Siamo grati al Dr. Magdalena Götz per il suo ingresso durante lo sviluppo di questo protocollo. Ringraziamo il Dr. Marius Wernig (Stanford University) per generosamente ci fornisce la sequenza codificante Sox2. Siamo anche molto grati al Dr. Alexandra Lepier per la produzione di virus. Questo lavoro è stato sostenuto da sovvenzioni della Deutsche Forschungsgemeinschaft (BE 4182/2-2) e il BMBF (01GN1009A) a BB e il Ministero bavarese delle Scienze, la Ricerca e le Arti a MK e BBCS ricevuto finanziamenti dal SYSTHER-binazionale INREMOS virtuale Institute (Ministeri federale tedesco e sloveno dell&#39;Istruzione e della Ricerca) e il DFG (SFB 824).

1. Isolamento e Coltura di cellule adulte cervello umano Gli esperimenti che coinvolgono il tessuto umano devono essere eseguite in conformità a tutte le normative governative e istituzionali pertinenti riguardanti l&#39;uso di materiale umano per scopi di ricerca. Il presente protocollo è stato sviluppato in conformità con l&#39;approvazione da parte del comitato etico della Facoltà di Medicina della LMU di Monaco di Baviera e il consenso informato per iscritto da tutti i pazienti. Questo protocollo di culture preparazione dell&#39;adulto corteccia cerebrale umana è stata stabilita utilizzando campioni di pazienti di entrambi i sessi affetti da epilessia del lobo temporale o di altre lesioni profonde non traumatiche, non maligne. Il tessuto ottenuto dalla sala chirurgica composto esclusivamente il canale di accesso alla lesione cerebrale e quindi è considerato sano. La fascia d&#39;età dei pazienti era di 19-70 anni. <ol><li> Preparare mezzo di crescita aggiungendo siero di vitello fetale inattivato al calore(FCS) ad alto glucosio DMEM con GlutaMAX per ottenere una concentrazione finale di 20% FCS. Aggiungere 5 ml di penicillina / streptomicina per un totale di 500 ml mezzo di crescita. Eseguire questa e di tutte le fasi successive che richiedono condizioni di coltura sterile in una cappa a flusso laminare adeguata. </li><li> Mantenere la biopsia del cervello umano adulto ottenuto dalla sala operatoria in soluzione salina bilanciata Hanks &#39;con CaCl 2 e MgCl 2 (HBSS) medio compreso HEPES (10 mM concentrazione finale) sul ghiaccio fino al trattamento. Avviare l&#39;elaborazione il più presto possibile. </li><li> Per avviare la dissociazione in celle singole, trasferire il tessuto in una capsula di Petri 65 millimetri e tritare a pezzetti utilizzando due sterili monouso bisturi. Per digestione enzimatica usare 3-6 ml TrypLE in un tubo da 15 ml ed incubare per 15-30 minuti a 37 ° C in un bagno d&#39;acqua. </li><li> Aggiungere 1 volume di mezzo di crescita preriscaldato ad agevolare dissociazione e triturare lentamente la soluzione contenente pezzi di tessuto su e giù utilizzando primauna pipetta monouso 5 ml, seguita utilizzando una pipetta Pasteur di vetro fino omogeneizzazione della sospensione cellulare. Tipicamente, alcuni pezzi di tessuto residui, per lo più costituiti da materia bianca, rimarranno in sospensione. </li><li> Centrifugare a 157 xg per 5 min e risospendere il pellet in quantità appropriata di mezzo di crescita (10 ml per non rivestito pallone di coltura T75). Utilizzare un pallone di coltura T75 per una biopsia di 5-10 mm di diametro dimensioni e estrapolare da questo nel caso di campioni più grandi. </li><li> Posizionare cellule in un incubatore a 37 ° C con 5% di CO 2 e 5% O 2. </li><li> Sostituire metà del mezzo ogni 3-4 giorni finché le cellule hanno raggiunto la confluenza. Questo richiede di solito fino a due settimane (di seguito il passaggio 0 [P0]). </li></ol> 2. Espansione in vitro e caratterizzazione di cellule cervello umano adulto <ol><li> Espansione in vitro: <ol><li> Aspirare il terreno di coltura e lavare con tampone fosfato salino (PBS), conservato a temperatura ambiente temperatura, prima di aggiungere 3 ml TrypLE. </li><li> Incubare a 37 ° C per 5-10 minuti finché la maggior parte delle cellule staccate. Battere delicatamente i palloni per facilitare il sollevamento delle cellule; dopo