Questo lavoro è stato sostenuto da una borsa di studio per ricercatori avanzati dal Fondo nazionale svizzero e attraverso sovvenzioni NYSTEM (C026446, C026447) e l&#39;iniziativa di cellule staminali Tri-istituzionale (Starr Foundation).

1. Preparazione della Cultura Media, piatti rivestiti e manutenzione di hPSCs Preparazione 1.1 supporti Nota: Filtrare tutti i media per la sterilizzazione e conservare a 4 ° C al buio per un massimo di due settimane. Numeri nomi dei reagenti, della società e catalogo sono elencati nella tabella dei materiali. <ol><li> % FBS DMEM/10: Unire 885 ml DMEM, 100 ml FBS, 10 ml Pen / Strep e 5 ml di L-glutammina. </li><li> HES-medio: Combine 800 ml DMEM/F12, 200 ml KSR, 5 ml di L-glutammina, 5 ml Pen / Strep, 10 ml di MEM minimo di soluzione aminoacidi essenziali, 1 ml β-mercaptoetanolo. Aggiungere 10 ng / ml di FGF-2 dopo filtrazione il mezzo. ATTENZIONE: β-mercaptoetanolo è tossico, evitare l&#39;inalazione, ingestione e contatto con la pelle. </li><li> Medio KSR-differenziazione: Combine 820 ml Knockout DMEM, 150 ml KSR, 10 ml L-glutammina, 10 ml Pen / Strep, 10 ml di MEM minima soluzione di amminoacidi essenziali e 1 ml di β-mercaptoetanolo. </li><li> N2-differentiatisu supporto: sciogliere 12 g DMEM/F12 polvere in 980 ml ​​di dH 2 O, aggiungere 1,55 g di glucosio, 2 g di bicarbonato di sodio e 100 mg APO transferrina umana. Mescolare 2 ml dH 2 O con 25 mg di insulina umana e 40 microlitri 1 N NaOH, aggiungere la soluzione disciolta al mezzo. Aggiungere 100 microlitri dicloridrato putrescina, 60 microlitri selenite, 100 microlitri progesterone e portare al volume di 1 litro con dH 2 O. </li></ol> 1.2 Rivestimento di piatti della cultura <ol><li> Rivestimento Matrigel: Thaw 1 ml congelati matrigel aliquota pipettando 19 ml DMEM/F12 sul aliquota fino a quando non si è sciolta. Togliere macchie passando attraverso un colino cella 40 micron e piastra 8 ml/10 cm piatto. Incubare i piatti per 1 ora a RT. Aspirare il matrigel immediatamente prima placcatura le cellule. Nota: Lavorate rapidamente con matrigel perché può aggregarsi a temperature superiori a 4 ° C. </li><li> Rivestimento PO / Lam / FN: Aggiungere 10 ml di 1x PBS contenente 15 mg / ml Poly-L ornitina hydrobromide ad un piatto 10 centimetri. Incubare il piatto per una notte a 37 ° C. Lavare le piastre con PBS 1x volta e aggiungere 10 ml/10 cm piatto di 1x PBS contenente 2 mg / ml topo laminina-I e 2 mg / ml di fibronectina. Incubare i piatti per una notte a 37 ° C. Prima placcatura le cellule, rimuovere completamente la soluzione e lasciare asciugare completamente le piastre a temperatura ambiente per 15-20 min da loro piedi sulla parete cappa coltura tissutale senza il coperchio. Nota: I piatti sono completamente asciutte e pronte per la placcatura cellulare quando si può vedere strutture cristalline sulla superficie (visibile ad occhio). Le piastre possono essere conservati a temperatura ambiente per qualche ora in questo stato essiccato. Piatti PO / Lam / FN devono essere preparati due giorni prima del tempo. In casi di emergenza Lam / FN può essere incubato per solo 2-4 ore. Tuttavia, questo può rischiare di non ottimali risultati differenziazione / sopravvivenza. </li></ol> 1.3 Manutenzione di hPSCs Nota: hPSCs sono mantenute su 0,1% di gelatina e mouse mitoticamente inattivato fibroblasti embrionali (MEFS) nelle HES-medium supplementato con 10 ng / ml di FGF-2 come descritto in precedenza 10,12. Le cellule devono essere divise ogni 6-8 giorni. <ol><li> Coat un piatto 10 centimetri con 8 ml di 0,1% di gelatina (in 1x PBS senza calcio o magnesio) a temperatura ambiente per 5 min. </li><li> Scongelare MEF congelati rapidamente in un bagno d&#39;acqua a 37 ° C. Aggiungi milione MEF a 10 ml% FBS DMEM/10. </li><li> Aspirare