Questo lavoro è stato finanziato (in parte) dal Helmholtz Alliance ICEMED - Imaging e Malattie Metaboliche polimerizzazione ambientale, attraverso il Fondo Initiative e la rete dell&#39;Associazione Helmholtz, una sovvenzione dalla Else Kroener-Fresenius Foundation, e un finanziamento della Deutsche Forschungsgemeinschaft ( SFB 655: cellule in tessuti).

Il lavoro aderisce alla Dichiarazione di Helsinski e l&#39;animale dichiarazione ARVO. Gli animali sono stati impiegati per la raccolta dei tessuti e tutti i metodi pertinenti sono state seguite secondo le istruzioni del stabulario dell&#39;Università di Dresda. Gli animali sono stati maneggiati e custoditi secondo le linee guida federali tedesche per l&#39;uso e la cura degli animali da laboratorio, e lo studio è stato approvato dal Landesdirektion Dresda. Si prega di consultare la politica veterinaria del proprio istituto (IACUC o l&#39;altro bordo) per quanto riguarda una metodologia appropriata e l&#39;eutanasia. Azione specifica e le concentrazioni di lavoro dei reagenti possono essere trovati nelle tabelle reagenti che accompagnano questo protocollo. 1. Culture Dish Preparazione <ol><li> Piatti cappotto con un volume sufficiente di 75 ug / ml di poli-L-ornitina (cioè 2 ml / pozzetto di una piastra da 6 pozzetti) durante la notte in incubatrice coltura cellulare 2 giorni prima della data dissezione programmata. </li><li&gt; Aspirare il poli-L-ornitina giorno lavare i pozzetti 2x con 3 ml di PBS per 5 min seguente. </li><li> Rimuovere il lavaggio finale, quindi aggiungere la fibronectina (diluito 1:250, 4 mg / ml in PBS) alla piastra e posizionare i piatti in incubatrice durante la notte. </li><li> Aspirare il fibronectina il giorno della dissezione e lavare i piatti 2x con PBS. Riportare le piastre ancora contenenti la finale PBS lavare per l&#39;incubatore fino a quando il tessuto dissociato è pronto per la coltura. A quel tempo, togliere la PBS prima di placcatura del tessuto. </li><li> Conservare le piastre rivestite con poli-L-ornithinein l&#39;incubatrice per un massimo di 3 settimane. Piastre rivestite con fibronectina possono essere conservati per un massimo di 1 settimana. Fibronectina rivestimento può essere fatto in meno di 2 ore se urgente. La fibronectina è suscettibile di denaturazione, non agitare la soluzione o lasciare asciugatura delle stoviglie. </li></ol> 2. Dissection / placcatura <ol><li> Questo protocollo si applica all&#39;uso di 3 ratti adulti (3-6 mesi di età). </li><li> Chill cervello di sezionamento blocco di ghiaccio prima di eutanasia ratto. </li><li> Posizionare un tubo Falcon da 15 ml che contiene 5 ml di N2 media con aggiunta di bFGF sul ghiaccio. </li><li> Euthanize i ratti in base alla politica veterinaria dell&#39;istituto. </li><li> Rimuovere il cervello dal cranio facendo un&#39;incisione lungo la linea mediana della testa. Utilizzare pinze a staccarsi l&#39;osso permettendo al cervello di essere attentamente estratto. </li><li> Mettere il cervello in un piatto di 10 centimetri di Petri contenente ghiacciata PBS. Questo consente di eliminare ogni residuo di capelli e di sangue. (Figura 1A) </li><li> Mettere il cervello nel blocco sezionamento refrigerata. (Figura 1B) </li><li> Inserire una nuova lama di rasoio pulito nel blocco, come illustrato nella figura 1C. </li><li> Inserire una seconda lama di rasoio pulito 3 millimetri caudale al primo, come illustrato nella Figura 1D. </li><li> Sollevare delicatamente le lame dal blocco, portando con loro la sezione di interesse. </li><li> Float l&#39;secZIONE fuori la lama