This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

In accordo con New South Wales, in Australia la legge, dopo il tessuto cerebrale mortem è stata acquisita da cani adulti eutanasia per ragioni non correlate allo studio. 1. Preparazione del terreno di coltura <ol><li> Preparare una soluzione di gelatina 0,1% aggiungendo 0,1 g di gelatina in polvere a 100 ml di acqua distillata e agitazione a 37 ° C fino a dissoluzione. Sterilizzare la soluzione per irraggiamento UV per 15 min. Questo supporto può essere conservato a 4 ° C per 1 mese. </li><li> Preparare un rapporto 3: 1 di Modified Eagle Medium di Dulbecco (DMEM) e nutrienti F12 miscela aggiungendo 167 ml di F-12 a 500 ml di DMEM (4,5 g / L D-glucosio). Aggiungere 6.7 ml di soluzione di penicillina / streptomicina (per una concentrazione finale 1%). Questo supporto può essere conservato al buio a 4 ° C per 1 mese. </li><li> Preparare 500 ml di media siero combinando 440 ml di DMEM (4,5 g / L D-glucosio), 5 ml di L-alanil-L-glutammina dipeptide (200 mM), 5 ml di penicillina / streptomicina e 50 ml di bovin fetalee siero (FBS). Questo supporto può essere conservato al buio a 4 ° C per 1 mese. </li><li> terreno di coltura completo è costituito da cellule staminali neurali (NSC) basale media e proliferazione Neural Supplemento-A (NS-A), 2% FBS, 2 mg / eparina ml, 20 ng / ml fattore di crescita epidermico (EGF) e 10 ng / ml fattore di crescita dei fibroblasti (bFGF). Rendere tale supporto fresco, immediatamente prima dissezione del cervello, e riscaldare a 37 ° C in un bagno d&#39;acqua. </li></ol> 2. Cervello Estrazione <ol><li> Inizia l&#39;estrazione entro 6 ore post mortem sterilizzando la superficie dorsale della testa del cane usando iodopovidone. </li><li> Usando un bisturi, fare una incisione longitudinale lungo la linea mediana sagittale, su tutta la superficie dorsale del cranio, per esporre i muscoli masseteri sottostanti. La lunghezza di questo taglio dipende dalla specie e l&#39;età del cane. </li><li> Ai confini rostrale e caudale del cranio, fare altri 5 - 10 cm tagli perpendicolari su entrambi i lati, passareing dietro gli occhi e le orecchie, rispettivamente, per permettere alla pelle di essere pienamente riflessa. </li><li> Usando un bisturi, separare i muscoli masseteri dal loro attaccamento alla cresta sagittale del cranio e raschiare qualsiasi tessuto connettivo rimanente dal cranio. </li><li> Utilizzando un osso oscillante visto tagliare il tappo del cranio in una linea circonferenziale. Usando una sonda o bisturi recidere ogni residuo di allegati con la dura, e poi rimuovere con attenzione la calotta per esporre la superficie dorsale del cervello. Attenzione: Lo spessore dell&#39;osso varia in diverse regioni del cranio, quindi si deve esercitare cautela per non penetrano troppo profonda e danneggiare il cervello sottostante. </li><li> Recidere il midollo spinale inserendo un bisturi tra le vertebre cervicali. </li><li> Con una mano per sollevare delicatamente il cervello, usare un bisturi per liberare con cura il cervello dalla fossa cranica e recidere i nervi e vasi sanguigni di collegamento situati sul suo lato ventrale. Sollevare delicatamente la bpioggia fuori del cranio e in un contenitore di DPBS. Attenzione: Le grandi bulbi olfattivi del cervello canino possono estendersi in profondità nella fossa cranica anteriore. Bisogna fare attenzione quando si rimuove il cervello in modo da preservare queste strutture. </li></ol> 3. ippocampale Dissection <ol><li> Sciacquare il cervello in