This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

1. Preparazione dei Materiali e terreno di coltura <ol><li> Per la dissezione e la dissociazione del SCZ, avvolgere la matrice del cervello, lama a doppio taglio di rasoio, e pinze con un foglio di alluminio, e poi sterilizzare in autoclave. </li><li> Preparare 50 ml di tampone PBS freddo per lavare l&#39;intero cervello di topo. </li><li> Impostare un microscopio dissezione e preparare gli strumenti chirurgici necessari per la dissezione del cervello (forbici e pinze in autoclave) e l&#39;isolamento del (ml siringa da 1, 30 ago G, matrice del cervello, e una pinza sottile) SCZ. </li><li> Preparazione di N2 Media: <ol><li> Preparare F-12 / DMEM (+ L-Glutamin, + bicarbonato di sodio), media con 2% B27, 1% integratori N2, e l&#39;1% penicillina-streptomicina. Nota: Il mezzo di crescita è costituito da N2 medie e fattori di crescita (20 ng / mL purificata fattore di crescita epidermico (EGF) e 20 ng / mL di base del fattore di crescita dei fibroblasti (bFGF2)). </li></ol></li><li> Preparare un tampone di digestione (40 unità / ml papaina, 2,4 unità / ml dispase II, e 2% di penicillina-streptomicina in PBS) per la digestione dei tessuti. </li><li> Preparazione del piatto del rivestimento e Coverslip: <ol><li> Preparare poli-L-ornitina (OLP; 0,01%) e laminina (10 mg / ml disciolti in DH 2 O). Per cappotto 6 pozzetti per la manutenzione del SCZ-aNSCs come un monostrato o 18 coprioggetto mm per immunocolorazione, incubare con OLP notte a 4 ° C. Poi lavare 3 volte con DH 2 O. Lasciare le piastre e coprioggetti ad asciugare dopo l&#39;ultimo lavaggio. </li><li> Successivamente, incubare le piastre con laminina notte a 4 ° C. Poi, lavarli 3 volte con DH 2 O. Attenzione: Non asciugare la laminina, che interesserà l&#39;adesione cellulare. Nota: Le soluzioni di rivestimento possono essere riutilizzati per 3 volte. </li></ol></li></ol> 2. Isolamento e la dissociazione della Adult SCZ <ol><li> Prima della preparazione della cultura, posizionare la matrice del cervello e rasoio a doppio taglio sul ghiaccio. Nota: Non congelare la matrice cerebrale, perché il brain potrebbe allegare alla stessa. </li><li> Sacrifica un mouse (8 settimane) di CO 2 asfissia o dislocazione cervicale. <ol><li> Tagliare la testa con le forbici affilate dopo la spruzzatura il 70% di etanolo. Per immobilizzare la testa, tenere entrambi i lati della testa ermeticamente. Tagliare la pelle con le forbici sulla linea mediana in una direzione caudale-rostrale. Questo promuove la rimozione completa della pelle dal cranio. </li><li> Rimuovere la parte caudale del cranio (posteriore al lambda) prima, e quindi inserire la pinza tra il cranio e il cervello in posizione dorsale linea mediana. Afferra il sinistro (o destro) osso parietale e con attenzione buccia fuori. Ripetere questa procedura per la rimozione dell&#39;altro osso parietale. Nota: disconnessione del nervo ottico e la rimozione delle meningi dal cervello renderlo più facile da staccare il cervello dal cranio. </li><li> Trasferire il cervello a tampone PBS freddo (25 ml) e sciacquarlo due volte per rimuovere il sangue in eccesso. </li></ol></li><li> Posizionare il cervello nella matrice cervello su ghiaccio e make tagli coronali per ottenere 1 mm fette spesse. Trasferire le fettine cerebrali (1 mm) contenenti le regioni SCZ che si trovano nella parte posteriore del cervello ad un PBS freddo in un piatto di plastica Petri da 35 mm. Attenzione: Al fine di ottenere un piano parallelo di sezioni coronali, verificare che la fessura cervello è posizionato sulla linea mediana della matrice cerebrale; è fondamentale