immergendolo in PBS. (Figura 1E) </li><li> Tessuto raccolto dalla zona di interesse (in questo esempio abbiamo usato commessura anteriore, (anteriore) ACA, Figura 1F) utilizzando # 5 pinze raccogli in una provetta conica da 15 ml contenente 1 ml di mezzo contenente N2 bFGF. </li><li> Spostare il tubo in una cappa a flusso laminare coltura cellulare, continuando il resto del protocollo in condizioni sterili. </li><li> Meccanicamente dissociare il tessuto con una punta di pipetta 1 ml collegato a una pipetta P1000. Pipetta su e giù diverse volte (circa 20x ad una velocità di 1 ciclo di pipettaggio al secondo) mentre si preme l&#39;apertura della punta della pipetta contro il fondo della provetta conica (figure 1-3) 1G. Smettere di triturazione quando la soluzione diventa omogenea. </li><li> Lasciare agire la soluzione per 2 minuti per consentire qualsiasi tessuto nondissociated più grande per depositarsi sul fondo. (Figura 1G 4) </li><li> Raccogliere il homogendell&#39;EOUS surnatante e posto in una provetta contenente 8,5 ml di mezzi N2 più 20 ng / ml bFGF, 500 ng / ml Dll4, 500 ng / ml Ang2 e 200 nMJAK inibitore. </li><li> Li piastra in 3 pozzetti di una piastra da 6 pozzetti con 3 ml di soluzione diluita di cellule / e. </li><li> Porre la piastra in un incubatore umidificato a cella 37 ° C, 5% CO 2, 5% O 2. </li></ol> 3. Manutenzione giornaliera <ol><li> Sostituire il supporto sulle piastre di coltura con N2 Piano contenenti bFGF, Ang2, Dll4, e JAK inibitore 24 ore dopo la placcatura. </li><li> Aggiungere un bolo di bFGF, Dll4, Ang2, e l&#39;inibitore JAK il giorno successivo. </li><li> Continuare questo processo alternato di cambiamenti dei media e integrazioni fattore per un totale di 10-14 giorni. </li></ol> 4. Induzione di differenziazione <ol><li> Ritirare bFGF, Dll4, Ang2, e JAK inibitore contenente il mezzo e sostituirlo con mezzo N2 che non contiene elementi aggiuntivi. </li><li> Cambia il mezzo ogni secondo giorno. </li><li> Fissare le cellule dopo 10 giorni ed effettuare immunocitochimica. </li></ol> 5. Rilevamento immunocitochimico <ol><li> Aspirare il mezzo e il fissaggio delle piastre di coltura cellulare con 2 ml di paraformaldeide al 4% per 20 min. </li><li> Lavare 2x con 3 ml di PBS per 5 min ciascuno. </li><li> Aspirare il PBS, poi permeabilize e bloccare le cellule aggiungendo 2 ml di PBS contenente 5% di siero normale asino (NDS) e 0,1% Triton X-100 per 20 min. </li><li> Preparare la miscela di anticorpo primario in PBS contenente 5% NDS. Anticorpo anti-nestin può essere utilizzato per identificare SC, mentre TUJ1 (classe III β-tubulina), anticorpi GFAP, e CNPase possono essere utilizzati insieme per una colorazione tripla di marcatori di differenziazione. </li><li> Aspirare la soluzione di saturazione e aggiungere 1,5 ml della miscela anticorpo primario a ciascun pozzetto. Incubare a temperatura ambiente per 90 min. </li><li> Rimuovere gli anticorpi primari e lavare 2x con 3 ml di PBS, e 1x con 3 ml di PBS contenente 5% NDS per 5 minuti ciascuna. </li><li> Prepae gli anticorpi secondari in PBS contenente 5% NDS. </li><li> Aspirare il lavaggio finale e aggiungere 1,5 ml di soluzione di anticorpo secondario in ciascun pozzetto. Incubare al buio per 40 minuti a temperatura ambiente. </li><li> Rimuovere la soluzione di anticorpo secondario e aggiungere 2 ml di una macchia DAPI preparata (500 ng / ml in PBS). Incubare per 3 min. </li><li> Aspirare la soluzione DAPI poi lavare 3x con 3 ml di PBS per 5 minuti ciascuna. Le cellule possono essere visualizzate con un microscopio a fluorescenza. </li></ol>

