Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.

1. Carta Preparazione NOTA: Il protocollo si basa sull&#39;utilizzo di una miscela di due distinti mezzi: DMEM / F12 addizionato con modificato supplemento N2 e Neurobasal supplementato con supplemento B27, tipicamente in un rapporto 1: 1. <ol><li> Preparare il supplemento N2 modificato mescolando i componenti in una provetta da 15 ml. Non vortice o filtrare questo; mescolare capovolgendo la provetta fino a quando la soluzione è limpida. <ol><li> Inizia pipettando 7,2 ml di DMEM / F12, quindi aggiungere 1 ml di 25 mg / ml di insulina (composta in 0,01 M HCl) e mescolare bene invertendo il tubo. Ci vogliono un paio di minuti fino a quando la soluzione è limpida. </li><li> Aggiungere 1 ml di 100 mg / ml apo-transferrina (preparati in acqua), 33 ml di 0,6 mg / ml progesternone (costituiti in etanolo), 100 ml di 160 mg / ml (1 M) putrescina (preparati in acqua ), 10 ml di 3 mM selenito di sodio (preparati in acqua) e 666 ml di 7,5% di siero albumina bovina e mescolare bene il tubo. Aliquota in 2,5 ml e conservare a -20 °C per un massimo di 2 mesi. </li></ol></li><li> Preparare il supporto. <ol><li> Per 250 ml di DMEM / F12 aggiungere 2,5 ml di N2. Mescolare bene ma non scuotere o filtro. </li><li> Per 250 ml di Neurobasal media aggiungi 5 ml di supplemento B27. Mescolare bene ma non scuotere o filtro. </li><li> Mescolare il supporto da operazioni 1.2.1 e 1.2.2 in un rapporto 1: 1. In alcuni casi un 1: 3 mix può essere richiesto (integrato come il 1: 1 mix al punto 1.2.4). </li><li> Per il mix di aggiungere 0,5 ml di L-glutammina (concentrazione finale 0,2 mm) e 3,5 ml di 2-mercaptoetanolo (concentrazione finale 0,1 mm) e mescolare bene senza agitare. Conservare il supporto a 4 ° C per un massimo di 3 settimane e non esporre alla luce. Questo mix finale si chiama N2B27. </li></ol></li><li> Preparare la soluzione di gelatina. <ol><li> Preparare una soluzione di gelatina 1% in acqua ultrapura e autoclave a 121 ° C, 15 psi, per 15 min. È importante che la bottiglia utilizzata per questo è completamente pulito da detergenti o disinfettanti, quindi si raccomanda una nuova bottiglia èutilizzato per questo ed è sempre risciacquato solo in acqua ultrapura tra usi. Aliquota della soluzione all&#39;1% in 50 ml aliquote e conservare a 4 ° C per mesi. </li><li> Scaldare un&#39;aliquota di 1% di gelatina in un bagno d&#39;acqua a 37 ° fino a scioglimento e aggiungere 500 ml di soluzione salina tamponata con fosfato calda (PBS). Mescolare bene e pipettare sufficiente a coprire il fondo di ogni bene o un piatto. Lasciare la gelatina per rivestire la superficie per almeno 30 min. </li></ol></li></ol> 2. placcatura celle NOTA: Questo protocollo vale anche per il mouse ESC coltivato in 10% di siero di leucemia fattore inibitorio (LIF), la sostituzione di siero con LIF o supporti privi di siero con LIF e proteina morfogenetica dell&#39;osso 4 (BMP4) o MEK e GSK3 inibitori (supporti 2i) con o senza LIF. Tuttavia, i tempi e l&#39;efficienza della differenziazione possono variare a seconda del supporto e le cellule (vedi la discussione). Per gli esperimenti riportati qui abbiamo usato la linea 46C del mouse ESC (che ha un reporter EGFP buttato in one degli alleli Sox1 endogeni), coltivati ​​in GMEM con 10% siero e LIF. Per ottenere risultati ottimali, è importante che le cellule sono dissociate e ripiastrate nei media N2B27; semplicemente cambiando di supporti da GMEM / siero / LIF a N2B27 si traduce sempre in efficienza differenziazione ridotto rispetto replating cellule. <ol><li> Far crescere le cellule in GMEM con 10% di siero, 1 mM piruvato di sodio, aminoacidi non essenziali 1x, 0,1 M 2-mercaptoetanolo, e LIF 13 100 unità / ml. Per avere una cultura subconfluenti di topo ESC, piatto 1 x 10 6 cellule in un pallone di coltura T25 che avrà 2-3 giorni. </li><li> Osservare le cellule al microscopio in campo chiaro. Quando le cellule sono subconfluenti e pronti per il passaggio, sciacquare due volte in PBS. Aggiungere 1 ml di cellule dissociazione reagenti e ritorno per l&#39;incubatore per 2 - 5 min. Sebbene