tampone fosfato di Dulbecco (DPBS) per rimuovere il sangue e posizionarlo lato dorsale verso il basso su un 150 millimetri piastra di Petri. </li><li> Lentamente bisect cervello longitudinalmente attraverso il piano sagittale con un coltello cervello bagnato e una sola fetta che utilizza l&#39;intera lunghezza della lama. Non applicare ulteriore pressione verso il basso, o utilizzare un movimento di taglio, per evitare di danneggiare il tessuto. </li><li> Posizionamento di ogni mediale dell&#39;emisfero superficie fino, sezionare l&#39;ippocampo con un bisturi e pinza sottile e le trasferisce ad un piatto di coltura di tessuti da 35 mm. </li></ol> 4. L&#39;isolamento di cellule neurali Precursore <ol><li> Tritare il temettere campioni utilizzando una lama di bisturi in circa 1 mm 3 pezzi. </li><li> Trasferire il tessuto in una provetta da 15 ml, 2 ml di 0,1% tripsina EDTA e incubare per 7 minuti in un bagno d&#39;acqua a 37 ° C. </li><li> Arrestare la reazione enzimatica aggiungendo 4 ml di mezzi di siero al tubo. Pellet la sospensione mediante centrifugazione a 100 xg per 7 minuti e rimuovere il surnatante. </li><li> Risospeso il pellet in 300 ml di DPBS, e meccanicamente dissociato pipettando gentilmente su e giù, con un puntale di 1.000 ml, fino a quando si forma una omogenato lisce. </li><li> Aggiungere 14 ml di mezzi di siero al tubo e passare la sospensione cellulare attraverso un filtro cella 40 micron. Pellet la sospensione mediante centrifugazione a 100 xg per 7 minuti, rimuovere il surnatante, e risospendere in terreno di coltura completo. </li></ol> 5. cultura neurosfere <ol><li> Dalla sospensione cellulare ottenuta in isolamento, contare il numero di cellule vitali utilizzando Trypan blue dye esclusione e un hemocitometro. </li><li> Diluire la sospensione cellulare in mezzo di crescita completo e sementi a 1 x 10 5 cellule / cm 2 nei pozzetti rivestiti di una piastra 6 bene. </li><li> Incubare a 37 ° C, 5% di CO 2 e&gt; 95% di umidità per 14 - 28 giorni, sostituire il 50% dei terreni di crescita ogni 7 giorni. Misurare il diametro delle neurosfere regolarmente. Se permesso di crescere oltre i 100 micron di diametro, la morte cellulare e la differenziazione spontanea possono verificarsi entro le neurosfere. </li></ol> 6. neurosfere Passage <ol><li> Per il passaggio da una cultura fluttuante, quando le neurosfere raggiungono circa 100 micron di diametro, unire tutti i media neurosfere contenente in un unico tubo. Pellet per centrifugazione a 100 xg per 7 minuti e rimuovere il surnatante. </li><li> Risospendere il pellet in 1 ml di 0,1% tripsina EDTA e incubare per 7 minuti in un bagno d&#39;acqua a 37 ° C. Delicatamente pipetta su e giù per 100 volte, usando un puntale 200 pl, al fine di garantire dissociatisu delle neurosfere. </li><li> Arrestare la reazione enzimatica aggiungendo 5 ml di media siero nella provetta e centrifugare a 100 xg per 7 minuti. </li><li> Rimuovere il surnatante, risospendere il pellet cellulare in 1 ml di mezzo di crescita completo e contare il numero di cellule vitali mediante trypan colorante blu esclusione e un emocitometro. </li><li> Diluire la sospensione cellulare in mezzo di crescita completo e sementi a 1 x 10 5 cellule / cm 2 nei pozzetti rivestiti di una piastra 6 bene. Incubare a 37 ° C, 5% di CO 2 e&gt; 95% di umidità per 14 - 28 giorni, sostituendo il 50% dei terreni di crescita ogni 7 giorni. </li></ol> 7. Cultura aderente cellule neurali Precursore <ol><li> Aggiungere un volume sufficiente di soluzione di gelatina 