per evitare variazioni inutili sezioni. Nota: Ottenere fettine di cervello a 2-3 mm posteriormente al bregma; ciò consente la separazione del SCZ dalla SVZ. </li><li> Sotto il microscopio da dissezione con un basso ingrandimento, micro-sezionare il SCZ dalla zona sostanza bianca della corteccia e ippocampo con un piegato 6,9 30G ago. Quindi, rimuovere le regioni della corteccia sopra la SCZ. Posizionare la regione SCZ sezionato dalle fette in un piatto di plastica 35 millimetri di Petri sul ghiaccio senza PBS freddo. Nota: La contaminazione delle regioni in più contengono neuroni maturi potrebbe influenzare la vitalità delle aNSCs e neurosfere formazione Because essi subiscono la morte delle cellule in condizioni di coltura ANSC. </li><li> Utilizzando un piegato 30 ago G, tagliare immediatamente i tessuti sezionati in piccoli pezzi. Nota: Se è necessario un tempo più lungo per sezionare il tessuto SCZ, immergere i tessuti dissezionati in PBS freddo prima di tagliare. </li><li> Risospendere il tessuto tritato con 1 ml di tampone di digestione, trasferire il tessuto ad una provetta da 15 ml contenente 2 ml di tampone di digestione, e incubare per 30 min in un bagno d&#39;acqua a 37 ° C. Nota: Agitare il tubo ogni 10 minuti per amalgamare bene. </li></ol> 3. Subcallosal Zone-derivato Cultura adulti cellule staminali neurali <ol><li> Toccare il tubo leggermente di dissociare il tessuto digerito, e poi centrifugare la provetta a 145 g per 5 min. Eliminare il surnatante, ri-sospeso il tessuto digerito con 1 ml di terreno di pre-riscaldato N2 per lavare il buffer di digestione, e pipettare delicatamente la soluzione del campione ad un massimo di 5 volte con una pipetta P1000. Nota: Over-triturazione con una strettapipetta punta può diminuire la vitalità delle cellule e la successiva crescita. </li><li> Centrifugare la provetta a 145 xg per 5 min. Dopo aver scartato il surnatante, risospendere il pellet cellulare in 1 ml di terreno N2. </li><li> Preparare 1 ml di terreno N2 in un piatto non rivestito da 6 pozzetti e aggiungere 1 ml di cellule sospese per fare un volume finale di 2 ml. </li><li> Aggiungere EGF (20 ng / ml) e bFGF (20 ng / ml) in ogni pozzetto. Agitare delicatamente il piatto cultura 6 pozzetti a mano per mescolare i fattori di crescita aggiunti con le cellule placcato. Mantenere il 6 pozzetti in un incubatore CO 2 37 ° C e 5%. </li><li> Aggiungere EGF (20 ng / ml) e bFGF (20 ng / ml) ad ogni pozzetto ogni giorno per 8 giorni. Ogni terzo giorno, aggiungere 200 ml di mezzi di N2 per mantenere la approssimata del volume 2 ml del mezzo. </li></ol> 4. Passaging di NSC come Neurosfere e colture monostrato <ol><li> Raccogliere le neurosfere e trasferirli in un nuovo tubo da 15 ml. Nota: Il numero di neurosfere (&gt; 50 micron di diametro) per bene dalla SCZ di un singolo cervello di topo era 64,3 ± 7,31, che era inferiore a quella della SVZ (190.5 ± 6,33) 9. </li><li> Incubare le neurosfere con 0,5 ml di tampone di dissociazione per 5 min in un bagno d&#39;acqua a 37 ° C per dissociare le neurosfere in singole cellule. neurosfere primarie possono essere dissociate in singole cellule e mantenuto per diversi passaggi come neurosfere o monostrato culture. Nota: Espansione SCZ-aNSCs come un monostrato è superiore a neurosfere perché SCZ-aNSCs possono essere diversi passaggi&gt; 10 volte in un formato cultura monostrato, ma &lt;5 volte in un formato cultura neurosfere. Attenzione: SCZ-aNSCs esporre forte aggregazione, ed è difficile per loro dissociarsi in singole cellule. Così, il formato la cultura neurosfere non è raccomandato per l&#39;espansione delle cellule di routine. </li><li> pipettare