la gelatina e piastra le cellule. Incubare a 37 ° C per almeno 6 ore. </li><li> Aspirare il FBS DMEM/10% dalla piastra MEF preparato, lavare piatto con 1x PBS una volta e aggiungere 10 ml HES-medium supplementato con 10 mM Y-27632 dicloridrato. Lasciar riscaldare a 37 ° C per 20 min. Nota: Per splitting manuale di hPSCs viene utilizzata una cappa a flusso laminare con un microscopio incorporato. Tuttavia, le cellule possono essere diversi passaggi utilizzando opportuni metodi alternativi. </li><li> Sotto una cappa a flusso laminare con un microscopio incorporato, staccare colonie separate utilizzando un sollevatore cella e far galleggiare. </li><li> Utilizzare unSiringa da 1 ml per aspirare le colonie galleggianti e li spedisce sul caldo piatto MEF fresca. Trasferire circa un quarto delle cellule al nuovo piatto. Incubare a 37 ° C. </li><li> Concime cellule quotidianamente con fresco medio HES. </li></ol> 2. Placcatura di hPSCs di Differenziazione Nota: hPSCs dovrebbero essere raggruppati o placcati per la differenziazione, quando le colonie sono grandi, ma hanno ancora margini taglienti con il meno possibile le cellule di differenziazione alle frontiere (vedi Figura 1B). Quando le cellule sono mantenuti utilizzando passaging manuale colonie dovrebbero essere abbastanza grandi da essere facilmente visibile ad occhio. Per ottenere la giusta sensazione per questo punto di tempo si può mantenere un piatto HPSC separata per due settimane senza passaging e guardare le cellule raggiungono e passare il punto di tempo ideale per passaging / li differenzia. <ol><li> Preparare 10 centimetri piatti con matrigel 1 ora prima di avviare la differenziazione. Rivestimento matrigel successo può essereverificate a 4 ingrandimenti. </li><li> Quando le hPSCs sono pronti per essere diviso, aspirare il terreno e aggiungere 4 ml di 0,05% tripsina-EDTA alle cellule. </li><li> Agitare il piatto orizzontale per 2 minuti energicamente fino a quando si può vedere il MEF come singole cellule sollevare la piastra sotto il microscopio. Le colonie HPSC rimangono attaccati come colonie. </li><li> Immediatamente e accuratamente, aspirare il tripsina e lasciare che la piastra di riposare a temperatura ambiente per 2-4 min. </li><li> Aggiungere 2 ml di HES-medio alla piastra e con una pipetta P1000, staccare le cellule. Nota: Se le cellule non possono essere sollevati dalla superficie facilmente, la piastra può essere incubato vuoto a temperatura ambiente per altri 2-3 min. </li><li> Trasferire le cellule a 8 ml HES-medium supplementato con 10 mM Y-27632 dicloridrato. </li><li> Aspirare il matrigel da 10 cm piatto precedentemente preparato. Piastra le cellule con un rapporto di 1:1 o 01:02 (per esempio: le cellule da un piatto 10 centimetri sono divisi in due 10 centimetri piatti) sulla piastra matrigel. Incubare a 37 °C durante la notte. Nota: Questo placcatura dovrebbe portare a circa 100.000 cellule / cm 2. </li></ol> 3. Induzione della differenziazione neurale Nota: La differenziazione può essere iniziata (giorno 0) quando le cellule sono 90-100% confluenti (vedere Figura 1C), di solito il giorno seguente. Se la confluenza preciso non è ancora raggiunto, le cellule possono essere alimentati quotidianamente con HES-medio finché non sono pronti per la differenziazione. In alternativa, il numero iniziale di cellule piastrate può essere aumentata. <ol><li> Il giorno 0-3, nutrire le cellule al giorno con 10 ml/10 cm servire a medio KSR-differenziazione contenente 0,1 micron LDN193189 e 10 micron SB431542. </li><li> Il giorno 4 e 5 alimenti le cellule con il 75% di media KSR-differenziazione e il 25% a medio N2-differenziazione entrambi contenenti LDN193189 e SB431542. </li><li> Il giorno 6 e 7 mangimi le cellule con 50% medio