Gli autori desiderano ringraziare il Dr. Y. Sasai generosamente per fornire l&#39;anticorpo Foxg1. Questo lavoro è stato sostenuto dalle concessioni ricerca con le cellule staminali (Connecticut 08-SCB-UCHC- 022 e 11-SCB24) e della Fondazione Paraplegia spastica.

1. Generazione di aggregati umani di cellule staminali pluripotenti (D1-D4) <ol><li> Le cellule staminali pluripotenti sono coltivati ​​in fibroblasti embrionali di topo (MEF) alimentatori in presenza di terreno hESC supplementato con fattore di crescita dei fibroblasti (bFGF, 4 ng / ml). Dopo 5-7 giorni di cultura, quando le colonie sono grandi, ma ancora indifferenziati, sono pronti per il passo successivo. </li><li> La soluzione enzimatica deve prima essere preparato. In una provetta da 50 ml, sciogliere il dispasi (o collagenasi) a mg / ml di concentrazione in 1 DMEM/F12 medie. Dal momento che queste soluzioni mescolare meglio quando riscaldato, porre la provetta contenente il mezzo e l&#39;enzima in un bagno d&#39;acqua a 37 ° C per 10-15 minuti e poi sterilizzare utilizzando un filtro Steriflip. </li><li> Aspirare il mezzo fuori dalle cellule, sciacquare con DMEM/F12, e aspirare il fuori pure. Aggiungere 1 ml di dispasi in ogni pozzetto e posto in incubatrice per 3-5 min per hESC (fino a 10 min per iPSCs). Guarda le cellule al microscopio, quandoarricciare i bordi leggermente / guardare un po &#39;più scuro, le cellule sono pronti per il passo successivo. Si consiglia di controllare le celle dopo 3 minuti e riporli in caso di necessità. Quando si utilizza questo protocollo per la prima volta, è meglio cominciare con solo una piastra di cellule in un momento in quanto è importante rimuovere le cellule dal dispasi più rapidamente possibile. </li><li> Aspirare il dispasi fuori dalle cellule. Delicatamente (le cellule sono molto vulnerabili in questa fase e verrà via della piastra con relativa facilità) aggiungere 1,5 ml di DMEM/F12 media in ciascun pozzetto in modo da risciacquare il dispasi. Aspirare fuori DMEM/F12 e aggiungere 1,5 ml di mezzo di hESC in ciascun pozzetto. </li><li> Per staccare e rompere le cellule, posizionare la punta di una pipetta 10 ml in vetro verso l&#39;angolo in basso a destra di un pozzo (toccando le cellule) e spostare la pipetta su e giù (in verticale) - verso l&#39;alto stoke dovrebbe essere nelle immediate vicinanze la corsa verso il basso procedimento. Una volta che il lato del bene è arrivato, ripetere la disastrosa orizzontalection. </li><li> Una volta che tutte le cellule sono in sospensione, metterli in provette da 15 ml e centrifugare loro a 200 xg per 2 min. Ci dovrebbe essere un pellet di cellule al fondo della provetta. Aspirare il mezzo fuori da sopra il pellet. </li><li> La dimensione del pallone di coltura cellulare che dovrebbe essere utilizzato per mantenere gli aggregati dipende dalla quantità originale di cellule. Da 6 pozzetti (1 piastra) di hESC, uno non-tessuto coltura trattata T75 pallone con 50 ml di mezzo hESC devono essere utilizzati. Trasferire le cellule dal passo 1,6 al pallone e incubare a 37 ° C. </li><li> Al fine di eliminare i detriti cellulari, il mezzo cella e pallone deve essere sostituito dopo circa 12 ore. Per fare questo, inserire tutti i medio / cellule in una provetta da 50 ml, far girare le celle in basso (200 xg per 2 minuti), e aspirare fuori il mezzo vecchio. Successivamente, aggiungere 50 ml di terreno fresco hESC alle cellule, e posto in un nuovo T75 pallone. Le cellule possono quindi essere alimentati quotidianamente seguendo la stessa procedura (fino al giorno 5). </li></ol><ol><li> Trasferire le cellule / mezzo di un tubo da 50 ml, e centrifugare le cellule per 2 minuti a 200 x g. Successivo, aspirare il surnatante, e trasferire le cellule ad una nuova (non-tessuto coltura trattata) T75 pallone con 50 ml di mezzo di induzione neurale (NIM). </li><li> Una volta che le cellule sono in NIM, mezzo del mezzo deve essere sostituito ogni