0,1% per coprire la superficie di un piatto di coltura tissutale e la polimerizzazione in un incubatore a 37 ° C per 1 ora prima di elaborare le neurosfere cultura aderente. </li><li> Dalla cultura neurosfere galleggiante, unire tutti i media in un unico tubo. Agglomerare le neurospheres per centrifugazione a 100 xg per 7 minuti e rimuovere il surnatante. </li><li> Risospendere il pellet in 1 ml di 0,1% tripsina EDTA e incubare per 7 minuti in un bagno d&#39;acqua a 37 ° C. Delicatamente pipetta su e giù per 100 volte, usando un puntale 200 l. </li><li> Aggiungere 5 ml di mezzi di siero per fermare la reazione enzimatica e centrifugare a 100 xg per 7 minuti. Eliminare il surnatante e risospendere il pellet cellulare in 1 ml di terreno di coltura completo e contare il numero di cellule vitali utilizzando Trypan colorante blu esclusione e un emocitometro. </li><li> Diluire la sospensione di cellule in terreno di coltura, rimuovere la colla di pesce dai fiaschi di coltura di tessuti e seminare le cellule a 1 x 10 4 cellule / cm 2. </li><li> Incubare le cellule a 37 ° C, 5% di CO 2 e&gt; 95% di umidità. Sostituire il supporto con, riscaldato, i media di crescita complete freschi ogni tre giorni fino a quando la cultura raggiunge circa l&#39;80% di confluenza (dopo circa 7 giorni). </li></ol> 8. Neural Colony Forming Assay <ol><li> Combinare neurale formanti colonia cellulari componenti multimediali (NCFC) del saggio: media NCFC siero libero, proliferazione NS-A, e la soluzione di collagene (3 mg / ml). Supplemento questo supporto con 2 mg / ml di eparina, 20 ng / ml EGF, e 10 ng / ml bFGF. </li><li> Dopo la dissociazione del tessuto dell&#39;ippocampo risospendere le cellule in colonia neurali che formano terreno di coltura, pre-riscaldato a 37 ° C in un bagno d&#39;acqua. Seme le cellule ad una densità clonale di 1,1 x 10 5 cellule / ml in un piatto di coltura da 35 mm. </li><li> Incubare le cellule in questa matrice di collagene semi-solido a 37 ° C, 5% di CO 2 e&gt; 95% di umidità. Culture per 28 giorni, sostituendo il 50% dei mezzi di crescita ogni 7 giorni. </li></ol> 9. differenziazione delle cellule neurali Precursore <ol><li> Aggiungere un volume sufficiente di 5 mg / cm 2 soluzione laminina per coprire la superficie di un piatto di coltura tissutale e la polimerizzazione in un incubatore a 37 ° C per 24 ore prima di elaborare le cellule per differentiazione. Nota: Rimuovere la soluzione laminina e risciacquare la superficie rivestita due volte in DPBS immediatamente prima della semina. </li><li> Quando passaging, seminare la sospensione cellulare a 1 x 10 4 cellule / cm 2 in DMEM-F12 (3: 1) i media (pre-riscaldato a 37 ° C) integrata con 20 ng / ml EGF e 40 ng / ml bFGF sul laminina rivestito piatto di coltura. </li><li> Incubare le cellule a 37 ° C, 5% di CO 2 e&gt; 95% di umidità e consentono alle cellule a proliferare per 3 giorni. </li><li> Per differenziare le cellule precursori neurali aderenti, dopo 3 giorni sostituire il supporto con i media differenziazione contenenti DMEM-F12 (3: 1) supporto pre-riscaldato a 37 ° C, supplementato con 10 ng / ml cervello derivato fattore neurotrofico (BDNF). </li><li> Durante differenziazione sostituire il 50% dei media ogni 3 giorni. La differenziazione avviene gradualmente nel corso di circa 21 - 28 giorni. Attenzione: Una volta che le cellule hanno iniziato a differenziarsi, sostituire solo il 50% dei mezzi a uno time come esposizione all&#39;aria può avere un effetto negativo sulla salute delle cellule. </li></ol>