KSR-differenziazione e 50% N2 medio-differenziazione entrambi contenenti LDN193189 e SB431542. </li><li> Il giorno 8 e 9 nutrire le cellule con il 25% di media KSR-differenziazione e il 75% a medio N2-differenziazione entrambi contenenti LDN193189 e SB431542. </li><li> Il giorno 9 e 10 preparare piatti PO / Lam / FN come indicato in 1.2.2 per replating delle cellule il giorno 11. </li><li> Il giorno 10 celle di alimentazione a media N2-differenziazione contenente LDN193189 e SB431542. </li></ol> 4. Replating in Goccioline per NC Specifica <ol><li> Il giorno 11 aspirato Lam / FN dalle piastre precedentemente preparati e lasciarli asciugare completamente a temperatura ambiente per 20-30 minuti, come spiegato in 1.2.2. </li><li> Rimuovere mezzo dalle cellule differenziazione, lavare le cellule una volta con PBS 1x e aggiungere 8 ml accutase/10 cm piatto. Incubare per 20 minuti a 37 ° C. </li><li> Utilizzando un sollevatore cella, staccare le cellule e risospendere in accutase con una pipetta da 5 ml. Trasferire la sospensione in una provetta da 15 ml. </li><li> Aggiungere 5 ml di PBS 1x e far girare le cellule per 5 min a 114 x g. </li><li> Risospendere le cellule in 10 ml 1x PBS. Per garantire una sospensione di cellule singole, li filtrare attraverso un colino cella 40 pm e contare le cellule utilizzando un emocitometro o tecnica equivalente. </li><li> Spin le cellule giù e risospendere in mezzo N2-differenziazione contenente 200 mM Acido ascorbico (AA), 20 ng / ml BDNF, 100 ng / ml FGF8, 20 ng / ml SHH, 10 pM Y-27632 dicloridrato alla concentrazione di 100.000 -150.000 cells/10 microlitri. </li><li> Utilizzando un repeat-pipetta, piatto 10 gocce microlitri vicini gli uni agli altri senza toccarli sul PO essiccati / Lam / FN 15 centimetri piatti. Nota: Un 10 centimetri piatto di cellule differenziate normalmente si traduce in piatti cm o circa 100 x 10 microlitri droplets/15 cm piatto circa 2-3 volte 15. </li><li> Lasciate che le goccioline stanno a temperatura ambiente per 20-30 minuti, poi con molta attenzione (per non disturbare le goccioline) aggiungere 30 ml di N2/AA/BDNF/FGF8/SHH/Y e incubare a 37 ° C. </li><li> Il giorno 12, con attenzione nutrire le cellule con 20 ml/15 cm piatto N2/AA/BDNF/FGF8/SHH. </li><li> Da day 14-17 nutrire le cellule ogni secondo o terzo giorno con N2/AA/BDNF/FGF8. </li><li> Il giorno 16 e 17 preparare PO / piastre Lam / FN per replate celle NC dopo FACS ordinamento. </li></ol> 5. Fluorescence Activated Cell (FACS) di celle NC Nota: La preparazione delle cellule per FACS richiede circa 2 ore. <ol><li> Il giorno 18 di media rimuovere, lavare le cellule una volta con PBS 1x nel piatto e aggiungere 12 ml accutase. Incubare le cellule per 20 min a 37 ° C. </li><li> Utilizzando un sollevatore cellulare, staccare le cellule dalla superficie e lasciarli galleggiare. Pipettare le cellule in sospensione con una pipetta da 5 ml, trasferirli in una provetta da 50 ml e aggiungere 30 ml di 1x PBS. Centrifugare le cellule per 5 min a 114 x g. </li><li> Usando una pipetta P1000, risospendere le cellule in 1 ml di 2% FBS / HBSS (il HBSS contenente 15 mM HEPES). Aggiungere 19 ml di 2% FBS / HBSS e filtrare le cellule attraverso un colino cella 40 micron per garantire una sospensione di cellule singole. Incubare le cellule in ghiaccio per 15 min per gli anticorpi di blocco e poi contare utilizzando un emocitometro. </li><li> Spin le cellule verso il basso e risospendere alla concentrazione di 10 milioni di cellule / ml a 2% FBS / HBSS. </li><li> Mettere da parte 1 milione di celle ciascuno per la macchia, single-macchiato (HNK-1 solo e soltanto p75) e anticorpo secondario macchiato solo (APC solo ed esclusivamente 488) FACS controlla. </li><li> Aggiungere 5 ml di HNK-1 e 5 ml di p75 anticorpo