The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

1 Preparazione della Cultura media <ol><li> Preparare topo fibroblasti embrionali (MEF) medium combinando il seguente: 445 ml di DMEM, 50 ml di siero fetale bovino (FBS), e 5 ml 100x penicillina / ampicillina soluzione stock. Tenere il filtro medio sterilizzato a 4 ° C per non più di 14 giorni. </li><li> Preparare siero contenente HPSC terreno di coltura combinando il seguente: 385 ml DMEM / F12, 100 ml di siero sostituzione knockout (KSR), 5 ml di 100x non essenziali aminoacidi soluzione madre, 5 ml 100x penicillina / ampicillina soluzione madre, 5 ml di 100x -mercaptoethanol soluzione stock, e 10 ng / ml bFGF. Tenere il filtro medio sterilizzato a 4 ° C per non più di 10 giorni. </li><li> Preparare mTeSR1 terreno privo di siero combinando il seguente: 400 ml mTeSR1 medio basale, 100 ml 5x supplemento, e 5 ml 100x penicillina / ampicillina soluzione stock. Mantenere il substrato a 4 ° C per non più di 10 giorni. </li><li> Preparare IV soluzione madre 10x collagenasi: pesare 0,5 g potere collagenasi IV, esciogliere con 50 ml di DMEM / F12 e filtro sterilizzare. Preparare delle aliquote e li scorta a -20 ° C per mesi. </li><li> Preparare la soluzione di gelatina allo 0,1%: pesare 0,5 g di gelatina (da pelle bovina, di tipo B), il potere e scioglierlo con acqua deionizzata. Mantenere soluzione sterilizzata in autoclave a temperatura ambiente per settimane. </li><li> Preparare KSR medio differenziazione combinando il seguente: 410 ml di DMEM, 75 ml KSR, 5 ml di soluzione 100X non essenziali aminoacidi magazzino, 5 ml 100x penicillina / soluzione madre ampicillina, 5 ml di soluzione madre 100X -mercaptoethanol. Tenere il filtro medio sterilizzato a 4 ° C per non più di 10 giorni. NOTA: KSR varia da lotto a lotto, che possono influenzare l&#39;efficienza di differenziazione. E &#39;quindi meglio provare diversi lotti di KSR per trovare quello migliore per la differenziazione. </li><li> Preparare N2 medio differenziazione combinando il seguente: 98 ml di DMEM, 1 ml 100x supplemento N2 e 1 ml 100x penicillina / ampicillina soluzione stock. Tenere il filtro medio sterilizzato a 4° C per non più di 10 giorni. </li><li> Preparare B27 medio differenziazione combinando il seguente: 480 ml di mezzo Neurobasal, 10 ml 50x B27 supplemento, 5 ml di 100x Glutamax soluzione madre, e 5 ml 100x penicillina / ampicillina soluzione stock. Tenere il filtro medio sterilizzato a 4 ° C per non più di 10 giorni. </li></ol> 2. Cultura di hESC e hiPSCs su cellule di alimentatore MEF Le hESC H9 sono stati ottenuti da WiCell Research Institute e hiPSCs sono stati stabiliti in laboratorio Reijo Pera attraverso retrovirus-mediata trasduzione di Yamanaka fattori Oct3 / 4, SOX2, Klf4, e c-MYC 18. <ol><li> Almeno un giorno prima del passaggio delle cellule, preparare alcuni piatti gelatina aggiungendo 1 ml di 0,1% di gelatina a 1 pozzetto di piastre da 6 pozzetti e incubare le piastre a 37 ° C per almeno 30 min. </li><li> Dopo l&#39;incubazione, aspirare gelatina dalle piastre e sementi irradiazione inattivato cellule MEF sulle piastre alla densità di 1,6 x 10 5 cellule/ Pozzetto in mezzo MEF utilizzando un emocitometro per contare le cellule. NOTA: In alternativa preparare alimentatori MEF fin da 3 giorni prima del passaggio delle cellule. </li><li> Cellule Passage Un giorno, dopo la placcatura alimentatori MEF: media aspirato da hPSCs, aggiungere 1 mg / ml di collagenasi IV a celle a 1 ml / pozzetto e incubare le cellule a 37 ° C per 1 ora. </li><li> Durante questa incubazione, lavare alimentatori MEF una volta con PBS, e quindi aggiungere calda (37 ° C) siero contenente HPSC terreno di coltura a 1,5 ml / pozzetto. </li><li> Dopo 1 ora, toccare la piastra di coltura un paio di volte in modo che la maggior parte delle colonie indifferenziate si staccherà dalle piastre, mentre