEM orthotopique dans le cerveau de la même portée syngéniques immunocompétentes 30. Ces modèles d&#39;allogreffe orthotopique permettent donc de tester in vivo non seulement de classique cytotoxique unee thérapies ciblées, mais immuno-modulateur et les thérapies ciblées stroma ainsi. Enfin, le rôle du micro-environnement sur l&#39;initiation et la progression de la tumeur peut être déterminée par la comparaison des résultats entre les modèles et nGEM GEM classiques en utilisant les mêmes mutations dans les mêmes types de cellules. Nous et d&#39;autres avons développé astrocytome nGEM utilisant des cellules primaires - les astrocytes, NSC, ou de cellules précurseurs d&#39;oligodendrocytes (OPC) - récoltées à partir de GEM 30-34. La raison d&#39;être de l&#39;élaboration d&#39;un astrocytome nGEM était de créer un modèle pour déterminer les conséquences phénotypiques de mutations oncogéniques dans des types cellulaires spécifiques qui pourraient être utilisés pour les essais préclinique in vitro et in vivo chez l&#39;animal immuno-compétentes. Nous avons récolté les astrocytes phénotype WT corticales et NSC à partir du non-Cre exprimant, GEM conditionnelle maintenu sur un&gt; 94% C57 / BL6 fond avec la voie RB floxé - Rb1 loxP / loxP, ou TgGZT121 - et Floxé RTK / RAS / PI3K voie - Nf1 loxP / loxP, Kras G12D, PTEN loxP / loxP - gènes dans diverses combinaisons 35-39. Nous avons provoqué la recombinaison génétique in vitro en utilisant des vecteurs adénoviraux codant pour la recombinase Cre. Étant donné que les récoltes d&#39;astrocytes corticaux contiennent un mélange de types de cellules, nous avons utilisé des vecteurs ou des transgènes Ad5GFAPCre oncogènes dominants, tels que TgGZT 121 entraîné par le promoteur de la GFAP humain, pour enrichir GFAP pour les astrocytes corticaux + dans ces cultures. Nous avons défini les conséquences phénotypiques de G 1 / S (Rb), MAPK, et les mutations de la voie de la PI3K dans les astrocytes corticaux et NSC in vitro et in vivo. MAPK et PI3K activée par voie G1 / S défectueux astrocytes moléculaire imitée de glioblastome humain proneural et, lors de l&#39;injection orthotopique, tumeurs formées dans un endroit pré-défini avec une cinétique de croissance uniformes, des latences courtes, et la histopathologiquescaractéristiques al de GBM humain 30. Surveillance longitudinale de la croissance tumorale in vivo facilite le dépistage des drogues à travers la normalisation préclinique de cohortes de traitement et d&#39;analyse quantitative de la croissance de la tumeur en réponse à un traitement 40. Nous avons déterminé la cinétique de croissance de la tumeur par imagerie par bioluminescence longitudinal de souris injectées avec les astrocytes corticaux exprimant la luciférase. Par conséquent, les astrocytes corticaux et NSC dérivées de GEM conditionnelle fournissent un système de modèle souple pour la définition des conséquences fonctionnelles des mutations d&#39;astrocytome-associé et un système de modèle potentiel pour le développement préclinique de médicaments.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:233px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">11995065</td>
			<td style="width:233px;">DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Fetal Bovine Serum, Regular</td>
			<td style="width:191px;">Cellgro</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">15140122</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">08-395</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Cartilage Thumb Forceps, Curved</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">1631</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4</td>
			<td style="width:191px;">Miltex</td>
			<td style="width:119px;">17-301</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Ethanol (200 proof)</td>
			<td style="width:191px;">Decon Labs</td>
			<td style="width:119px;">2710</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Razor Blades</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14175-095</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">TrypLE Express (1X), Phenol Red</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">12605-010</td>
			<td style="width:233px;">Other Trypsin solutions are also suitable for cortical astrocyte havest and culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">CULTURE DISH, 60 x 15 mm</td>
			<td style="width:191px;">Thomas Scientific</td>
			<td style="width:119px;">9380H77</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">15 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">50 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352070</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Adenovirus stock</td>
			<td style="width:191px;">Gene Transfer Vector Core, U. Iowa</td>
			<td style="width:119px;">Ad5CMVCre</td>
			<td style="width:233px;">Store in 5 &#181;l aliquots at -80 C. Hazardous.&#160; Use Bsl2 safetyy&#160; precautions</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium sulfate (Na2SO4)&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">238597</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Potassium sulfate (K2SO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">221325</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium chloride (MgCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">HEPES potassium salt</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0527</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">D-(+)- Glucose</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Phenol Red</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3532</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Sodium hydroxide (NaOH)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Papain</td>
			<td style="width:191px;">Worthington</td>
			<td style="width:119px;">LS003127</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">L-Cysteine-HCl</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1276</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Syringe filter (0.22 &#181;m pore size)</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLGP033NS</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:241px;">Neurocult proliferation kit, mouse</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5702</td>
			<td style="width:233px;">This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">0.2% Heparin solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">7980</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PMG8041</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">bFGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (10X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">E-64</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3132</td>
			<td style="width:233px;">Make 10 mM stock in DMSO, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">6-well plates</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-83</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Cell strainer (40 &#181;m pore size)</td>
			<td style="width:191px;">Corning</td>
			<td style="width:119px;">352340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Stem cell dissociation solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5707</td>
			<td style="width:233px;">Alternatively, use gentle enzyme solutions such as Accutase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Methyl cellulose 15 cP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7027</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dulbecco&#39;s Modified Eagle&#39;s Media 2X</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLM-202-B</td>
			<td style="width:233px;">For making 5% methyl cellulose solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">1.7mL Snap Cap Microcentrifuge Tube</td>
			<td style="width:191px;">Corning</td>
			<td align="right" style="width:119px;">3620</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hamilton syringe, 250 &#181;l LT no needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-815-92</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">PB600-1 Antigen Dispenser</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:119px;">&#160;83700</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Disposable 18 ga needles&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9015638&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">27 ga 1/2&#34; luer tip needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-48</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">T48402</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Betadine</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9386574</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Puralube Opthalmic Ointment</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9689910</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">&#160;Model 900 Stereotaxic frame</td>
			<td style="width:191px;">Kopf Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">VETBOND</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9259532&#160;</td>
			<td style="width:233px;">Tissue adhesive&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Lidocaine&#160;</td>
			<td style="width:191px;">ShopMedVet</td>
			<td style="width:119px;">RXLIDO-EPI</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;">G3580</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Anti-Sox2</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">AB5603</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)</td>
			<td style="width:191px;">Dako</td>
			<td style="width:119px;">Z0334&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">IVIS Kinetic</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td style="width:233px;">For in vivo imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">122796</td>
			<td style="width:233px;">For in vivo bioluminescence imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EdU Imaging Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">C10340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">MSCV Luciferase PGK-hygro</td>
			<td style="width:191px;">Addgene</td>
			<td style="width:119px;">18782</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Nous avons développé un système modèle nGEM avec des astrocytes corticaux et NSC récoltées à partir néonatale GEM l&#39;hébergement floxé allèles oncogènes conditionnels qui peuvent être caractérisées phénotypiquement in vitro et in vivo (figure 1). Afin d&#39;étudier les conséquences de mutations oncogéniques spécifiquement dans les astrocytes corticaux in vitro, il est essentiel de d&#39;abord enrichir pour les astrocytes. Récoltes astrocytes corticaux c...