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A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li><li>Goffredo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li></ol>

The authors have nothing to disclose.

I protocolli descritti qui sono ottimizzati per mantenere condizioni di coltura favorevoli per massimizzare la vitalità cellulare. La velocità e la cura durante l&#39;estrazione, l&#39;isolamento e l&#39;espansione è di importanza critica. Un passo fondamentale per stabilire l&#39;espansione monostrato aderente è la dissociazione efficace delle neurosfere primarie. A seguito di passaggio, non sufficientemente dissociate neurosfere possono generare neurosfere galleggianti secondarie. Durante il cambio dei media, questi neurosfere possono essere rimossi, dissociate e reseeded per il passaggio monostrato aderente. Al contrario, se la vitalità delle cellule post-dissociazione è inferiore all&#39;80%, questo può essere indicativo di pipettaggio eccessivo durante la dissociazione. cellule precursori neurali, e in particolare le loro controparti differenziati, sono sensibili alle variazioni di pH al di fuori del range fisiologico, e l&#39;esposizione all&#39;aria. L&#39;osservazione della improvvisa morte cellulare diffusa può essere attribuibile a questi fattori. Di conseguenza, un passo fondamentale nel supporto chanGES è per i supporti da fatti freschi ogni volta, e solo il 50% del volume da sostituire. Infine, mentre i fattori mitogeni EGF e bFGF sostengono la sopravvivenza e la proliferazione dei precursori neurali dell&#39;ippocampo in condizioni di assenza di siero in vitro 22,23, la risposta cellulare a questi mitogeni è fortemente dipendente dall&#39;età evolutiva delle cellule e la concentrazione mitogenica 24,25. Pertanto, assicurando variazioni sperimentali minima questi componenti è cruciale per generare coerenti, colture cellulari riproducibili. Per la massima vitalità, le cellule devono essere seminate per la cultura entro 6 ore post mortem. Tuttavia, il cervello estratto può essere conservato temporaneamente in PBS a 4 ° C. Questa modifica può consentire l&#39;isolamento di essere ritardata fino a 24 ore con una minima riduzione della resa delle cellule 26,27. Il fattore di crescita integrazione dei terreni di coltura può anche essere modificato per aumentare la proliferazione o per favorire la differentiation di specifici tipi di cellule neurali. Fattore di crescita insulino-simile 1 (a 100 ng / ml) ha mostrato di avere un effetto favorevole sulle cellule staminali roditore neurali in vitro 28,29, mentre sotto differenziazione condizioni fattore pro-neuronale BDNF 30 può essere utilizzato in combinazione con fattori tali come interleuchina 7 (a 500 ng / ml) o acido retinoico (a 0,1 pM) per indurre una polarizzazione verso sottotipo neuronale o differenziazione gliale 31,32. La decisione di espandere le cellule precursori neurali come neurosfere galleggianti o come un monostrato aderente dipende in gran parte delle applicazioni a valle desiderati e le caratteristiche della cultura. Mentre il sistema di coltura neurosfere è semplicistico e altamente riproducibile, che è limitato nella sua capacità di generare popolazioni omogenee di cellule. Il microambiente complesso all&#39;interno di ogni sfera può promuovere l&#39;apoptosi e la differenziazione spontanea, favorendo una popolazione eterogenea 33, mentre repfusion orted di neurosfere che possono confondere quantificazione delle popolazioni di cellule precursori neurali 34. Inoltre, la dissociazione meccanica ripetuta richiesta per neurosfere passaggio può anche portare a stress dannoso, senescenza o addirittura la morte delle cellule. L&#39;uso di enzimi di dissociazione alternative, come ad esempio la papaina, può influenzare la vitalità delle cellule risultante 16. Al contrario, il sistema di coltura monostrato aderente, con l&#39;esposizione più uniforme a mitogeni ambientali, favorisce una maggiore omogeneità nel tipo di cellula e divisione più simmetrica. Questo sistema è stato dimostrato di produrre una nicchia frazione indipendente cellule, sulla base dell&#39;espressione di marcatori di cellule staminali chiave 35,36. Questo sistema richiede l&#39;utilizzo di recipienti di coltura pre-rivestiti di promuovere l&#39;adesione cellulare. La scelta di substrato cultura dovrebbe essere considerato con attenzione, in quanto può avere effetti significativi sul profilo differenziazione dei precursori neuronali in coltura 25,37. Here presentiamo protocolli efficaci per l&#39;isolamento, espansione e la differenziazione delle cellule precursori adulte canine dell&#39;ippocampo neurali dal tessuto post mortem fresco. Adulti precursori neurali canini sono sotto-rappresentate nella letteratura 18; con i protocolli completi per il loro isolamento e l&#39;espansione, tanto meno 17. I nostri protocolli possono poi servire per aumentare la consapevolezza di questo modello animale prezioso e rappresentano una significativa aggiunta a questo modesto numero di studi. Di rilievo, con il nostro metodo unico, adulti canini precursori neurali dell&#39;ippocampo può essere espansa con successo più di 10 raddoppi popolazione. Uno studio simile utilizzando adulto canino precursore neurale dell&#39;ippocampo riferito che neurosfere più grandi, diversi passaggi a 100 - 150 micron di diametro, hanno cessato la proliferazione di là della quinta generazione 17. Come notato nel nostro protocollo, eccessiva crescita neurosfere può incoraggiare la differenziazione spontanea e apoptosi, che nel corso del passaggio di serie può avere lEd a questa ridotta capacità proliferativa. Forniamo anche un protocollo per espansione monostrato aderente di questa popolazione cellulare, in alternativa al sistema di espansione neurosfere classica 17-21. Questo sistema alternativo permette all&#39;utente maggiore controllo sull&#39;ambiente cellulare e amplia notevolmente la gamma di applicazioni a valle di queste cellule, con un potenziale di omogeneizzazione fenotipica e diretto differenziazione neuronale 35,38. Colta adulti canini dell&#39;ippocampo cellule precursori neurali hanno applicazioni in una vasta gamma di campi di ricerca cellulari e neurobiologici. Il cervello canino possiede una omologia più vicino al cervello umano di modelli di roditori esistenti. Come tale, adulti canini cellule precursori neurali rappresentano un importante, ma poco studiato, risorsa. Queste cellule possono essere utilizzati per ottenere ulteriori informazioni sui meccanismi che stanno dietro neurogenesi adulta negli esseri umani, e per lo sviluppo di terapie rigenerative che Target questo processo.