l&#39;aggiunta di 3 volumi di mezzo di crescita, trasferire le cellule in una provetta conica da 15 ml e centrifugare a 157 xg per 5 min e risospendere le cellule in quantità appropriata di terreno di crescita. </li><li> Passaggio le celle in un rapporto di 1:3 per la resa ottimale in nuove fiasche di coltura cellulare T75. Nota: Abbiamo diversi passaggi queste cellule fino a P5, senza alcun segno di ridotta efficienza riprogrammazione. Mentre a P0 la coltura contiene una considerevole frazione di cellule endoteliali (valutata mediante espressione CD34), a passaggi successivi il loro numero diventa trascurabile. </li></ol></li><li> Caratterizzazione delle culture cervello umano adulto: <ol><li> Immunocytochemistry: <ol><li> Per la caratterizzazione delle cellule, seme ~ 60.000 cellule su poli-D-lisina rivestite con copertura in vetro scivola in 500 microlitri fresca Mediu crescitam in 24 pozzetti piastre di coltura tissutale e incubare a 37 ° C con 5% di CO 2 e 5% O 2. </li><li> Per l&#39;analisi immunocitochimica delle cellule coltivate cervello umano rimuovere il supporto e lavare 3x con 1 ml di PBS. </li><li> Fix cellule aggiungendo 500 ml di paraformaldeide al 4% in PBS per 15 minuti a temperatura ambiente. </li><li> Trasferire la copertura in vetro scivola in un apposito camera di colorazione umidificata e bloccare con 80 microlitri di PBS contenente il 3% di albumina di siero bovino (BSA) e 0,5% Triton X-100 per 1 ora a temperatura ambiente. </li><li> Aggiungere anticorpi primari diluiti nella stessa soluzione (per fattori di diluizione vedi tabella di reagenti e attrezzature specifiche) e incubare una notte a 4 ° C o 1 ora a temperatura ambiente. Nota: Una frazione sostanziale ma variabile delle cellule in questa fase della coltura sono immunoreattivi per β del recettore del fattore di crescita derivato dalle piastrine (PDGFRβ), cluster di differenziazione 146 (CD146), NG2 condroitin solfato proteoglicani, e actina muscolo liscio (SMA), vale a dire i marcatori caratteristici pericytes microvasali associati. Solo una minoranza (&lt;1%) esprimono il marker gliale fibrillare astrogliali proteina acida (GFAP) e S100β. Inoltre, in questa fase la coltura deve essere privo di cellule che esprimono il marcatore neuronale βIII-tubulina. Utilizzare più combinazioni di anticorpi primario per co-etichettatura dei diversi marcatori. Un&#39;ulteriore conferma dell&#39;espressione di gliali, neuronali, endoteliali e periciti marcatori può essere ottenuto mediante trascrizione inversa e tempo reale PCR quantitativa (non descritto in questo protocollo). </li><li> Dopo l&#39;incubazione con l&#39;anticorpo primario lavata tre volte con 1 ml di PBS e aggiungere i corrispondenti anticorpi secondari (nelle opportune diluizioni) alla soluzione colorante e incubare 1 ora a temperatura ambiente al buio. Lavare le cellule 3 volte con PBS. Se necessario, prima del montaggio includono colorazione con DAPI per 5 min a temperatura ambientetemperatura. </li><li> Utilizzare antifading mezzo di montaggio e analizzare i vetrini coprioggetto utilizzando un microscopio a epifluorescenza o un microscopio confocale. </li></ol></li><li> Citometria a flusso: <ol><li> Per l&#39;analisi di espressione marcatore di superficie con citometria a flusso, crescere le cellule a confluenza in T25 non rivestito o T75 fiasche di coltura cellulare. </li><li> Staccare celle utilizzando TrypLE come descritto prima e risospendere in soluzione colorante costituito da PBS + 0,5% BSA. Per reazione a colorazione, risospendere 100.000-200.000 cellule in 100 ml di soluzione colorante. Preparare le reazioni di colorazione singoli per anticorpi utilizzati (CD146-FITC, CD140b-PE, CD34-APC, CD13-FITC, rispettivi anticorpi di controllo isotipo). </li><li> Aggiungere anticorpi coniugati con fluorocromi per ogni soluzione colorante (per diluizioni vedi tabella di reagenti e attrezzature specifiche) direttamente nella sospensione cellulare e incubare a 4 ° C per 30 minuti al buio. </li><li> Lavare le cellule tre volte