primario per 10 milioni di cellule in 1 ml di sospensione ai campioni adeguati e incubare per 20 min in ghiaccio. </li><li> Lavare le cellule in 10 ml di PBS 1x (centrifuga 5 min a 114 xg) e risospendere le cellule in 1 ml 2% FBS / HBSS. Aggiungere 2 ml di APC e 1 ml di 488 anticorpo secondario per 10 milioni di cellule in 1 ml di sospensione ai campioni adeguati e incubare per 20 min in ghiaccio al buio. Nota: APC e 488 sono gli anticorpi secondari utilizzati qui per la HNK-1 e p75 colorazione, rispettivamente. </li><li> Lavare le cellule in 10 ml 1x PBS due volte, risospendere 10 milioni di cellule in 500 ml di2% FBS / HBSS contenente DAPI (a 0.5 ng / ml) o opportuno macchia cellule vive alternativa di escludere le cellule morte dalla analisi FACS. </li><li> Trasferire i campioni di appropriarsi tubi FACS. </li><li> Preparare tubi FACS con 0,5 ml 2% FBS / HBSS per la raccolta delle cellule. Nota: Le celle e tubi di raccolta sono tenuti in ghiaccio al buio durante il tempo di ordinamento. </li><li> Utilizzando una macchina di smistamento delle cellule con il laser in grado di rilevare DAPI, APC e 488 specie DAPI-/HNK-1 + / P75 + cellule doppie positive. Nota: Tempo di preparazione e manipolazione delle cellule porta ad una piccola percentuale di cellule morte, macchie DAPI cellule morte permette solo e quindi per queste cellule per essere esclusi dalla popolazione ordinato. Qualsiasi alternativa macchia dal vivo / morto funziona altrettanto bene per questo scopo. DAPI sé non causa citotossicità nella popolazione di cellule vivo. </li></ol> 6. Replating di cellule ordinati, NC manutenzione ed espansione Nota: le cellule FACS ordinati dovrebbero essere a manocondotto con particolare cura per garantire la sopravvivenza ottimale. Tenerli in ghiaccio fino al loro replating. Non vortex o pipettare loro duramente. Le cellule possono essere risospese muovendo il tubo. <ol><li> Dopo FACS ordinamento, contare le celle NC, poi girare giù e risospendere in mezzo N2 differenziazione integrato con 10 ng / ml FGF2, 20 ng / ml EGF e 10 micron Y-27632 dicloridrato nel volume appropriato per loro replate a 30.000 cellule / 10 gocce microlitri. </li><li> Asciugare accuratamente preparata in precedenza PO piastre / Lam / FN e piatto circa 50-70 gocce per 10 centimetri piatto. Lasciate che i piatti si distinguono a temperatura ambiente per 20-30 min. </li><li> Aggiungere molto attentamente 20 ml/10 cm piatto di N2/FGF2/EGF/Y-27632 senza disturbare le goccioline. Incubare le cellule a 37 ° C. </li><li> Nutrire le cellule ogni 2-3 giorni con N2/FGF2/EGF. Nota: le cellule NC vengono espanse o diversi passaggi circa ogni 4-5 giorni o quando iniziano ad accumularsi all&#39;interno delle goccioline. </li><li> Per il passaggio delle cellule NC, rimuovere il mezzo, wash le cellule una volta con PBS 1x e aggiungere 8 ml accutase/10 cm piatto. Incubare per 20 minuti a 37 ° C. </li><li> Pipettare le cellule dalla piastra con una pipetta 5 ml e trasferirli in una provetta da 15 ml. Aggiungere 6 ml di PBS 1X. </li><li> Centrifugare le cellule per 5 min a 114 x g. </li><li> Risospendere le cellule in mezzo N2-differenziazione addizionato con 10 ng / ml FGF2, 20 ng / ml EGF e 10 pM Y-27632 dicloridrato per avere 20.000 cells/10 microlitri gocciolina. </li><li> Replate le cellule in 10 gocce microlitri sulle secche piatti PO / Lam / FN. Lasciate che i piatti si distinguono a temperatura ambiente per 20-30 min. </li><li> Aggiungere con cautela 20 ml/10 cm piatto di N2/FGF2/EGF/Y-27632. Incubare a 37 ° C. Nota: le cellule NC possono essere mantenuti o ampliati per un massimo di due settimane come una popolazione progenitore relativamente omogenea. Cultura più può portare loro di differenziarsi in vari tipi di cellule progenie. </li></ol>