tripsina e di altri enzimi possono essere utilizzati anche per la dissociazione cellulare, possono avere un impatto negativo sulla adesione cellulare così le densità di placcatura dovranno essere regosted. </li><li> Una volta che le cellule iniziano a sollevare dalla piastra, toccare il vaso loro rimuovere in una sospensione di cellule singole. Raccogliere le cellule in un totale di 10 ml di mezzi di comunicazione e di dissociazione delle cellule del reagente e pipetta in una provetta da centrifuga da 15 ml. </li><li> Spin le cellule a 300 xg per 3 minuti a temperatura ambiente. </li><li> Con attenzione aspirare il surnatante e risospendere il pellet in 10 ml di N2B27 preriscaldata pipettando su e giù 3 - 5 volte. Quando pipettando sospensione giù, pipetta contro il lato del tubo per evitare la creazione di bolle. Contare le cellule con un contatore di cellule automatico o un emocitometro e registrare la concentrazione. </li><li> Preparare una sospensione cellulare contenente il numero desiderato di cellule per unità di volume da placcare per pozzetto in N2B27 pre-riscaldato. Una densità di 10.500 cellule per cm 2 in un piatto da 6 pozzetti (cioè, 1 x 10 5 cellule per pozzetto di un piatto 6 pozzetti) è ottimale. Vedi Tabella 1 per il numero consigliato di cellule per cm 2 e plating volumi di media per una serie di recipienti di coltura cellulare. </li><li> Aspirare la gelatina dal recipiente di coltura e la piastra della sospensione cellulare in funzione della densità suggerito e volume in Tabella 1. Non agitare le piastre in quanto ciò concentrare le cellule al centro. Collocare le culture in un incubatore umido a 37 ° C, 5% CO 2. </li><li> Modificare i media ogni 1 - 2 giorni, sostituendo tutti i media con N2B27 fresca. Pipetta delicatamente come vampate di calore le cellule potrebbero incidere densità e diminuire l&#39;efficienza differenziazione nei giorni successivi. Dove vitalità iniziale dopo la placcatura è povero, piastra le cellule in un 1: 3 mix dei media N2 e B27-integrati e cambiare questo N2B27 dopo i primi due giorni. Le cellule cominceranno a differenziare e dovrebbero mostrare espressione Sox1 (visibile fluorescenza verde del reporter nella linea cellulare 46C usato qui) entro 4 - 6 giorni. </li></ol> 3. immunofluorescenza NOTE: Il protocollo può essere applicato a qualsiasi tipo di imbarcazione. Il volume di ogni reagente è descritta per pozzetto di 6 pozzetti e può essere installato su qualsiasi superficie. Replating è sconsigliato a causa della bassa redditività in seguito. <ol><li> Rimuovere terreno di differenziamento e fissare le cellule aggiungendo 500 ml di 4% (v / v) paraformaldeide, lasciare per 15 min. </li><li> Lavare e permeabilize le cellule con 2 ml di PBS-T (tampone fosfato salino con 0,5% (v / v) di Tween-20) due volte. Le cellule possono essere mantenute in PBS-T fino a 2 mesi a 4 ° C. </li><li> Sostituire PBS-T con 1 ml di PBS-T con 10% (v / v) di siero (dalla stessa specie come anticorpo secondario). Incubare 1 ora sulla sedia a dondolo piatto a temperatura ambiente per bloccare qualsiasi legame non specifico. </li><li> Dopo 1 ora, lavare le cellule due volte con 2 ml di PBS-T, e incubare in 500 microlitri IgG topo contro βIII-tubulina (diluito 1: 1000 in PBS-T con siero 10%). Mettere sul rocker piatto, e lasciare O / N a 4 ° C. </li><li> Da questo punto, proteggere sempre il recipientedalla luce. Lavare le cellule due volte con PBS-T, quindi aggiungere 500 ml di fluorescenza marcato anti-topo IgG (diluito a 2 mg / ml in PBS-T con siero 10%). Mettilo sul bilanciere piatto per 1 ora a temperatura ambiente. </li><li> Lavare le cellule due volte con PBS-T, quindi aggiungere 500 ml di DAPI (diluito a 0,5 mg / ml in PBS-T) e lasciar riposare per 5 minuti. </li><li> Lavare nuovamente le cellule e tenerli in PBS-T. Le cellule sono pronte per essere fotografata e possono essere conservati per un massimo di 2 settimane a 4 ° C. Si noti che il segnale GFP svanisce nel tempo in modo non è opportuno confrontare l&#39;intensità GFP delle cellule fisse e in diretta o di cellule fissate in tempi diversi. </li></ol>