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.
In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.
In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

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			<td height="21" style="height:21px;width:337px;">1000 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF1000</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm petri dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well culture plate</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm tissue culture dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well culture plate</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">B-27 Supplement (50X) serum free</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">13256029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
			<td style="width:252px;">GF029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen solution</td>
			<td>Stem Cell Technologies</td>
			<td>04902</td>
			<td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (4.5g/L, D-glucose) 500mL</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">11960044&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">DPBS</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor (EGF)</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">354001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F-12 nutrient mixture (Ham) (1X) Liquid</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">31765035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">16141079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from Porcine Skin Type A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-alanyl-L-glutamine dipeptide (GlutaMAX)</td>
			<td>Life Technologies</td>
			<td style="width:252px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt from (porcine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">H314950KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin (mouse)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">23017015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCFC serum free medium (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td>5720</td>
			<td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proliferation NS-A (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">05773</td>
			<td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&#160; from Stem Cell Technologies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSC basal medium (Rat; NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">5770</td>
			<td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Penicillin/Streptomycin (5000 U/mL)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-iodine</td>
			<td style="width:196px;">Munipharma</td>
			<td style="width:252px;">Betadine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Trypan blue (0.4%)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Class II biological safety cabinet</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">Safe 2020 1.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain knife (disposable)</td>
			<td style="width:196px;">Macroknife</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Cell culture incubator</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">HERAcell 150i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Centrifuge</td>
			<td style="width:196px;">Hettich</td>
			<td style="width:252px;">Universal 320R</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps</td>
			<td style="width:196px;">Dumont</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Easypet Electric pipette</td>
			<td style="width:196px;">Eppindorf&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:196px;">Boeco</td>
			<td style="width:252px;">Bright-Line Improved Neubauer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Manual pipettes</td>
			<td style="width:196px;">Eppindorf research</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oscillating bone saw&#160;</td>
			<td style="width:196px;"></td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blades (No.21)</td>
			<td style="width:196px;">Paramount</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Scissors&#160;</td>
			<td style="width:196px;">Delta</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:196px;">Grant</td>
			<td style="width:252px;">JB Aqua 18 plus</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and expansion of adult canine hippocampal neural precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