altro giorno. Gli aggregati dovrebbero diventare grande abbastanza per cui si depositano sul fondo di un tubo da 50 ml in proprio dopo pochi minuti (senza centrifugazione richiesto). Dopo 2-3 giorni di NIM (D7-D8 totale), le cellule devono essere pronti a piastra. Laminina o siero bovino fetale (FBS) può essere utilizzata per aiutare le cellule attaccano alle piastre. </li><li> Il numero di coltura di tessuti trattati piastre da 6 pozzetti necessari dipende dalla densità delle cellule come ogni aggregato deve essere in grado di crescere sul piatto per parecchi giorni senza toccare nessuna delle altre. In media, le celle da unaT75 pallone produrrà circa 4-6 6 piatti e vale la pena di cellule. </li><li><ol class="ualpha"><li> Se si utilizza laminina, diluirlo NIM al 10-20 ug / ml per rivestire la piastra da 6 pozzetti (~ 0,5 ml / pozzetto). Dopo il rivestimento per 2-3 ore, aggregati piastra cellulare in piastre da 6 pozzetti (~ 20-30 aggregati per pozzetto). Dopo gli aggregati hanno attaccato, aggiungere 1,5 ml di NIM. </li><li> Se si utilizza FBS, preparare una miscela di 90% NIM e 10% FBS (1,5 ml / pozzetto). Aggiungere 1,5 ml di terreno con le cellule di ogni pozzetto, scuotere delicatamente il piatto avanti e indietro per diffondere gli aggregati, e permettere alle cellule di attaccarsi O / N in un incubatore. Assicurarsi di cambiare il mezzo (2 ml / pozzetto di NIM) la mattina seguente per rimuovere la FBS. </li></ol></li><li> Dopo un giorno o due di essere placcato, ogni aggregato si allargano a formare un monostrato. Nel giro di un paio di giorni, i centri della maggior parte di queste cellule morfologicamente sembrano simili alle cellule colonnari (allungata). Durante questo periodo, modificare il NIM ogni altro giorno. Alcune linee di PSC (especially IPSC), possono formare cellule colonnari meno efficiente. In questo caso, l&#39;aggiunta di inibitori BMP e TGF (Dorsomorphin 29 SB431542 19 a 2 pm) durante la fase 2 può essere utile. </li><li> Intorno al giorno 14-17, le cellule colonnari apparirà più compatta. A volte queste cellule formano creste o anelli a lume visibili all&#39;interno. Oltre alla morfologia colonnare, le cellule in questa fase esprimere marcatori neuroepiteliale, come PAX6 e SOX1 12,26. </li><li> Prima di iniziare l&#39;isolamento, si deve prima esaminare le cellule per determinare se ci sono cellule presenti non neuroepiteliale. Un marcatore obiettivo può essere utilizzata per indicare i non-neuroepiteliale colonie. Questi possono poi essere eliminati raschiando via con una pipetta. </li></ol> 3. Generazione di Progenitori telencefalici (D17-D24) <ol><li> I centri delle colonie contengono le cellule neuroepiteliali, e deve essere isolata dal resto della colonia. Questo può essere fatto utilizzandouna micropipetta e un puntale sterile da 1 ml di filtrato. Quando una piccola quantità di pressione è applicata alla parte centrale della colonia, solleva generalmente fuori abbastanza facilmente. Essere prudenti per non lasciare la porzione esterna della colonia staccare pure. Se i centri sono testardo e non verranno per conto loro, essi possono essere rimossi ablazione utilizzando un ago al microscopio in una cappa sterile. Trasferire le porzioni centrali delle colonie in una provetta da 15 ml e centrifugare a 200 xg per 2 min. </li><li> Il terreno deve essere aspirato fuori delle cellule. Due piastre di valore di cellule può poi essere trasferito in un non-tessuto coltura trattata T25 pallone contenente 10 ml di NIM con l&#39;aggiunta di B27 (senza vitamina A, 1:50). Utilizzare 5-10 ml di terreno per una piastra di cellule. </li><li> Quando le cellule sono visti il ​​giorno seguente, essi dovrebbero avere una sfera come l&#39;apparenza. È necessario trasferire queste cellule in un matraccio nuova (in presenza di NIM e B27, 1:50) come è spesso cellule perifericheche furtivamente e allegare al fondo del pallone. </li><li> Il neurosfere dovrebbe