<ol>
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Molti dei passaggi critici all&#39;isolamento successo, l&#39;espansione e la differenziazione delle cellule staminali neurali dal cervello adulto sono condivise in comune con le tecniche di coltura di tessuti standard. L&#39;obiettivo da tenere a mente è che le cellule staminali neurali isolate dal cervello adulto dovrebbe mantenere come molti dei loro caratteristiche in vivo come possibile (Figura 2J). Spesso un equilibrio tra la necessaria prudenza e velocità adeguata deve essere colpito. ...

Il dogma centrale della neurobiologia, stabilito dalle osservazioni fondamentali del citoarchitettura cervello fatto da Ramón Y. Cajal oltre un secolo fa, ha dichiarato che la neurogenesi è improbabile dopo l&#39;adolescenza data la complessità delle reti neurali presenti nel sistema nervoso centrale 1. Nonostante il lavoro di Altman nel 1960, e successivamente Kaplan, dimostrando che 3 H-timidina è stato trovato in neuroni maturi che indicano che in realtà neuroni venivano generati in zone distinte del cervello adulto, il dogma continuato a tenere 2, 3. La prova ha continuato a montare con la ricerca di Nottebohm descrivere i cambiamenti stagionali nel numero di neuroni presenti nel cervello Songbird 4. Non è stato fino al 1999, quando Gould et al. Lavoro pubblicato sulla generazione di neuroni nell&#39;ippocampo aumenta con l&#39;esecuzione di compiti di apprendimento associativo nel ratto, nonché le osservazioni di Kornack e Rakic ​​dimostrazioneting continuato neurogenesi nel macaco adulto che il concetto di una meno rigida, cervello più plastico, è stato riconosciuto 5, 6. La ricerca della sorgente cellulare di questi neuroni de novo generati portato alla scoperta di una popolazione discreta di cellule staminali (SC) che risiedono in aree del cervello denominato nicchie 7. La zona subventricular e sub zona granulari dell&#39;ippocampo sono considerate le due principali regioni neurogenici 8,9. Le cellule isolate da questi luoghi mostrano la classica caratteristica del SCs derivate embrionali o fetali, self-renewal e potenziale di differenziazione. Nel caso di cellule staminali neurali (NSC), possono essere differenziati in neuroni, astrociti, oligodendrociti e. Inoltre, questi SC macchia positivo per fetali marcatori NSC come il filamento intermedio proteina nestin 10. Lavori più recenti sottolinea che SC potrebbero non essere limitate a queste two aree, e sono infatti localizzati in tutto il cervello come una popolazione in gran parte quiescente di cellule strettamente associati al sistema vascolare 11. Le osservazioni che la SCS sono mobilitati in risposta al danno suggerisce la possibilità di essere in grado di utilizzare queste cellule per scopi rigenerativi per aiutare nel recupero dalla malattia neurodegenerativa e ictus 12, 13. Questo non è dissimile il ruolo che le cellule staminali mesenchimali (MSC) svolgono nella guarigione dei tessuti connettivi, che si trovano come cellule perivascolari che hanno il potenziale di diventare osteoblasti, condrociti e cellule adipose 14. Tuttavia, NSC non possono essere raccolti nello stesso modo come MSC da midollo osseo di aspirazione routine e densità gradiente tecniche di centrifugazione e successivamente utilizzati in terapie cellulari basate autologhe. Di conseguenza, altre fonti di cellule, come l&#39;uso di cellule staminali neurali fetali e precursori neuronali derivate da embLe cellule staminali ryonic sono state ampiamente esplorate in malattie animali e modelli di pregiudizio con vari gradi di successo 15. Tecnologie di cellule staminali pluripotenti indotte che utilizzano fonti di cellule somatiche offrono un&#39;altra viale potenziale per la produzione di terapie cellulari terapeuticamente utili per una vasta gamma di applicazioni, superando la limitata disponibilità e preoccupazioni etiche riguardanti l&#39;uso di cellule embrionali e tessuti fetali 16. Tuttavia, la traduzione clinica di questi risultati ha dimostrato di essere un compito difficile, come dimostrato nelle lotte di trattamento di varie condizioni neurologiche con terapeutico basato-SC approcci 17,18, così come un percorso tortuoso di gioco regolamentare. Come approccio alternativo, l&#39;introduzione di specifici trattamenti farmacologici in grado di modulare i numeri NSC e facilitare il recupero in modelli di malattia di Parkinson e ictus 19. Qualunque sia la strategia potrebbe essere, capire come manipolare efficacemente queste cellulerichiede un sistema accessibile in vitro. Culture del NSC possono essere eseguite sia come culture aggregati, noto anche come neurosfere, o come un monostrato 8,20. Entrambe le tecniche sono stati ampiamente utilizzati, permettendo la creazione di condizioni di coltura definiti, cioè uso di Epidermal Growth Factor (EGF) o fattore fondamentale di crescita dei fibroblasti (bFGF) come fonte mitogeno, che prevedono l&#39;espansione di precursori multipotenti. Mentre colture di neurosfere può essere più adatto per studiare capacità propagazione clonale di un tipo di cellula isolata, il sistema ha dimostrato di produrre una popolazione mista di cellule durante l&#39;espansione 21. Inoltre, la struttura chiusa di neurosfere rende manipolazione farmacologica delle cellule impraticabile, e l&#39;interpretazione dell&#39;influenza questi fattori possono avere potrebbe essere confuso a causa del microambiente stabilito nella neurosfere stesso. Colture monostrato, d&#39;altro canto, possono essereimpiegato in schermi ad alta produttività di librerie di piccole molecole, fornendo un potente strumento per esplorare i meccanismi di trasduzione del segnale che regolano la crescita e la differenziazione SC e si apre la possibilità di scoprire nuovi composti che colpiscono specificamente questa popolazione di cellule. Di conseguenza, la capacità di generare riproducibile colture di NSC adulte provenienti da diverse regioni di interesse nel cervello può essere utilizzato in una vasta gamma di applicazioni di ricerca comprendono studi di sviluppo del sistema nervoso centrale (CNS) per esplorare nuovi approcci di medicina rigenerativa . Il protocollo presentato qui dimostra come sezionare e valutare il potenziale di differenziazione della SC SNC isolate dal cervello di roditori adulti.