le colonie differenziate rimangono attaccati. Raccogliere accuratamente le colonie galleggianti, e trasferirli in provetta 15 ml. </li><li> Lavare delicatamente la piastra una volta con acqua calda (37 ° C) DMEM / F12 e trasferire DMEM / F12 nello stesso tubo da 15 ml. </li><li> Centrifugare le provette a 179 xg per 3 min. </li><li> Aspirare il terreno dai tubi, facendo attenzione a non disturbare il pellet. Aggiungi guerram HPSC terreno di coltura, le cellule pipettare su e giù più volte per romperli in piccoli gruppi e mescolare bene. </li><li> Trasferire le cellule su nuovi alimentatori MEF a 1: 3-1: 6 rapporto. </li><li> Cambiare la media ogni giorno e il passaggio delle cellule ogni 5-6 giorni. </li></ol> 3 Preparazione di cellule di Differenziazione <ol><li> Almeno un giorno prima della preparazione delle cellule, preparare alcuni piatti Matrigel (tipicamente 3 pozzetti di una piastra da 6 pozzetti). Diluire il Matrigel con il freddo (4 ° C) DMEM / F12 a 01:40 e aggiungere 1 ml di Matrigel per pozzetto di 6 pozzetti. Incubare le piastre Matrigel a 4 ° C durante la notte. NOTA: Si può sigillare le piastre Matrigel con Parafilm e scorta a 4 ° C per tutto il tempo una settimana. </li><li> Utilizzare cellule (vedi punto 2) 4 giorni dopo il passaggio. Rimuovere tutte le colonie differenziate dalle piastre. Aspirare il terreno dalla piastra di coltura, aggiungere 1 ml accutase per pozzetto e incubare le cellule a 37 ° C per 5 min. </li><li> Durante l&#39;incubazione, preparare alcuni gelAtin piatti con l&#39;aggiunta di 1 ml di gelatina a 1 pozzetto di 6 pozzetti. Posizionare questi piatti e le Matrigel piastre preparate il giorno prima in incubatrice. </li><li> Dopo 5 min di incubazione o quando le cellule sono diventati semi-flottante, le cellule pipetta su e giù più volte per farli in cellule singole. </li><li> Raccogliere singole cellule in provette da 15 ml e centrifugare a 258 xg per 3 min. </li><li> Risospendere le cellule con un&#39;adeguata quantità di siero contenente HPSC mezzo di coltura, e aggiungere Thiazovivin alla concentrazione finale di 2 micron. </li><li> Trasferire le cellule su piastre di gelatina rivestite in rapporto 1: 1, e incubare le cellule a 37 ° C per 30 minuti per rimuovere alimentatori MEF. </li><li> Trasferire le cellule in una provetta da 15 ml, aggiungere siero appropriato contenente HPSC medio e contare le cellule con un emocitometro. </li><li> Centrifugare le cellule a 258 xg per 3 min. </li><li> Durante centrifuga, aggiungere Thiazovivin appropriarsi mTeSR1 mezzo per effettuare una concentrazione di 2 mM. </li><li> Dopo centrifuga, unaspirano medio e aggiungere mezzo appropriato mTeSR1 con Thiazovivin modo che ci sono 1,8 x 10 5 cellule / ml medie. </li><li> Estrarre le piastre Matrigel dal termostato, aspirare il Matrigel e aggiungere 2 ml di cellule miste su 1 bene dei 6 pozzetti. NOTA: La densità delle cellule dovrebbe essere 3,6 x 10 4 cellule / cm 2 di superficie. </li><li> Riportare le piastre torna l&#39;incubatrice, e lasciare che le cellule attaccano per 24 ore. </li><li> 24 ore dopo la placcatura le cellule, aspirare vecchio medio e aggiungere 2 ml nuovo mezzo mTeSR1 per pozzetto e lasciare le cellule crescono per un altro 24 ore prima di iniziare la differenziazione. </li></ol> Differenziazione cellulare <ol><li> Inizia differenziazione 48 ore dopo replating le cellule su piastre Matrigel. </li><li> Aspirare il terreno di coltura dalla piastra, lavare le cellule una volta con PBS, e aggiungere 2 ml della seguente mezzo di differenziazione per pozzetto: medio differenziazione KSR supplementato con 10 mM SB431542 e 100 nM LDN-193189. Segna questo giorno come D0 di differenziazione. </li><li> Modificare media ogni giorno da D0 a D20. NOTA: Si può preparare mezzo per uso non più di 2 giorni alla volta. </li><li> Utilizzare il seguente mezzo per D1 e