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Per la differenziazione di successo di cellule R-NC da hESC / hiPSCs devono essere effettuate le seguenti considerazioni. E &#39;fondamentale per lavorare in condizioni di coltura sterili in ogni momento. In particolare, è importante testare culture HPSC per contaminazione micoplasma regolarmente, poiché questa contaminazione ostacolerà differenziazione successo, ma non può essere facilmente rilevata visivamente in culture HPSC. La differenziazione R-NC deve essere iniziato al 90-100% della densità cellulare; densi...

Le cellule staminali embrionali umane (hESC) e le cellule staminali umane pluripotenti indotte (hiPSC) hanno mostrato un potenziale immenso, in particolare per la ricerca e il trattamento futuro delle malattie umane per i quali sono disponibili né modelli animali buoni né i tessuti primari. Esempi di applicazione per la tecnologia hESC / hiPSC sono i seguenti: Celle di particolare interesse possono essere generati da hESC / hiPSCs per la medicina rigenerativa in quantità illimitata 1. Le cellule possono essere prodotti da pazienti portatori di una malattia specifica e usati per stabilire in modelli di malattia vitro 2,3. Tali modelli di malattia possono poi essere utilizzati per lo screening di stupefacenti su larga scala nella ricerca di nuovi composti farmacologici 4, nonché la sperimentazione di farmaci esistenti per l&#39;efficacia e la tossicità 5. Modelli in vitro di malattia può portare alla identificazione di nuovi meccanismi di malattia. Per tutte le applicazioni della tecnologia hESC / iPSC è importante lavorare con specifici e ben definiretipi d cellule colpite nella malattia di interesse. Pertanto, la disponibilità di protocolli di differenziazione solidi e riproducibili in vitro è fondamentale per tutte le applicazioni della tecnologia hESC / hiPSC. I protocolli sono desiderabili che mostrano la variabilità minima, spese tempo, fatica, difficoltà e costi, nonché la riproducibilità massima tra le linee hESC / hiPSC e diversi ricercatori. Cresta neurale (NC), le cellule emergono durante neurulazione vertebrati tra l&#39;epidermide e l&#39;epitelio neurale. Essi proliferano e migrano ampiamente in tutto l&#39;embrione in via di sviluppo e danno luogo a una varietà impressionante di tipi di cellule progenie, tra cui ossa / cartilagine, lo scheletro cranio-facciale, nervi sensoriali, cellule di Schwann, melanociti, cellule muscolari lisce, i neuroni enterici, i neuroni autonomici, cellule cromaffini , cellule setto cardiaco, denti e surrenale / tiroide cellule ghiandolari 6. Così, le cellule NC sono un tipo cellulare attraente per il campo delle cellule staminali e importante per lamodellazione di una varietà di malattie, come la malattia di Hirschsprung 7, familiare Disautonomia 8 nonché tumori come il neuroblastoma 9. Inoltre, offrono la possibilità di studiare aspetti dello sviluppo embrionale umano in vitro. Il attualmente disponibili e ampiamente applicato in protocollo di differenziazione in vitro per la derivazione di cellule NC da hESCs 10,11 richiede fino a 35 giorni di differenziazione e comporta l&#39;induzione neurale delle cellule stromali di alimentazione come le cellule MS5 e viene così eseguita in condizioni scarsamente definiti. Mentre può essere up-scalata a generare grandi quantità di celle NC, ad esempio necessaria per high throughput screening farmacologico 4, questo è il lavoro e costosi. Inoltre, esso comporta passaging manuale di rosette neurali, che possono essere difficili da riprodurre e quindi è soggetta a variabilità complessiva, in particolare quando viene applicato ad una grande varietà di hESC o hiPSClinee. Qui, viene mostrata la derivazione graduale delle cellule NC in un protocollo di 18 giorni che è privo di cellule di alimentazione. Questo metodo è più breve e più definito rispetto al protocollo attualmente utilizzato. Inoltre, è molto robusto nel generare celle NC tra diverse linee hiPSC. Importante, è dimostrato che le cellule CN prodotti dai entrambi i protocolli emergono al confine di rosette neurali (qui di seguito denominato rosetta-NC o R-NC). Le cellule derivate utilizzando uno dei due protocolli appaiono morfologicamente identici, esprimono gli stessi marcatori NC e si raggruppano in analisi di microarray. Celle NC derivate utilizzando il nuovo protocollo (R-NC) sono funzionali, simili alle cellule derivate NC utilizzando il vecchio protocollo (MS5-R-NC) tale che possono migrare e ulteriormente differenziarsi in neuroni. Pertanto, le cellule possono essere usati contemporaneamente con le cellule MS5-R-NC. Il protocollo di cellule R-NC per la derivazione di cellule NC da cellule staminali embrionali umane / iPSC sarà utile per tutte le applicazioni della tecnologia hESC / iPSC che coinvolge il lignaggio NC. &lt;/ P&gt;