<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

Un recente lavoro dimostra che il sistema nervoso centrale (SNC) rigenerazione e tumorigenesi coinvolge popolazioni di cellule staminali (SCS) residenti all&#39;interno del cervello adulto. Tuttavia, i meccanismi di queste cellule normalmente quiescenti impiegano per assicurare il corretto funzionamento delle reti neurali, così come il loro ruolo nel recupero da un infortunio e la mitigazione dei processi neurodegenerativi sono poco capito. Queste cellule risiedono nelle regioni denominate &quot;nicchie&quot; che forniscono un ambiente di sostegno che coinvolgono i segnali modulanti sia dal vascolare e immunitario. L&#39;isolamento, la manutenzione e la differenziazione di CS del sistema nervoso centrale in condizioni di coltura definite che escludono fattori sconosciuti, li rende accessibili al trattamento con mezzi farmacologici o genetici, fornendo così spaccato il loro comportamento in vivo. Qui offriamo informazioni dettagliate sui metodi per la generazione di colture di SC SNC da regioni distinte del cervello adulto e approcci per valutare la loro dpotenziale ifferentiation in neuroni, astrociti, oligodendrociti e in vitro. Questa tecnica produce una popolazione omogenea di cellule come una coltura monostrato che può essere visualizzato per studiare individuale SC e la loro progenie. Inoltre, può essere applicato a diversi sistemi modello animale e campioni clinici, utilizzato in precedenza per prevedere le risposte di rigenerazione nel sistema nervoso adulto danneggiato.

Identificare la regione di interesse che per raccogliere le cellule staminali neurali è il primo punto critico e definirà la quantità di tempo necessario per ottenere un piatto confluenti di cellule. Ad esempio, la SVZ è una zona neurogena classico e ha quindi una relativa proporzione elevata di cellule staminali neurali. Tuttavia, la tecnica qui presentata può essere utilizzato con altre regioni spesso non riconosciuti come aventi un potenziale neurogena robusto nell&#39;età adulta. Ai fini del presente protocoll...

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

Cellule staminali neurali raccolte dal cervello adulto sono in aumento utilizzati in applicazioni che vanno dalla ricerca di base dello sviluppo del sistema nervoso ad esplorare potenziali applicazioni cliniche in medicina rigenerativa. Questo rende rigoroso controllo delle condizioni di isolamento e di coltura utilizzate per coltivare queste cellule fondamentali per suonare i risultati sperimentali.

Crescere cellule staminali neurali da Regioni convenzionali e non convenzionali del cervello adulto Roditore

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 Views.  University of Dresden.  Cellule staminali neurali raccolte dal cervello adulto sono in aumento utilizzati in applicazioni che vanno dalla ricerca di base dello sviluppo del sistema nervoso ad esplorare potenziali applicazioni cliniche in medicina rigenerativa. Questo rende rigoroso controllo delle condizioni di isolamento e di coltura utilizzate per coltivare queste cellule fondamentali per suonare i risultati sperimentali. 

Video: 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.

Crescita e la differenziazione degli adulti ippocampale suolo artico Cellule Staminali neurali Squirrel

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

Ricerca

Neuroscienze

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.

Isolamento ed espansione della cellule staminali neurali del mouse utilizzando il saggio neurosfere

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.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

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.

Un mouse, due culture: isolamento e la coltura di Adult Neural Stem Cells dalle due zone neurogena Mice individuale

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.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

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.

Ordinamento delle cellule staminali neurali e di cellule progenitrici del topo adulto subventricolare Zone e Live-imaging loro dinamiche del ciclo cellulare

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.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

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.

Uso di zinco sinaptica istochimica per rivelare diverse regioni e lamine nel cervello in via di sviluppo ed adulto

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.

Raccolta di regioni cerebrali del roditore congelato per analisi a valle

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

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.