D2: media differenziazione KSR supplementato con 10 mM SB431542, 100 nM LDN-193.189, 0,25 mM SAG, 2 mM purmorphamine, e 50 ng / ml FGF8b. </li><li> Utilizzare il seguente mezzo per D3 e D4: media differenziazione KSR supplementato con 10 mM SB431542, 100 nM LDN-193.189, 0,25 mM SAG, 2 mM purmorphamine, 50 ng / ml FGF8b, e 3 mM CHIR99021. </li><li> Utilizzare il seguente mezzo per D5 e D6: 75% KSR terreno di differenziamento più il 25% medio di differenziazione N2 integrato con 100 nM LDN-193.189, 0,25 mM SAG, 2 mM purmorphamine, 50 ng / ml FGF8b, e 3 mM CHIR99021. </li><li> Utilizzare il seguente mezzo per D7 e D8: 50% KSR terreno di differenziamento più il 50% medio di differenziazione N2 integrato con 100 nM LDN-193.189 e 3 micron CHIR99021.</li><li> Utilizzare il seguente mezzo per D9 e D10: 25% KSR terreno di differenziamento più il 75% medio di differenziazione N2 integrato con 100 nM LDN-193.189 e 3 micron CHIR99021. </li><li> Utilizzare il seguente mezzo per D11 e D12: media B27 differenziazione integrato con 3 mM CHIR99021, 10 ng / ml BDNF, 10 ng / ml GDNF, 1 ng / ml TGF3, acido ascorbico 0,2 mM e 0,1 mM cAMP. </li><li> Utilizzare il seguente mezzo per il resto della differenziazione: B27 medio differenziazione supplementato con 10 ng / ml BDNF, 10 ng / ml GDNF, 1 ng / ml TGF3, acido ascorbico 0,2 mM e 0,1 mM cAMP. </li><li> A circa D16 di differenziazione, preparare alcuni piatti / laminina / fibronectina Poly-L-ornitina. Diluire Poly-L-ornitina soluzione madre con il freddo (4 ° C) PBS a 15 mcg / ml, aggiungere 1 ml a 1 pozzetto di 6 pozzetti e incubare le piastre a 37 ° C durante la notte. </li><li> Aspirare la soluzione di poli-L-ornitina, lavare i piatti 3 volte con acqua sterile e poi asciugare le piastre. </li><li> Diluire laminina magazzino solution con il freddo (4 ° C) PBS a 1 mcg / ml, aggiungere 1 ml di un pozzetto di 6 pozzetti e incubare le piastre a 37 ° C durante la notte. </li><li> Aspirare la soluzione laminina, lavare i piatti 3 volte con acqua sterile e poi asciugare i piatti. </li><li> Diluire la soluzione fibronectina magazzino con il freddo (4 ° C) PBS per 2 mcg / ml, aggiungere 1 ml a 1 pozzetto di 6 pozzetti e incubare le piastre a 37 ° C durante la notte. </li><li> Chiudere le piastre con parafilm e metterli a 4 ° C per la durata di due settimane. </li><li> Dopo 20 giorni di differenziamento, le cellule replate alle piastre / laminina / fibronectina Poly-L-ornitina. Aspirare il terreno di differenziazione, aggiungere 1 ml di acqua calda (37 ° C) Accutase a 1 pozzetto di 6 pozzetti e incubare le cellule a 37 ° C per 5 min. </li><li> Durante l&#39;incubazione, posizionare le piastre / laminina / fibronectina Poly-L-ornitina nell&#39;incubatrice. </li><li> Dopo 5 min di incubazione o quando le cellule sono diventati semi-flottante, delicatamente le cellule pipetta su e giù diverse volte pertrasformarli in cellule singole. </li><li> Raccogliere le singole cellule in 15 ml tubi conici, e aggiungere la giusta quantità di mezzo Neurobasal per il conteggio delle cellule, che variano a seconda espansione (dopo 11 giorni di differenziamento, le cellule possono espandersi 15-20 volte). Contare le cellule utilizzando un emocitometro. </li><li> Centrifugare le cellule a 258 xg per 3 min. Aspirare il surnatante e risospendere le cellule con terreno di differenziamento B27 in modo che la concentrazione cellulare è di 1 x 10 6 cellule / ml medie. </li><li> Aspirare fibronectina dalle piastre, lavare due volte con PBS e aggiungere 2 ml di cellule di 1 pozzo di 6 pozzetti. NOTA: La densità delle cellule per replating dovrebbe essere 2 x 10 5 cellule / cm 2 di superficie. </li><li> Modificare media 24 ore più tardi per rimuovere tutte le cellule morte senza legami. </li><li> Modificare media ogni giorno fino a quando i punti di tempo desiderati per un dato esperimento. </li></ol>