quindi essere coltivati ​​in sospensione per una settimana con NIM integrato con B27 (1:50). Al fine di promuovere la resistenza e la proliferazione cellulare, AMP ciclico (cAMP, 1 pM) e fattore di crescita insulina (IGF1, 10 ng / ml) può essere aggiunto nel terreno di coltura durante la sospensione. A questo punto, le neurosfere contengono progenitori telencefalici Foxg1 (+) e sono pronti per essere piastrate su vetrini per differenziazione terminale. </li></ol> 4. Un&#39;ulteriore differenziazione nei neuroni telencefalici (D25 +) <ol><li><ol class="ualpha"><li> Preparare i coprioggetti poly-ornithine/laminin rivestite (in piastre da 24 pozzetti) per la placcatura cellule. Coprioggetti vengono prima puliti, e viene rivestito da manovra-ornitina (a 0,1 mg / ml, 37 ° C, O / N, e conservate nel congelatore). Poly-ornitina rivestimento: <ol><li> Sterilizzare i coprioggetti: Posizionare i vetrini in unbecher contenente acido nitrico e scuotere delicatamente sotto cappa per un&#39;ora. Versare l&#39;acido nitrico, risciacquare più volte con acqua deionizzata, e lasciare i coprioggetti sotto l&#39;acqua deionizzata per almeno 15 minuti. Posizionare i vetrini in etanolo al 95% e sia agitare per 30 minuti (se si utilizza subito) o conservare in etanolo a temperatura ambiente. </li><li> In una cappa sterile, versare il contenuto della provetta da 50 ml contenente coprioggetti sterilizzati nel coperchio di una piastra da 6 pozzetti. Utilizzando pinze sterili, prendere uno vetrino alla volta e in posizione verticale in un unico pozzetto di una piastra a 24 pozzetti. Ripetere in modo che ogni bene ha un coprioggetto. </li><li> Dopo aver asciugare completamente, toccare le piastre fino coprioggetti sono caduti appartamento in ciascun pozzetto. Aggiungere quindi 100 pl poli-ornitina (0,1 mg / ml in sterile dH 2 O) a ciascun vetrino coprioggetto e incubare le piastre O / N a 37 ° C. </li><li> Il giorno dopo, rimuovere le piastre dal termostato e lasciare raffreddare a temperatura ambiente. Aspirare poly-ornitina off di ciascun coprioggetto (dal bordo di tlui coprioggetto) facendo attenzione a non toccare o graffiare i coprioggetti nel processo. Lasciare i coprioggetti asciugare per circa 30 minuti prima del lavaggio. </li><li> Aggiungere 1 ml sterile dH 2 O a ciascun pozzetto, lasciate riposare per 10 min, e aspirare l&#39;acqua da ogni pozzetto. Ripetere questa fase di lavaggio altre due volte. Dopo le piastre asciugare completamente, (congedo copre fuori nella cappa) posizionare i coperchi su, coprire con un foglio di alluminio, e conservare a -20 ° C. </li></ol></li><li><ol><li> Per rivestire i coprioggetti con laminina, aggiungere 50 microlitri di una miscela di terreno di differenziamento neurale e laminina (concentrazione finale di 20 pg / ml) ad ogni poli-ornitina coprioggetto trattati facendo attenzione ad avere solo il liquido tocca la stessa coprioggetto (e non spargimento off). Incubare a 37 ° C per 1-2 ore prima di preparare le cellule per il fissaggio. </li></ol></li></ol></li><li> Raccogliere e centrifugare neurosfere a 200 xg per 2 minuti in una provetta da 15 ml. Aspirare il sopranatante. </li> &lt;li&gt; <ol class="ualpha"><li> Lavare le cellule con DMEM-F12 e centrifugare le cellule di nuovo. Se le neurosfere sono troppo grandi per collegare correttamente, possono essere dissociati con Accutase in questa fase. </li><li> Se si utilizza Accutase: Dopo aver lavato le cellule con DMEM-F12, le neurosfere sono dissociati per piccoli cluster aggiungendo Accutase (~ 2 ml) e incubando le cellule a 37 ° C per 3 min. Successivamente, aggiungere la stessa quantità di inibitore della tripsina (~ 2 ml) e centrifugare le cellule a 200 xg per 3 min. Le cellule devono quindi essere risospeso in NDM e rotto utilizzando una pipetta P200. I cluster di piccole dimensioni possono essere piastrate su vetrini pre-verniciati. </li></ol></li><li> Aspirare off la maggioranza del mezzo e posizionare