<ol>
	<li>Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo-derived+stem+cells:+of+mice+and+men.">Embryo-derived stem cells: of mice and men.</a> Annu Rev Cell Dev Biol. 17, 435-462 (2001).</li><li>Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+express+neuronal+properties+in+vitro.">Embryonic stem cells express neuronal properties in vitro.</a> Dev. Biol. 168, 342-357 (1995).</li><li>Fraichard, A., 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=In+vitro.+differentiation+of+embryonic+stem+cells+into+glial+cells+and+functional+neurons.">In vitro. differentiation of embryonic stem cells into glial cells and functional neurons.</a> J. Cell Sci. 108, 3181-3188 (1995).</li><li>Strubing, C., 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=Differentiation+of+pluripotent+embryonic+stem+cells+into+the+neuronal+lineage+in+vitro+gives+rise+to+mature+inhibitory+and+excitatory+neurons.">Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.</a> Mech. Dev. 53, 275-287 (1995).</li><li>Stavridis, M. P., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+mouse+embryonic+stem+cells.">Neural differentiation of mouse embryonic stem cells.</a> Biochem Soc Trans. 31, 45-49 (2003).</li><li>Ying, Q. L., Stavridis, M., Griffiths, D., Li, M., 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=Conversion+of+embryonic+stem+cells+into+neuroectodermal+precursors+in+adherent+monoculture.">Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.</a> Nat Biotechnol. 21, 183-186 (2003).</li><li>Ying, Q. L., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+conditions+for+neural+commitment+and+differentiation.">Defined conditions for neural commitment and differentiation.</a> Methods Enzymol. 365, 327-341 (2003).</li><li>Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+period+of+FGF-induced+Erk1/2+signalling+is+required+for+vertebrate+neural+specification.">A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.</a> Development. 134, 2889-2894 (2007).</li><li>Stavridis, M. P., Collins, B. J., Storey, K. G. <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+orchestrates+fibroblast+growth+factor+signalling+to+drive+embryonic+stem+cell+differentiation.">Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation.</a> Development. 137, 881-890 (2010).</li><li>Speakman, C. M., 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=Elevated+O-GlcNAc+Levels+Activate+Epigenetically+Repressed+Genes+and+Delay+Mouse+ESC+Differentiation+Without+Affecting+Naive+to+Primed+Cell+Transition.">Elevated O-GlcNAc Levels Activate Epigenetically Repressed Genes and Delay Mouse ESC Differentiation Without Affecting Naive to Primed Cell Transition.</a> Stem Cells. 32, 2605-2615 (2014).</li><li>Kunath, T., 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=FGF+stimulation+of+the+Erk1/2+signalling+cascade+triggers+transition+of+pluripotent+embryonic+stem+cells+from+self-renewal+to+lineage+commitment.">FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.</a> Development. 134, 2895-2902 (2007).</li><li>Dani, C., 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=Paracrine+induction+of+stem+cell+renewal+by+LIF-deficient+cells:+a+new+ES+cell+regulatory+pathway.">Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.</a> Dev Biol. 203, 149-162 (1998).</li><li>Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+differentiation+of+embryonic+stem+cells.">Culture and differentiation of embryonic stem cells.</a> Journal of Tissue Culture Methods. 13, 89-94 (1991).</li><li>Aubert, J., 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=Screening+for+mammalian+neural+genes+via+fluorescence-activated+cell+sorter+purification+of+neural+precursors+from+Sox1-gfp+knock-in+mice.">Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 11836-11841 (2003).</li><li>Niwa, H., Burdon, T., Chambers, I., 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=Self-renewal+of+pluripotent+embryonic+stem+cells+is+mediated+via+activation+of+STAT3.">Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.</a> Genes Dev. 12, 2048-2060 (1998).</li><li>Silva, J., 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=Capturing+pluripotency.">Capturing pluripotency.</a> Cell. 132, 532-536 (2008).</li></ol>