Attraverso l&#39;uso di precursori neuronali in vitro saggi, neurogenesi e popolazioni di cellule progenitrici sono stati caratterizzati e confrontati attraverso l&#39;asse dorsoventrale dell&#39;ippocampo cane adulto. cellule precursori neurali derivate da tessuto dell&#39;ippocampo isolato formarono neurosfere galleggiante entro 14 giorni di isolamento, raggiungendo un diametro di 100 micron entro il 28 giorni di coltura. Neurosfere derivate da dorsali e ventrali isolati hanno mostrato alcuna differenza nella dimensione media, e dopo dissociazione enzimatica in singole cellule, potrebbero essere diversi passaggi come galleggiante culture neurosfere. neurosfere galleggianti secondari di entrambe le regioni dell&#39;ippocampo sono stati in grado di formare entro 5 giorni dal passaggio. Quando seminate a 1 x 10 4 cellule / cm 2 sul 0,1% ricoperto di gelatina fiasche di coltura, le cellule precursori neurali sono stati anche in grado di proliferare monostrati come aderenti (Figura 1A). Nessuna differenza è stata osservata morfologiche between dorsale e cellule precursori neurali dell&#39;ippocampo ventrali e entrambe le culture aderenti sono stati in grado di sottoporsi a più di 10 raddoppi popolazione senza alcuna osservato un rallentamento nel passaggio-tempo (Figura 1B). Nestin e Sox2 espressione genica di cellule staminali neurali in coltura aderente non era significativamente diversa lungo l&#39;asse dorsoventrale dell&#39;ippocampo (Figura 2). Una più ampia caratterizzazione genica e proteica, confermando l&#39;identità precursori neurali di queste cellule, è riportata in nostri dati pubblicati 7. L&#39;utilizzo di un adeguato Neural formanti colonia saggio 4,6 abbiamo valutato la frequenza di cellule precursori neurali al fine di quantificare i potenziali differenze di popolazioni di cellule precursore neuronali attraverso la dorsale e ventrale sottoregioni dell&#39;ippocampo canina. Le cellule sono state seminate ad una densità clonale in un semi-solido matrice di collagene che preclude fusione cellulare, e coltivate per 28 giorni (Figura 3A </strong&gt;). All&#39;interno confronti soggette rivelato una significativa maggiore frequenza di sfere più grandi per unità di superficie a dorsale (7.3 ± 2.0) rispetto alle colture ippocampali di derivazione ventrale (3.6 ± 1.7; n = 5; misure ripetute ANOVA F = 96,8; p = 0.001; figura 3B ). In condizioni di differenziazione, aderenti canine cellule precursori neurali hanno mostrato alterazioni progressive nella morfologia lordo, in via di sviluppo più a lungo e processi più elaborate. Espressione della proteina per neuronale (βIII-tubulina) e gliale (proteina silicea fibrillare gliale; GFAP) marcatori anche aumentato a seguito di differenziazione (Figura 4). Nessuna differenza significativa osservata nel numero di cellule marcate positivamente tra dorsali e ventrali isolati. I nostri dati pubblicati in precedenza avvalora questi cambiamenti di espressione proteica, dimostrando up-regolazione dei loro geni associati con down-regulation di neurale<html> geni precursori durante il periodo di differenziazione 28 giorni 7. <img alt="Figura 1" src="/files/ftp_upload/54953/54953fig1.jpg" /> Figura 1:. In Vitro espansione di cellule adulte Canine ippocampale neurale precursori adulti canini dell&#39;ippocampo cellule precursori neurali isolate dalla dorsale e regioni dell&#39;ippocampo ventrali sono espansi come (A) neurosfere galleggianti o come (B) un monostrato aderente (C) Come. monostrato aderente queste cellule precursori neurali potrebbero subire il passaggio di oltre 10 volte senza calo significativo raddoppiando la velocità. n = 5; scala = 50 micron. Modificata dalla figura pubblicato 7. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> /ftp_upload/54953/54953fig2.jpg &quot;/&gt; Figura 2:. L&#39;espressione genica in cellule proliferanti Canine Adult ippocampale neurale Precursore espressione di geni di cellule staminali neurali (A) Nestin e (B) Sox2 è stata confermata in cellule adulte canine dell&#39;ippocampo neurali precursori in condizioni di coltura proliferative aderenti. Espressione di questi geni era equivalente sia dorsale e cellule dell&#39;ippocampo derivato ventrale. fibroblasti Canine Adult sono state usate come una linea di controllo per confrontare le differenze di espressione genica relativa. n = 5; barre di errore = SEM. Modificata dalla figura pubblicato 7. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54953/54953fig3.jpg" /> Figura 3:. Neural formanti colonia Assay (A)Adulti cellule canine neurali precursori, seminate a densità clonale in una matrice di collagene semi-solido, neurosfere modulo entro 28 giorni della cultura. (B) le cellule precursori neurali derivate dalle dell&#39;ippocampo dorsale generano significativamente un maggior numero di neurosfere per unità di superficie rispetto a quelle derivate da l&#39;ippocampo ventrale. n = 5; barre di errore = SEM; scala = 50 micron. Modificata dalla figura pubblicato 7. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54953/54953fig4.jpg" /> Figura 4:. Immunocitochimica di cellule differenziate Canine Adult ippocampale neurale precursori Quando coltivate in BDNF per 28 giorni, per adulti canino precursori neurali sia dalla dorsale e ventrale ippocampo si differenziano in neuroni maturi (po βIII-tubulinaallo snervamento) e gliali (tipi positivo) di cellule GFAP. n = 5; scala = 50 micron. Modificata dalla figura pubblicato 7. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Il cervello canino è un modello valido in cui studiare neurogenesi adulta. Presentato qui sono i protocolli per isolare ed espandere adulti canini dell&#39;ippocampo cellule precursori neurali dal tessuto cerebrale primario.