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The authors declare that they have no competing financial interests.

Le cellule staminali pluripotenti umane (inclusi sia hESC e hiPSCs) possono essere differenziate in vitro per generare la maggior parte, se non tutti, i tipi di cellule del nostro corpo tra cui neuroni DA, che è stata dimostrata in studi precedenti 19. Qui, sulla base delle conoscenze da dopaminergici embrionale neurone sviluppo 20 e protocolli pubblicati da altri laboratori 14,15, abbiamo ottimizzato le condizioni di coltura per la generazione di neuroni DA provenienti da hESC...

Dopaminergica (DA), i neuroni possono essere trovati in diverse regioni del cervello, tra cui il mesencefalo, l&#39;ipotalamo, retina, e bulbi olfattivi. I neuroni A9 DA nella pars compacta della substantia nigra (SNPC) comportamento di controllo e il movimento proiettando alla striato nel proencefalo e formare il sistema motorio extrapiramidale. La degenerazione dei neuroni A9 DA conduce alla malattia di Parkinson (PD), che è la seconda malattia neurodegenerativa umana più comune e attualmente incurabile. Terapia di sostituzione cellulare è una delle strategie più promettenti per il trattamento della malattia di Parkinson 1, quindi, c&#39;è stato un grande interesse derivante neuroni A9 DA da cellule staminali umane pluripotenti (hPSCs), includendo sia le cellule staminali embrionali umane (hESC) e il recenti cellule staminali umane pluripotenti indotte (hiPSCs). Molta ricerca ha cercato di ricavare neuroni A9 DA da hPSCs utilizzando vari metodi. Le prime relazioni di tutti i neuroni DA generati attraverso una neuralefase progenitore rosetta. In co-coltura con MS5 2 o PA6 3-5 cellule stromali con o senza fattori di crescita esterna per 2-4 settimane, L. Studer e colleghi e di altri tre gruppi indotte con successo hESC per produrre rosette neurali. Hanno poi arricchito queste rosette di dissezione meccanica o digestione enzimatica per un&#39;ulteriore differenziazione. In altri rapporti, i ricercatori hanno generato rosette neurali attraverso il corpo embrioide (EB) galleggianti cultura differenziazione 6-9. In seguito, i ricercatori hanno stabilito un monostrato basato metodo differenziazione 10,11, dove si placcati hESC e hiPSCs sulla matrice extracellulare, aggiunti diversi fattori di crescita nella cultura di indurre la differenziazione di hPSCs di neuroni DA, che imita il vivo DA sviluppo embrionale dei neuroni in . Anche se tutti questi studi ottenuti tirosina idrossilasi (TH) cellule esprimente con alcune caratteristiche di neuroni DA, l&#39;intero processo di differenziazione è il tempo e il lavoro consumo,generalmente inefficiente, e, soprattutto, l&#39;identità A9 di questi neuroni non sono state dimostrate nella maggior parte degli studi, eccetto quella con Lmx1a espressione ectopica 12. Recentemente, un piatto nuovo piano (FP) basata su protocollo è stato sviluppato 13-16, in cui i precursori FP con DA potenziale del neurone sono stati generati dall&#39;attivazione del riccio Sonic e canoniche vie di segnalazione Wnt durante la fase iniziale della differenziazione, e quindi queste cellule FP sono stati ulteriormente specificati per neuroni DA. Anche se questo protocollo è più efficiente, ci sono ancora alcuni problemi; per esempio, l&#39;intero processo di differenziazione richiede molto tempo (almeno 35 giorni) ed è dipendente cella alimentatore 15, o è dipendente EB 16 o l&#39;identità A9 non è stato dimostrato 14. Qui, sulla base delle conoscenze di sviluppo embrionale in vivo DA neurone e risultati pubblicati di altri ricercatori, abbiamo ottimizzato le condizioni di coltura per l&#39;effgenerazione icient di DA neuroni da entrambi hESC e hiPSCs. In primo luogo abbiamo generato cellule FP precursori dalla attivazione del segnale Wnt canonica con piccola molecola CHIR99021 e sonic hedgehog segnalazione con piccole molecole SAG e purmorphamine. Queste cellule esprimono FP Foxa2, Lmx1a, CORIN, OTX2 e nestina. Abbiamo poi specificato queste cellule FP a neuroni DA con fattori di crescita, tra cui BDNF, GDNF, ecc. I neuroni DA generati sono di tipo cellulare A9 come sono positivi per GIRK2 mentre negativo per calbindin 17. Questo protocollo è cella alimentatore o EB indipendente, altamente efficiente e riproducibile. Usando questo protocollo, si può ricavare neuroni DA in meno di 4 settimane dalla hESC o hiPSCs di persone normali per lo studio trapianto di cellule, o da hiPSCs di pazienti PD per la modellazione in vitro di PD o testare potenziali agenti terapeutici per il PD.