delicatamente la soluzione campione su e giù con una pipetta P1000 inferiore a 5 volte e centrifugare la provetta a 145 xg per 5 min. Eliminare il surnatante e ri-Sutrascorrere le neurosfere con 1 ml di terreno N2. Nota: Nel corso triturazione con una stretta punta della pipetta può diminuire la vitalità cellulare e la successiva crescita. </li><li> Per contare le cellule, fare una miscela 1: 1 di sospensione cellulare (10 microlitri) e il trypan blue soluzione 0,4%, e poi contare il numero di cellule vive in un hematocytometer. Dopo rivestimento di un 6-pozzetti con OLP / laminina, piastra le cellule a 2,5 x 10 5 cellule / ml con 2 ml di terreno N2 per ogni bene. Nota: Le variazioni di densità delle cellule possono influenzare la loro condizione e potenziale di differenziazione. </li><li> Mantenere le SCZ-aNSCs con un trattamento giornaliero di fattori di crescita (2 ml, 20 ng / ml) per 5 giorni, e il passaggio di loro. </li></ol> 5. La differenziazione di Subcallosal Zone-derivati ​​cellule staminali adulte neurali <ol><li> ANSCs piatto sulla un&#39;OLP / laminina rivestite coprioggetto 18 mm con filetto 1 x 10 5 cellule / ml in 1 ml N2 con fattori di crescita (20 ng / ml) per la differenziazione dei SCZ-aNSCs. </li><li> Il giorno successivo,quando le cellule sono saldamente attaccati al vetrino, scambiare il mezzo di crescita con N2 per rimuovere i fattori di crescita. </li><li> Dopo 6 giorni, lavare le cellule differenziate con 1 ml di PBS per rimuovere i detriti cellulari e fissarle per immunocolorazione. Nota: BrdU può essere incorporato nel DNA di nuova sintesi di cellule proliferanti. Pertanto, se desiderato, BrdU (10 ug / ml) può essere aggiunto a vivere cellule prima del fissaggio delle cellule. </li></ol> 6. cellule staminali neurali Immunocolorazione adulti e differenziata Progenitori <ol><li> Per immunostaining, lavare le cellule con PBS e fissarle con 4% PFA per 20 minuti a temperatura ambiente. Attenzione: PFA è altamente tossico; evitare il contatto con la pelle e gli occhi. </li><li> Rimuovere il 4% PFA, lavare le cellule fissate con PBS 3 volte, e poi conservarli a 4 ° Cuntil sono necessari per immunocolorazione. </li><li> Incubare le cellule sul vetrino con una soluzione bloccante (3% di albumina sierica bovina e 0,1% Triton X-100 in 1x PBS) per almeno 30 minuti a temperatura ambiente. </li><li> Preparare gli anticorpi primari in soluzione di blocco fresco e incubare i campioni notte a 4 ° C. <ol><li> Immunocolorazione i aNSCs <ol><li> Macchiare le aNSCs utilizzando anti-Nestin e anticorpi anti-BrdU. Prima di fissazione, aggiungere 20 mg di BrdU alle aNSCs e incubare per 2 ore. Eseguire il passo denaturazione usando 2 N HCl per 20 min a 37 ° CBefore eseguire il passo di blocco. </li></ol></li><li> Immunocolorazione i progenitori differenziati: <ol><li> Usare gli anticorpi anti-proteina silicea fibrillare gliale (GFAP) anti-O4, anti-bIII-tubulina, e per etichettare i progenitori differenziati. </li></ol></li></ol></li><li> Lavare i campioni con PBS 3 volte e incubare con anticorpi secondari coniugati ai coloranti fluorescenti (1: 500) in soluzione bloccante per 30 minuti a temperatura ambiente. Poi, lavare i campioni 3 volte con PBS. Nota: utilizzare anticorpi secondari che corrispondono i padroni di casa del primarioanticorpi. Hoechest33343 (1: 2.000) è utilizzato per la colorazione nucleare. </li><li> Aggiungere la soluzione di montaggio di un vetrino e procedere con il montaggio. Osservare e immagine campione su un microscopio confocale a più lunghezze d&#39;onda: FITC (488 nm), Cy3 (543 nm), Cy5 (647 nm), e Hoechest33343 (405 nm). </li></ol>