The authors have nothing to disclose.

Il protocollo di differenziamento neurale monostrato è stato in uso per oltre un decennio 6. Il protocollo è altamente efficiente, composto terreno specifico, e fatto in un sistema monostrato che rende il sistema più applicabile per preclinica (ad esempio, la selezione della droga) utilizza. Tuttavia, vi sono alcuni fattori critici che determinano l&#39;efficienza differenziazione. Questo articolo sottolinea questi fattori e la soluzione per ogni ostacolo. Densità del...

Le cellule staminali embrionali sono cellule pluripotenti derivate da embrione precoce con la capacità di proliferare indefinitamente in vitro, pur mantenendo la capacità di differenziarsi in tutti i tipi cellulari adulti seguenti reintroduzione in un apposito embrione stadio (formando una chimera), iniezione in singenici o immunocompromessi host (formando un teratoma) o in vitro soggetto a segnali appropriati 1. La differenziazione in vitro di cellule staminali embrionali di topo in lignaggi neuronali è stato descritto nel 1995 e prevedeva la formazione di aggregati multicellulari di sospensione (corpi embrionali, EBS) in mezzi di siero contenenti integrati con l&#39;acido retinoico morfogeno 2-4. Da allora una serie di protocolli sono stati sviluppati per permettere la differenziazione neuronale 5. Molti ancora utilizzare l&#39;aggregazione, gli altri co-coltura con induzione tipi di cellule e molti prevedono l&#39;utilizzo di mezzi privi di siero. Tutti i protocolli hanno vantaggi and svantaggi e la natura precisa neurali o cellule neuronali prodotte anche varia secondo il protocollo usato. Il protocollo ideale sarebbe robusto, scalabile e fare uso di mezzi di comunicazione e substrati completamente definiti, essere suscettibili di monitoraggio non invasivo del processo di differenziazione e causare la generazione delle popolazioni pure di cellule progenitrici neurali in grado di essere modellato da stimoli esterni e per differenziare in tutti i sottotipi neuronali e gliali con elevata efficienza e la resa in un tempo relativamente breve. Negli ultimi dodici anni abbiamo utilizzato un metodo per la generazione di progenitori neurali e neuroni di topo CES in una bassa densità, privo di siero cultura monostrato aderente 6-10. Questo protocollo soddisfa molti dei criteri di cui sopra: nelle nostre mani l&#39;efficienza della differenziazione è stato abbastanza costante nel corso di molti anni, e una varietà di linee cellulari, si può essere scalato verso l&#39;alto o verso il basso (usiamo con successo navi di piastre da 96 pozzetti per 15 centimetri di diametro dishes) ei supporti utilizzati sono ben definiti. Il processo è suscettibile di microscopia timelapse per il monitoraggio della differenziazione e una varietà di segnali patterning può essere aggiunto per indurre la generazione di tipi distinti di sottotipi neuronali (es Shh e Fgf8 per i neuroni dopaminergici 6). Tuttavia, ci sono alcune sfide per l&#39;efficace applicazione di questo protocollo. Uno degli aspetti chiave è un&#39;attenta preparazione dei media. Abbiamo sempre prepariamo il supporto in-house nonostante la disponibilità di alternative commerciali. Uno degli integratori utilizzati (N2; vedi Protocol) ha modifiche nel corso dello standard versioni disponibili in commercio. Infine, una delle fasi più importanti per il successo di questo metodo è la densità cellulare ottimale placcatura. Questo è principalmente perché mentre la natura autocrina di uno dei segnali che inducono (Fgf4 11) richiede che le cellule sono presenti sufficienti per consentire viabil ottimalelità e la differenziazione, a troppo elevate densità di differenziazione è compromessa (forse in parte a causa della produzione autocrina di LIF 12). È quindi importante che la preparazione sia supporti e placcatura cellule vengono eseguite accuratamente e costantemente per garantire risultati ottimali.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Reconstitute with sterile 0.01M HCl</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Reconstitute with ethanol</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V</td>
			<td>Life Technologies</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Life Technologies</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukaemia inhibitory factor</td>
			<td colspan="3">prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Mouse anti betaIII tubulin IgG antibody</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence-labelled anti-Mouse IgG antibody</td>
			<td>Life Technologies</td>
			<td>A31571</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>25cm2 tissue culture flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
	</tbody>
</table>