Watch this Scientific Journal Video about Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice at JoVE.co

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modélisation astrocytome pathogenèse<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em&gt; Utilisation corticale astrocytes ou cellules souches neurales à partir de, souris génétiquement modifiées conditionnelle

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

modeling-astrocytoma-pathogenesis-vitro-vivo-using-cortical

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JoVE Journal

Neuroscience

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

13.3K vues.  University of North Carolina School of Medicine. Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Vidéo: Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Génération de cellules souches neurales à partir de tissus humains mis au rebut corticale fœtale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Recherche

Neurosciences

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Modélisation Neural signalisation immunitaire de migraine épisodique et chronique utilisant Dépression épandage In Vitro</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Utilisation de neuro-imagerie fonctionnelle par ultrasons rupture de la barrière hémato-encéphalique et le manganèse-IRM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

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

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

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

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

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

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

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

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

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

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo Imagerie biphotonique d&#39;expérience qui dépendent des changements moléculaires dans les neurones corticaux

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imagerie intracellulaire de Ca<sup&gt; 2+</sup&gt; Signaux dans Striatale astrocytes de souris adultes utilisant des indicateurs de calcium génétiquement codés

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

La détection de 5-Hydroxymethylcytosine dans les cellules souches neurales et le cerveau des souris

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Différenciation neuronale des cellules souches embryonnaires de souris in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Modélisation in vitro pour les maladies neurologiques à l’aide de la conversion directe de fibroblastes en cellules progénitrices neuronales et différenciation en astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...