le cellule su un piatto Petri 6 centimetri in poche gocce del mezzo in modo che selezionandoli diventa più facile. Aggiungi circa 4-5 neurosfere di ogni pre-rivestito coprioggetto. Non vi è alcuna necessità di aspirare largo della laminina prima. </li><li> Lasciate che il neurosfere attaccare per almeno 2-4 ore. Una volta che hanno unttached bene, più a medio (0,5 ml / vetrino) dovrebbe essere aggiunto. Si dovrebbe consistere di terreno di differenziamento neurale supplementato con B27 (01:50), cAMP (1 pM), e fattori neurotrofici (BDNF, GDNF, e IGF, il tutto a 10 ng / ml). A questo punto mezzo del mezzo può essere cambiato ogni altro giorno e queste cellule possono essere mantenute per diversi mesi. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+enhancement+of+neural+differentiation+from+human+ES+and+iPS+cells+regardless+of+their+innate+difference+in+differentiation+propensity.">Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity.</a> Stem Cell Rev. 6, 270-281 (2010).</li><li>Zhang, X., Huang, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+is+a+human+neuroectoderm+cell+fate+determinant.">Pax6 is a human neuroectoderm cell fate determinant.</a> Cell Stem Cell. 7, 90-100 (2010).</li><li>Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+patterning+of+the+central+nervous+system:+how+many+organizers.">Initial patterning of the central nervous system: how many organizers.</a> Nat. Rev. Neurosci. 2, 92-98 (2001).</li><li>Ma, L., Hu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+GABA+neurons+correct+locomotion+deficits+in+quinolinic+acid-lesioned+mice.">Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice.</a> Cell Stem Cell. 10, 455-464 (2012).</li><li>Li, W., Wei, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Lowry, W. E., Richter, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Dimos, J. T., Rodolfa, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Ebert, A. D., Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+from+a+spinal+muscular+atrophy+patient.">Induced pluripotent stem cells from a spinal muscular atrophy patient.</a> Nature. 457, 277-280 (2009).</li><li>Lee, G., Papapetrou, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461, 402-406 (2009).</li><li>Park, I. H., Arora, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</li><li>Chamberlain, S. J., Chen, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+models+of+the+genomic+imprinting+disorders+Angelman+and+Prader-Willi+syndromes.">Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 17668-17673 (2010).</li><li>Kiskinis, E., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+toward+the+clinical+application+of+patient-specific+pluripotent+stem+cells.">Progress toward the clinical application of patient-specific pluripotent stem cells.</a> J. Clin. Invest. 120, 51-59 (2010).</li><li>Koch, P., Tamboli, I. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin-1+L166P+mutant+human+pluripotent+stem+cell-derived+neurons+exhibit+partial+loss+of+gamma-secretase+activity+in+endogenous+amyloid-beta+generation.">Presenilin-1 L166P mutant human pluripotent stem cell-derived neurons exhibit partial loss of gamma-secretase activity in endogenous amyloid-beta generation.</a> Am. J. Pathol. 180, 2404-2416 (2012).</li><li>Walsh, R. M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Rett+syndrome+with+stem+cells.">Modeling Rett syndrome with stem cells.</a> Cell. 143, 499-500 (2010).</li><li>Egawa, N., Kitaoka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+Screening+for+ALS+Using+Patient-Specific+Induced+Pluripotent+Stem+Cells.">Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells.</a> Sci. Transl. Med. 4, (2012).</li><li>Kola, I., Landis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+pharmaceutical+industry+reduce+attrition+rates.">Can the pharmaceutical industry reduce attrition rates.</a> Nature Reviews Drug Discovery. 3, 711-716 (2004).</li></ol>