Isolamento ed espansione di Canine Adult Hippocampal neurali Precursori

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.0K Views.  University of Sydney.  Il cervello canino è un modello valido in cui studiare neurogenesi adulta. Presentato qui sono i protocolli per isolare ed espandere adulti canini dell'ippocampo cellule precursori neurali dal tessuto cerebrale primario. 

Video: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Ricerca

Biologia dello sviluppo

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Stabile ed efficiente Modificazione genetica di cellule nel topo adulto V-SVZ per l&#39;analisi di cellule staminali neurali autonoma ed effetti non autonomi

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex Cultura Vivo del Chick cerebellare fette e spazialmente mirata elettroporazione di granuli precursori delle cellule

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolamento e la coltura di Neurosfere Zebrafish adulti Brain-derived

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Coltura umane pluripotenti e neurali cellule staminali in un sistema di coltura cellulare chiuso per base e preclinica di ricerca

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolamento e l&#39;espansione delle staminali mesenchimali stromali derivate / cellule da placenta umana Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

La stimolazione di Notch segnalazione in mouse osteoclasti Precursori

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

L'isolamento e coltura di cellule primarie dell'epicardio ricavate da campioni di cuore umano adulto e fetale

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Derivazione e differenziazione delle cellule mesenchimali ovarico canini

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

In tempo reale Live-cella flusso Cytometry per indagare l'afflusso del calcio, la formazione del poro e fagocitosi dai recettori P2X7 in cellule progenitrici neurali adulto

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...