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The authors declare no conflicts of interest.

Questo documento descrive un protocollo dettagliato per generare NSCs dal SCZ topo adulto e mantenerli per varie applicazioni. Ci sono tre passi fondamentali per determinare lo vitro sistema di cultura necessaria per purificare ed espandere SCZ-NSC. In primo luogo, è importante assicurare che la regione SCZ è proprio sezionato da altri potenziali regioni neurogena (Figura 1B). Sezioni spesse e precisi contenenti le regioni SCZ sono stati ottenuti con una matrice cerebrale intervallo di 1 mm, e quindi un ago è stato utilizzato per il micro-dissezione del SCZ da altre regioni corticali (Figura 1A). Quando non NSC da tessuti adiacenti, come la corteccia cerebrale, sono messi in coltura con SCZ-aNSCs, si verifica la morte catastrofica, che lede la vitalità e la sfera-formazione di SCZ-NSC 16-18,22. Il caudale SCZ (2-3 mm posteriormente al bregma) è stato confermato come il migliore regione per la generazione di distinte SCZ-aNSCs. In secondo luogo, il approptrattamento enzimatico riate nei passaggi di raccolta e passaging è fondamentale per ottenere un alto rendimento di cellule. Dispasi II e papaina sono stati più efficaci per isolare aNSCs di tripsina. La dissociazione di cellule per passaging con tampone di dissociazione, invece di tripsina migliorato la loro vitalità 19. dissociazione meccanica e triturazione con una pipetta dovrebbe essere minimo. Terzo, un filtro cella viene generalmente utilizzato in sistemi di coltura primaria per rimuovere i detriti cellulari dopo la digestione tessutale. Tuttavia, a causa della localizzazione delle SCZ-aNSCs, queste cellule si ottengono dopo la rottura della sostanza bianca mediante digestione enzimatica e triturazione meccanica. Durante la filtrazione con un filtro di cellule, una notevole quantità di cellule sarebbe perso. Pertanto, la coltura SCZ-aNSCs senza utilizzare un colino cellula è un modo migliore per ottenere un alto rendimento di cellule. Mentre entrambi colture di neurosfere e colture monostrato possono essere applicati al mantenimento della NSC, uno limitazione della neurocultura sfera è che i singoli NSC dissociati dopo la divisione può essere aggregato in modo casuale. I risultati di aggregazione di diverse dimensioni di neurosfere. Quando la dimensione di un neurosfere raggiunge un certo valore critico, la neurosfere cresce come una struttura eterogenea, a causa di una mancanza di nutrienti, fattori di crescita, e ossigeno al cuore 20. Inoltre, neurosfere con grandi dimensioni non sono facilmente dissociate con tampone di dissociazione e richiedono tempi di trattamento più lungo enzimatici con una vasta triturazione meccanica, con conseguente minore vitalità cellulare. Pertanto, il sistema di coltura monostrato raccomanda di mantenere SCZ-aNSCs attraverso passaggi multipli. In un sistema di coltura monostrato, aNSCs sono stabilmente mantenuti come NSC (tipo B) senza differenziazione spontanea in cellule specifici, quali progenitori (tipo C e A) (Figura 3A). SCZ-aNSCs sono stati diversi passaggi per lunghi periodi, &lt;5 passaggi in un formato neurosfere e&gt; 10 passaggi come un monostrato. Questo è consstente con i risultati precedenti, che suggeriscono che il sistema di coltura monostrato mantiene NSC in vitro in colture a lungo termine 21. Tuttavia, la velocità proliferanti diminuita, e la porzione di cellule morenti aumentato più di 5 passaggi. Passaging estesa colpisce il multipotenza e la differenziazione neuronale con maggiore aberrazione cromosomica 22. Pertanto, per evitare effetti passaging estese, si raccomanda di utilizzare il passaggio precoce (&lt;passaggio 5) cellule per la proliferazione e la differenziazione analisi. Anche se il potenziale di auto-rinnovamento delle SCZ-aNSCs è simile a SVZ-aNSCs, la differenziazione neuronale meno è stato mostrato in SCZ-aNSCs 9. I metodi per isolare aNSCs dalle regioni neurogena del cervello adulto, compreso il SVZ e giro dentato (DG), sono stati stabiliti 22. Sebbene tali protocolli hanno promosso l&#39;isolamento e la coltura di aNSCs in vitro, esistono diverse limitazioni per ottenere un elevato numero dicellule. Molti protocolli utilizzano un chopper tessuto cerebrale che può causare la perdita di tessuto cerebrale durante la procedura di taglio. Un altro approccio per isolare aNSCs dalle regioni neurogena utilizza un taglio coronale attraverso il cervello utilizzando un bisturi. Questo è seguito dal micro-dissezione del SVZ o della DG lungo la fessura longitudinale 23. La presenza di altre regioni del cervello può causare altri tipi di cellule di contaminare la cultura ANSC, che potrebbe influenzare la vitalità delle cellule in vitro. Con il protocollo attuale, molti campioni diversi possono essere gestiti in un singolo esperimento, compresi i controlli contro una varietà di gruppi sperimentali. Inoltre, affettare utilizzando una matrice cervello è superiore ad uno strumento di taglio del cervello, in quanto consente il conseguimento di NSCs da diverse regioni del cervello con una resa elevata cellulare. Esso consente inoltre il confronto delle aNSCs provenienti da diverse regioni del cervello stesso. Trapianti e ingegnerizzazione di NSC endogeni sono stati considered come possibili strategie per la terapia con cellule staminali. Per questo, studi in vitro sulle caratteristiche di aNSCs dovrebbero essere ampiamente esplorato. Pertanto, la creazione di un ben caratterizzato sistema di coltura in vitro sarà utile per promuovere l&#39;applicazione di NSC. In questo sistema di coltura, le proprietà di cellule staminali di SCZ-aNSCs erano ben mantenute, come evidenziato da auto-rinnovamento e la differenziazione multi-lignaggio in condizioni appropriate. Pertanto, questo sistema di coltura può essere utilizzato per l&#39;espansione del SCZ-aNSCs per studi biologici e applicazioni terapeutiche.