Gli autori dichiarano di non avere conflitto di interessi finanziari.

Ci sono diversi passaggi critici durante il processo di differenziazione neurale. È importante garantire che i PSC umani sono pluripotenti perché altrimenti le cellule possono già essere polarizzato verso diventare un non-lineage neuronale. Ciò può essere confermato dalla colorazione dei PSC umani con anticorpi contro marcatori pluripotenza come Oct4, Sox2, Nanog, e Tra-1-60 1-3. Se i PSC umani non attribuiscono molto bene dopo passaging di loro, inibitore ROCK (Y27632) può essere aggiunto per aiutare. ...

Le cellule staminali pluripotenti (PSC), includendo sia le cellule staminali embrionali umane (hESC) e cellule staminali pluripotenti indotte (iPSCs), hanno la capacità di generare ogni tipo di cellula del corpo, compresi i neuroni 1-3. Differenziazione diretta dei vari sottotipi neuronali da CSP umani è la chiave per l&#39;applicazione di queste cellule nella medicina rigenerativa. La generazione di funzionali sottotipi neuronali durante lo sviluppo è un processo complesso che coinvolge l&#39;induzione di lignaggio neurale, la specificazione dei progenitori regionali lungo l&#39;asse rostro-caudale, e la differenziazione di post-mitotici tipi di neuroni dai progenitori regionali 4,5. A partire dal 2001, i sistemi sono stati creati vari per generare discendenza da hESC neurale, che hanno fornito una piattaforma per la generazione successiva di sottotipi neuronali 6,7. Basato su principi di sviluppo, i tipi di neuroni diversi, come spinale motoneuroni 8-12, mesencefalo dopneuroni aminergici 13-15, e le cellule neurali della retina 16,17 sono stati efficacemente specificato da CSP umani. Ciò è stato fatto applicando morfogeni critici che sono importanti per la specifica di questi tipi di neuroni durante lo sviluppo in vivo. Altri protocolli sono stati sviluppati anche per promuovere la differenziazione delle hESC in neuroni utilizzando sia fattori addizionali 18-20 come piccole molecole o mediante co-coltura con altri tipi cellulari per promuovere la differenziazione 21. La neocorteccia umana è altamente sviluppato e contiene molti tipi di cellule, tra cui i neuroni glutamatergici che svolgono un ruolo importante per l&#39;apprendimento, la memoria e le funzioni cognitive 22,23. Il primo passo nel generare neuroni glutamatergici in coltura è specificare cellule progenitrici telencefalici. Gruppo Yoshiki Sasai primo riportato la differenziazione dei precursori telencefalici diretto da mouse CES (mESCs) con un siero privo suspension coltura in presenza di DKK1 (che inibisce segnalazione Wnt) nonché LeftyA (che inibisce nodale segnalazione) 24. In seguito, diversi gruppi tra cui i nostri hanno anche riportato la specificazione dei precursori telencefalici dal PSC umani nel siero terreno privo di 25-27. La generazione di precursori telencefalici da CSP umani non richiede l&#39;uso di morfogeni esogeni e l&#39;efficienza nella generazione di questi precursori è molto superiore a quella da mESCs 26,27. Qui, un sistema chimicamente definito per induzione neurale che è stato ben stabilito dal gruppo di Zhang 7 è stato descritto. Senza l&#39;aggiunta di fattori esogeni caudalizing, questo protocollo genera in modo efficiente precursori telencefalici dal PSC umani 27. Questi progenitori possono essere differenziate in progenitori dorsale o ventrale regolando la segnalazione di Wnt e sonic hedgehog (SHH). I progenitori dorsali possono ulteriormente differenziarsi in neuroni e glutamatergicafficiently 27. Inoltre, questo protocollo funziona bene anche per la generazione di neuroni glutamatergici da umano iPSCs 28, che consente la generazione di paziente-specifici neuroni che possono essere utilizzati per esplorare il meccanismo di azione e terapie potenziali per una vasta gamma di malattie . Inoltre, il nostro sistema fornisce anche una piattaforma per esplorare lo sviluppo e le specifiche dei diversi tipi di neuroni nel telencefalo.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>1140-050</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (14.3 M)</td>
			<td>Sigma</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine)</td>
			<td>Sigma</td>
			<td>116K5103</td>
		</tr>
		<tr>
			<td>Laminin (human)</td>
			<td>Sigma</td>
			<td>L-6274</td>
		</tr>
		<tr>
			<td>Laminin (mouse)</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Stemgent</td>
			<td>04-0012</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
		</tr>
		<tr>
			<td>Foxg1 antibody</td>
			<td>Dr. Y. Sasai</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoxb4 antibody (1:50)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>I12</td>
		</tr>
		<tr>
			<td>Pax6 antibody (1:5000)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>PAX6</td>
		</tr>
		<tr>
			<td>Nkx2.1 antibody (1:200)</td>
			<td>Chemicon</td>
			<td>MAB5460</td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
		</tr>
		<tr>
			<td>vGLUT1 antibody (1:100)</td>
			<td>Synaptic Systems</td>
			<td>135302</td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-02</td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (GDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-10</td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>PrepoTech Inc.</td>
			<td>100-11</td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma</td>
			<td>D-0260</td>
		</tr>
		<tr>
			<td>Sonic hedgehog (SHH)</td>
			<td>R&amp;D</td>
			<td>1845-SH</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Becton Dickinson (BD)</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352097</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>BD</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>BD</td>
			<td>353047</td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>BD</td>
			<td>353009</td>
		</tr>
		<tr>
			<td>T75 flasks (untreated)</td>
			<td>BD</td>
			<td>353133</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>BD</td>
			<td>353004</td>
		</tr>
		<tr>
			<td>9'' glass pipetes</td>
			<td>Fisher</td>
			<td>13-678-20D</td>
		</tr>
		<tr>
			<td>Steriflip filters (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>Stericup filters 1,000 ml (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
		</tr>
		<tr>
			<td>Phase contrast microscope (Observer A1)</td>
			<td>Zeiss</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (Hera Cell 150)</td>
			<td>Thermo Electron Corporation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety hood (Sterilgard III Advance)</td>
			<td>The Baker Company</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge (5702 R)</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