con 500 microlitri di PBS e spin giù tra ognifase di lavaggio a 157 xg per 5 min. </li><li> Risospendere il pellet di cellule in 0,5-1 ml di soluzione di colorazione e le cellule di trasferimento attraverso un colino cella (70 micron) per tubi FACS. Proteggere il campione dall&#39;esposizione alla luce. </li><li> Campioni Vortex prima di metterli in strumento FACS. </li><li> Per analizzare le cellule viventi ed escludere detriti e di cella aggregati, cancello per FSC-A e SSC-A. Duine cellulari sono specificamente gated da FSC-A e FSC-W. Per determinare le condizioni gating giuste per i marcatori di superficie cellulare impostare le porte con il controllo isotipo-abbinato coniugato al rispettivo fluorocromo. Quindi determinare la proporzione di cellule anticorpo-bound. </li></ol></li></ol></li></ol> 3. Arricchimento per le celle pericyte-derivate da cellule Fluorescence Activated Ordinamento <ol><li> Purificare la popolazione di cellule periciti (risospese in terreno di crescita) prima trasduzione tramite FACS classificare usando un ugello di 70 micron. Utilizzare CD34-APC in combinazione con la CD140b-PE e CD146-FITC per selezionare per pericytes. Ordina per, cellule CD140b-e CD146-CD34 positivi-negativi. </li><li> Raccogliere le cellule filtrate in mezzo di crescita e piastra su poli-D-lisina rivestite con copertura in vetro scivola in piastre di coltura tissutale a 24 pozzetti e incubare a 37 ° C con 5% di CO 2 e 5% O 2. </li><li> 48 ore dopo l&#39;ordinamento sostituire il supporto con mezzo di crescita fresco ed eseguono trasduzione come descritto nel protocollo 4. </li></ol> 4. Retrovirale trasduzione delle cellule pericyte-derivati ​​e Riprogrammazione in neuroni indotti Nota: Fare riferimento alle linee guida di biosicurezza locali durante la manipolazione di particelle retrovirali. In Germania l&#39;uso dei retrovirus impiegati qui richiede livello di biosicurezza 2. Per la produzione di particelle retrovirali riferimento al protocollo 6. Per costrutti retrovirali usati per la riprogrammazione, fare riferimento a Karow et al. (2012). I retrovirus sono stati pseudotyped con VSV-G (stomatite vescicolare virus-glicoproteina) 5. Per la produzione di consultare Gavrilescu et al 6. <ol><li> 24 ore prima della trasduzione retrovirale seme ~ 60.000 cellule su poli-D-lisina rivestite con copertura in vetro scivola in 500 microlitri terreno di coltura fresco in piastre da 24 pozzetti di coltura tissutale e incubare a 37 ° C con 5% di CO 2 e 5% O 2 . </li><li> Il giorno seguente, sostituire il mezzo di crescita con 500 microlitri fresco mezzo di crescita preriscaldata, e aggiungere 1 ml di rispettivi surnatanti concentrati contenenti particelle retrovirali (pCAG-IRES-DsRed per il controllo, specialmente durante stabilisce il protocollo in laboratorio), pCAG-ascl1 -IRES-DsRed, pCAG-Sox2-IRES-GFP. </li><li> Un giorno dopo, rimuovere il mezzo comprese le particelle virali dalle cellule e aggiunge 1 ml freschi, preriscaldata medio B27-differenziazione, composto da 49 ml DMEM con elevato glucosio GlutaMAX e 1 ml integratore privo di siero B27. Mantenere le cellule in coltura a 37 ° C in 5% CO 2 e 5% O 2 per 4-8 settimane senza cambiare il mezzo come variazioni medie ripetitive diventano dannose come INS maturare. </li></ol> 5. Caratterizzazione dei neuroni indotta da Immunocytochemistry <ol><li> Per determinare l&#39;identità cellulare delle cellule trasdotte, in particolare per dimostrare l&#39;acquisizione di un fenotipo neuronale, effettuare immunocitochimica contro antigeni neuronali come bIII-tubulina, MAP2, e NeuN (per gli anticorpi e le loro rispettive diluizioni, vedere tabella di reagenti specifici e attrezzature; seguire la stessa procedura indicata al punto 2.2.1). </li><li> Per migliorare la maturazione di PdiNs, co-coltura queste cellule con neuroni derivati ​​da E14 topo cortecce cerebrali. A tal fine, sezionare la corteccia cerebrale e dissociare il tessuto meccanicamente con una pipetta Pasteur di vetro lucidati a fuoco. Aggiungi 10,000-50,000 neuroni