<table border="0" cellpadding="0" cellspacing="0" width="778">
	<tbody>
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			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">26140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
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			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Collagenase IV</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Growth factor reduced matrigel</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:308px;">Accutase</td>
			<td style="width:184px;">MP Biomedicals</td>
			<td style="width:119px;">1000449</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SB431542</td>
			<td style="width:184px;">Tocris Bioscience</td>
			<td style="width:119px;">1614</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
			<td style="width:184px;">Stemgent</td>
			<td style="width:119px;">04-0074</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
			<td style="width:184px;">R&#38;D Systems</td>
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			<td style="width:167px;"></td>
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			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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.

Una panoramica del protocollo differenziazione è mostrato in Figura 1. L&#39;efficienza del protocollo differenziazione presentato qui invoca lo stato delle celle di partenza. Pertanto, è fondamentale fare in modo che, prima rimuove tutte le colonie differenziate prima di dissociare hPSCs in celle singole per la differenziazione, e la seconda, una esaurisce la maggior parte, se non tutte, le cellule di alimentazione MEF incubando le cellule su piastre di gelatina rivestite per 30 minuti, e il terzo, u...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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 ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.3K Views.  Stanford University School of Medicine. 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’s disease.

Video: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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 ...

Ricerca

Neuroscienze

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 ...

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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

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

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Replatazione post-differenziazione dei neuroni derivati da cellule staminali pluripotenti umane per lo screening ad alto contenuto di neuritogenesi e maturazione delle sinapsi

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Differenziazione neuronale dalle cellule staminali embrionali del topo in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Derivazione, espansione, crioconservazione e caratterizzazione di cellule endoteliali microvascolari cerebrali da cellule staminali pluripotenti indotte dall'uomo

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Generazione di neuroni umani e oligodendrociti da cellule staminali pluripotenti per la modellazione delle interazioni neurone-oligodendrociti

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Cellule simili alla microglia umana: differenziazione da cellule staminali pluripotenti indotte e saggio di fagocitosi di cellule vive in vitro utilizzando sinaptosomi umani

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Colture organotipiche della corteccia umana adulta come modello ex vivo per il trapianto e la validazione di cellule staminali umane

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...