Qui, una procedura graduale per generare efficacemente telencefalici neuroni glutamatergici da cellule staminali pluripotenti (PSC) è stato descritto. Il processo di differenziazione è iniziato rompendo i PSC umani in ciuffi che completano fino a formare aggregati quando le cellule sono posti in coltura in sospensione. Gli aggregati vengono poi coltivate in terreno hESC da 1-4 giorni per consentire differenziazione spontanea. Durante questo periodo, le cellule hanno la capacità di diventare uno dei tre strati germinali. Da 5-8 giorni, le cellule sono posti in un mezzo di induzione neurale per spingerli nella discendenza neurale. Circa 8 giorni, le cellule vengono fatte attaccare su piastre da 6 pozzetti e differenziare durante i quali la forma neuroepiteliale cellule. Queste cellule possono essere isolate neuroepiteliale al giorno 17. Le cellule possono quindi essere mantenuta come neurosfere finché non sono pronti per essere piastrate su vetrini. Utilizzo di un supporto di base senza alcun fattore caudalizing, cellule neuroepiteliali sono spécifiéd in precursori telencefalici, che possono poi essere ulteriormente differenziate in progenitori telencefalici dorsali e neuroni glutamatergici in modo efficiente. Nel complesso, il nostro sistema fornisce uno strumento per generare neuroni umani glutamatergici per i ricercatori per studiare lo sviluppo di questi neuroni e le malattie che li riguardano.

Qui, un protocollo per differenziare PSCs umani in telencefalici neuroni glutamatergici attraverso diverse fasi fondamentali: la formazione di aggregati PSC, l&#39;induzione di cellule neuroepiteliali, la generazione di progenitori telencefalici, e la differenziazione terminale di questi progenitori in neuroni telencefalici (Figura 1) ha stato descritto. Questo sistema è robusto ed efficiente nella generazione dei progenitori telencefalici e neuroni glutamatergici. Come esempio (Figura 2),</str...

Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Questa procedura produce neuroni telencefalici passando attraverso i punti di controllo che sono simili a quelli osservati durante lo sviluppo umano. Le cellule possono differenziarsi spontaneamente, sono esposti a fattori che li spingono verso il lineage neurale, sono isolati, e vengono piastrate su vetrini per consentire la differenziazione terminale e maturazione.

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 ...

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10.8K Views.  The University of Connecticut Health Center. Questa procedura produce neuroni telencefalici passando attraverso i punti di controllo che sono simili a quelli osservati durante lo sviluppo umano. Le cellule possono differenziarsi spontaneamente, sono esposti a fattori che li spingono verso il lineage neurale, sono isolati, e vengono piastrate su vetrini per consentire la differenziazione terminale e maturazione.

Video: The Specification of Telencephalic Glutamatergic Neurons 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 ...

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Neuroscienze

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 ...

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 ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Semplice generazione di una cultura ad alto rendimento dei neuroni indotti da fibroblasti umani della pelle adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

Conversione di cellule staminali pluripotenti indotte dall'uomo (IPSC) in neuroni motore spinale e cranico funzionale utilizzando vettori PiggyBac

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Analisi del trasporto mitocondriale e della morfologia nei neuroni derivati dalle cellule staminali indotte dall'uomo in paraplegia ereditaria

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

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 ...