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>+L-glutamin, +Sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td>1X dilution</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociattion buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>04-942-078-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>WPI</td>
			<td>555229F</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Storz</td>
			<td>E3321-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain matrix (1mm)</td>
			<td>RWD</td>
			<td>68707</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-edged razor</td>
			<td>DORCO</td>
			<td>ST-300</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</td>
			<td>Deckglaser</td>
			<td>111580</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well dish</td>
			<td>SPL</td>
			<td>32012</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>6 well dish</td>
			<td>SPL</td>
			<td>32006</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>Coating materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Primary antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>Abcam</td>
			<td>ab2430</td>
			<td>rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>DCX</td>
			<td>Santa Cruz</td>
			<td>SC8066</td>
			<td>goat (1:500)</td>
		</tr>
		<tr>
			<td>Tuj1</td>
			<td>Sigma</td>
			<td>T2200</td>
			<td>rabbit (1:2000)</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>rat (1:1000)</td>
		</tr>
		<tr>
			<td>O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>mouse (1:500)</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>Rat (1:500)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td rowspan="12">1:500</td>
		</tr>
		<tr>
			<td>anti-mouse cy3</td>
			<td>Jackson</td>
			<td>715-165-151</td>
		</tr>
		<tr>
			<td>anti-mouse 647</td>
			<td>Jackson</td>
			<td>715-606-150</td>
		</tr>
		<tr>
			<td>anti-rabbit 488</td>
			<td>Alexa</td>
			<td>A21206</td>
		</tr>
		<tr>
			<td>anti-rabbit cy3</td>
			<td>Jackson</td>
			<td>711-165-152</td>
		</tr>
		<tr>
			<td>anti-rabbit 647</td>
			<td>Jackson</td>
			<td>711-605-152</td>
		</tr>
		<tr>
			<td>anti-goat 488</td>
			<td>Alexa</td>
			<td>A11055</td>
		</tr>
		<tr>
			<td>anti-goat cy3</td>
			<td>Jackson</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>anti-goat 647</td>
			<td>Invitrogen</td>
			<td>A21447</td>
		</tr>
		<tr>
			<td>anti-rat 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
		</tr>
		<tr>
			<td>anti-rat cy3</td>
			<td>Jackson</td>
			<td>712-166-150</td>
		</tr>
		<tr>
			<td>anti-rat 647</td>
			<td>Jackson</td>
			<td>712-605-153</td>
		</tr>
		<tr>
			<td>Immunostaining materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Millipore</td>
			<td>82-100-6</td>
			<td>3% BSA with 0.1% Triton X-100 in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>usb</td>
			<td>22686</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Biosesang</td>
			<td>P2031</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechest33342</td>
			<td>Life Technology</td>
			<td>H3570</td>
			<td>Dye for staining nuclei</td>
		</tr>
	</tbody>
</table>

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.