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			<td height="21" style="height:21px;width:353px;">Regents</td>
			<td style="width:219px;">company</td>
			<td style="width:160px;">cataloge number</td>
			<td style="width:165px;">comments</td>
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			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Gibco-Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum Replacement</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td>Lot should be tested</td>
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			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
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			<td height="20" style="height:20px;">Penicinlin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM minimum essential amino acids solution&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-050</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">&#946;-Mercaptoethanol&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>toxic</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human FGF basic (FGF2)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
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			<td height="20" style="height:20px;">DMEM/F12 powder&#160;</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
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			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APO human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
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			<td height="20" style="height:20px;">Human insulin&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Putriscine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-L Ornithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase in Hank&#39;s Balanced Salt Solution 5U/ml</td>
			<td>Stem Cell Technologies</td>
			<td>7013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">FGF8</td>
			<td>R&#38;D Systems</td>
			<td>423-F8</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Mouse recombinant sonic hedgehog (SHH)</td>
			<td>R&#38;D Systems</td>
			<td>464SH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco-Life Technologies</td>
			<td>14170-112</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Gibco-Life Technologies</td>
			<td>1563-080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant EGF</td>
			<td>R&#38;D Systems</td>
			<td>236EG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti HNK-1/N-Cam (CD57) mIgM</td>
			<td>Sigma</td>
			<td>C6680-100TST</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-p75 mIgG1 (Nerve Growth factor receptor)</td>
			<td>Advanced Targeting Systems</td>
			<td>AB-N07</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APC rat anti-mIgM</td>
			<td>BD Parmingen</td>
			<td>550676</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 goat anti-mouse IgG1&#160;</td>
			<td>Invitrogen</td>
			<td>A21121</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Anti Oct4 mIgG2b (used at 1:200 dilution)</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Material/Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-6105M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture dishes: 10cm and 15 cm plates, centrifuge tubes, FACS tubes,&#160; pipetts, pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass hematocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture incubator (CO2, humidity and temperature controlled)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture laminar flow hood with embedded microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture biosafety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell sorting machine, i.e. MoFlo</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml TB syringe 27Gx1/2</td>
			<td>BD Biosciences</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell lifter Polyethylene</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td></td>
		</tr>
	</tbody>
</table>

feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells

Le cellule umane staminali pluripotenti (hPSCs) hanno un grande potenziale per lo studio dello sviluppo embrionale umano, per modellare malattie umane nel piatto e come fonte di cellule trapiantabili per applicazioni rigenerative dopo malattie o incidenti. Cresta (NC), le cellule neurali sono i precursori per una grande varietà di cellule somatiche adulte, come le cellule del sistema nervoso periferico e glia, melanociti e cellule mesenchimali. Essi sono una preziosa fonte di cellule per studiare gli aspetti dello sviluppo embrionale umana, inclusa la specificazione destino delle cellule e la migrazione. Ulteriore differenziazione delle cellule progenitrici NC in tipi di cellule terminalmente differenziate offre la possibilità di modellare malattie umane in vitro, studiare meccanismi di malattia e di generare cellule per la medicina rigenerativa. Questo articolo presenta l&#39;adattamento di un protocollo vitro attualmente disponibile nella differenziazione per la derivazione di cellule NC da hPSCs. Questo nuovo protocollo richiede 18 giorni di differentiation, è facilmente scalabile e altamente riproducibile tra cellule staminali embrionali umane (hESC) linee così come cellule staminali pluripotenti indotte umane (hiPSC) linee privo di alimentatore. Entrambi i protocolli vecchi e nuovi producono celle NC di uguale identità.

I due più importanti miglioramenti del protocollo R-NC tramite il protocollo MS5-R-NC 11 sono le condizioni differenziazione definite libero-feeder e la riduzione complessiva del requisito tempo. Cellule di alimentazione MS5 13 sono di midollo osseo cellule stromali derivate murine che hanno dimostrato di sostenere differenziamento neurale da hESC 14. HESCs coltivati ​​su cellule di alimentazione MS5 presso le strutture epiteliali bassa densità di forma e rosette neurali 15,</...

Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Cresta neurale (NC), le cellule derivate da cellule umane staminali pluripotenti (HPSC) hanno un grande potenziale per modellare lo sviluppo umano e la malattia e per le terapie di sostituzione cellulare. Qui, un libero adattamento-feeder della tuttora ampiamente utilizzate<em&gt; In vitro</em&gt; Protocollo di differenziazione per la derivazione di cellule NC da hPSCs è presentato.

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

feeder-free-derivation-neural-crest-progenitor-cells-from-human

Research

JoVE Journal

Neuroscience

14.1K Views.  Sloan-Kettering Institute for Cancer Research. Cresta neurale (NC), le cellule derivate da cellule umane staminali pluripotenti (HPSC) hanno un grande potenziale per modellare lo sviluppo umano e la malattia e per le terapie di sostituzione cellulare. Qui, un libero adattamento-feeder della tuttora ampiamente utilizzate In vitro Protocollo di differenziazione per la derivazione di cellule NC da hPSCs è presentato.

Video: Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-formanti colonia saggio Cell: un test per discriminare Bona Fide cellule staminali neurali da cellule progenitrici neurali

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Ricerca

Neuroscienze

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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

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

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

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

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

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivazione efficiente di progenitori neuronali umane e Neuroni pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

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

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

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

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

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

La specifica di telencefalici neuroni glutamatergici di cellule staminali umane pluripotenti

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Un metodo di espansione cGMP-applicabile per Aggregati di neurali umane staminali e cellule progenitrici derivate da cellule staminali pluripotenti o fetale tessuto cerebrale

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Regia neuroni dopaminergici differenziazione da cellule staminali pluripotenti umane

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Derivazione di adulti fibroblasti umani e la loro conversione diretta in celle espandibile progenitrici neurali

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificazione dei marcatori di superficie cellulare delle cellule staminali primarie e delle cellule progenitrici mediante l'etichettatura metabolica di Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...