neural differentiation of mouse embryonic stem cells in serum-free monolayer culture

La capacità di differenziare le cellule staminali embrionali di topo (ESC) per progenitori neurali consente lo studio dei meccanismi di controllo specifica neurali nonché la generazione di tipi di cellule neuronali mature per ulteriori studi. In questo protocollo si descrive un metodo per la differenziazione di ESC per progenitori neurali usano senza siero, cultura monostrato. Il metodo è scalabile, efficiente e si traduce in produzione di circa il 70% di cellule progenitrici neurali entro 4-6 giorni. Esso può essere applicato a ESC da vari ceppi coltivate sotto una varietà di condizioni. Progenitori neurali possono essere autorizzati a differenziare ulteriormente in neuroni funzionali e glia o analizzati al microscopio, il flusso delle tecniche di citometria o molecolari. Il processo di differenziazione è suscettibile di microscopia time-lapse e può essere combinato con l&#39;uso di linee giornalista per monitorare il processo di specifica neurale. Noi forniamo istruzioni dettagliate sulla preparazione dei media e l&#39;ottimizzazione della densità cellulare per consentire il processo di Be applicato alla maggior parte delle linee ESC e una varietà di recipienti di coltura cellulare.

In questo esperimento, abbiamo utilizzato la linea cellulare 46C 14, le cellule staminali embrionali di topo con un endogena Sox1-reporter GFP, per monitorare la differenziazione neuronale. Utilizzando questa linea cellulare, espressione di Sox1, un marker per progenitore neuronale, può essere rilevata da fluorescenza verde. Placcatura densità è un fattore critico per raggiungere la differenziazione neuronale. Cellule staminali embrionali di topo sono stati placcati in 6 pozzetti a diverse densità che var...

Watch this Scientific Journal Video about Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture at JoVE.com

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Differenziamento neurale delle cellule staminali embrionali di topo in siero privo monostrato Cultura

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

neural-differentiation-mouse-embryonic-stem-cells-serum-free

Research

JoVE Journal

Developmental Biology

14.0K Views.  University of Dundee. This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Video: Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

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 ...

Ricerca

Biologia dello sviluppo

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Regolazione epigenetica del Cardiac differenziazione delle cellule staminali embrionali e Tessuti

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Un modello enzima e Neural Stem Cell Culture siero-libero per EMT Investigation Adatto per Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Regia di differenziazione dei progenitori ematopoietici primitivi e definitivo da cellule staminali pluripotenti umane

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Analisi di acido retinoico-indotta neurale differenziazione di cellule staminali embrionali di topo a organi embrionali due e tridimensionali

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Generazione di cellule staminali neurali indotte da cellule mononucleari periferiche e differenziazione verso i precursori dei neuroni dopaminergici per gli studi di trapianto

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

Produzione senza siero di epofere umane tridimensionali da cellule staminali pluripotenti

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Differenziazione neurale efficiente utilizzando la coltura a cella singola di cellule staminali embrionali umane

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...