Definizione del sistema di coltura per aNSCs dalla regione neurogena ignoto è essenziale per la comprensione di queste cellule e di sviluppare il loro potenziale utilizzo nella riparazione del cervello 12. È noto che le NSC a diversi stadi di sviluppo o in regioni diverse si comportano in modo diverso 3,4. Recentemente, è stato riportato che le cellule SCZ derivate presentano potenziali differenziali di differenziazione neuronale in vivo e in vitro rispetto alle cellule SVZ-derivate 7-9. Pertanto, per isolare precisamente ciascuna regione neurogenica, fette di cervello che includono il SCZ stati sezionati utilizzando una matrice cerebrale di 1 mm (Figura 1A). Dopo 8 giorni di coltura con fattori di crescita, aNSCs derivati dalla SCZ possono formare neurosfere, in cui le cellule possono essere mantenute in seguito (Figura 1B). Dal momento che un sottoinsieme di cellule staminali neurali nelle neurosfere può essere spontaneously differenziato 13, un sistema di coltura monostrato è anche utile per il mantenimento della popolazione relativamente omogenea di SCZ-aNSCs. I fattori di crescita ed enzimi di dissociazione come la tripsina non permeano facilmente in profondità all&#39;interno del neurosfere 14,15. colture monostrato offrono le condizioni più anche per l&#39;espansione delle cellule staminali neurali. Da neurosfere primarie, SCZ-aNSCs erano dissociate in singole cellule da un trattamento di buffer di digestione. Un giorno dopo la semina cellulare, aNSCs erano attaccati alla piastra rivestita e la proliferazione delle cellule esposte (Figura 2A). Per confermare la loro potenza della proliferazione, BrdU è stato aggiunto nella media. Dopo l&#39;incubazione con BrdU per 2 ore, le cellule sono state prontamente colorati con anti-BrdU (un marker di proliferazione) e anti-Nestin (un marker per le cellule staminali neurali) di anticorpi, indicando che SCZ-aNSCs sono attivamente proliferando e mantenendo le proprietà chiave di Le cellule staminali (Figura 2b). Di conseguenza, non hanno exhibit marcatori per cellule differenziate, come EGFR (espressi in cellule transientemente-amplificazione) e DCX (espressa in neuroblasti) (Figura 2C). Per confermare il potenziale di differenziazione multiplo di SCZ-aNSCs, fattori di crescita sono stati rimossi dal terreno di coltura. Dopo 6 giorni, le cellule sono state immunostained con i vari responsabili di cellule differenziate. Per esporre le diverse progenie di aNSCs, marcatori per i neuroni (TUJ1), astrociti (GFAP), e oligodendrociti (O4) sono stati impiegati; tutti questi tipi cellulari sono stati generati dal SCZ-aNSCs (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/54929/54929fig1.jpg" /> Figura 1: Isolamento della regione SCZ e formazione del neurosfere. (A) Procedimento per la dissezione della regione SCZ dal cervello di topo adulto. Per la cultura SCZ-ANSC, a 8 settimane di età cerebrale mouse è posizionato su una matrice cerebrale (1 mm intervalli). Dopo sezionamento, 1 mm fettine cerebrali che comprendeva la regione SCZ (2-3 mm dal bregma) sono stati sezionato (indicata dalla linea tratteggiata rossa). Formazione (B) neurosfere. Otto giorni dopo la coltura in vitro, neurosfere primarie (passaggio 0) si sono formati e diversi passaggi. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54929/54929fig2.jpg" /> Figura 2: Manutenzione delle SCZ-aNSCs come cultura monostrato. (A) Un giorno dopo la semina delle cellule sezionato, SCZ-aNSCs fosse collegata ed espanso su un piatto rivestito in modo monostrato (a sinistra). Tre giorni dopo la manutenzione, è stato aumentato il numero di SCZ-aNSCs (a destra). (B) Immunocolorazione con BrdU (rosso, un marcatore per cellule proliferanti), Nestin (verde, un marker per le cellule staminali neurali), e Hoechest33343 (blu, un marker per nuclei). (C) immunocolorazione con staminali neurali / marcatori di cellule progenitrici Nestin (rosso, un marker per le cellule staminali di tipo B neurali), EGFR (verde, un marker per le cellule transitori di amplificazione di tipo C), e DCX (blu, un marker di tipo A neuroblasti). I nuclei sono stati di contrasto con Hoechest33343 (bianco). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54929/54929fig3.jpg" /> Figura 3: Immunocolorazione delle cellule differenziate dalle SCZ-aNSCs. Immunocolorazione con marcatori di differenziazione TUJ1 (verde, un marcatore per i neuroni immaturi), O4 (rosso, un marker per gli oligodendrociti), e GFAP (urloOW, un marker per astrociti). immagini ingrandite sono mostrati come inserti. Hoechest33343 (blu) è stato utilizzato per la contro-colorazione dei nuclei. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

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

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 ...

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.0K Views.  Korea University College of Medicine. 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. 

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

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 ...

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Neuroscienze

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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

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

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

L&#39;isolamento e la coltura di cellule staminali neurali Crest di follicoli dei capelli umani

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

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

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

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 ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

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

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

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

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

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 Embryonic Mouse Neural Stem Cells

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

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