murini di colture di cellule pericyte derivate da 2-3 settimane dopo trasduzione con re-Sox2 e ascl1 di codificatroviruses e continuare la coltura. Cellule trasdotte possono essere distinte dai neuroni murini per la loro reporter (GFP e DsRed) espressione, sia per epifluorescenza dal vivo o in seguito immunocitochimica per la GFP e DsRed. </li><li> Per valutare la formazione di sinapsi su PdiNs di neuroni murini, eseguire immunocitochimica contro recettori per i neurotrasmettitori vescicolari come vescicolare del glutammato trasportatore 1 (VGLUT1). </li><li> Sonda cellule trasdotte per le loro proprietà elettriche (cioè la capacità di generare potenziali d&#39;azione) e la creazione di sinapsi funzionali da patch-clamp elettrofisiologia. Per i dettagli della procedura vedi Heinrich et al. (2011). </li></ol>

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Il presente protocollo descrive l&#39;espansione in vitro e l&#39;arricchimento di cellule pericyte-derivate a seguito isolamento dal adulta corteccia cerebrale umana e la successiva conversione in Ins dall&#39;espressione retrovirus-mediata della trascrizione neurogena fattori Sox2 e Ascl1. Tale protocollo prevede un sistema sperimentale in vitro per studiare la stirpe di conversione delle cellule cerebrali residente in neuroni e potenzialmente anche glia, con l&#39;obiettivo in mente di tradurre in u...

Al contrario di riprogrammazione di cellule somatiche in cellule staminali pluripotenti indotte (iPSCs), che a loro volta sono dotati di una pletora di potenziali di differenziazione, riprogrammare diretto mira per conversione diretta di uno specifico tipo cellulare in un altro. Per quanto riguarda la loro applicazione nel contesto della modellazione malattie e potenziali terapie a base di cellule, entrambi gli approcci di riprogrammazione hanno vantaggi e svantaggi specifici. Riprogrammazione in iPSCs (i) fornisce una fonte pressoché infinita di cellule; (Ii) consente di ingegneria genetica; (Iii) conferisce con un potenziale di differenziazione quasi illimitate. Tuttavia, principali svantaggi di iPSCs comportano il rischio di tumorigenicità delle cellule indifferenziate (formazione teratoma in vivo) e la necessità di coltura ex vivo e successivo trapianto se queste cellule devono essere utilizzati per terapie cellulari. Viceversa, diretto lignaggio riprogrammazione è limitata dalla minore resa delle cellule desiderateche correla direttamente con il numero di cellule bersaglio della popolazione di partenza, ma possiede il vantaggio che cellule della linea-riprogrammato sembrano mostrare alcun rischio oncogeno dopo trapianto 1,2; Inoltre, riprogrammazione diretta può anche essere realizzato in situ, cioè all&#39;interno dell&#39;organo dove sarebbero necessarie queste cellule, evitando così la necessità di trapianto. Con questo in mente, il nostro laboratorio ha perseguito la possibilità delle cellule cerebrali residente-lignaggio riprogrammazione in Ins come un nuovo approccio verso terapie cellulari delle malattie neurodegenerative. Le cellule cerebrali residente che possono essere potenzialmente considerati bersagli cellulari per lignaggio riprogrammazione comprendono diversi tipi di macroglia (astrociti, cellule NG2 e oligodendrociti), microglia e cellule dei microvasi associate (cellule endoteliali e periciti). Abbiamo ampiamente studiato il potenziale riprogrammazione in vitro della astroglia del cor cerebraletex dei primi topi postnatale 3-5. Alla ricerca di fonti di cellule simile adatti per lignaggio riprogrammazione diretta nel cervello umano adulto, abbiamo incontrato una popolazione di cellule che può essere riprogrammato con successo in Ins e caratteristiche espositive di periciti. Qui si descrive un protocollo di come raccogliere queste cellule adulte da biopsie del cervello umano, per espandere e arricchire queste cellule in vitro, e infine riprogrammare successo una frazione sostanziale di queste cellule espanse in vitro (nell&#39;intervallo 25-30%) in Ins. Riprogrammazione può essere ottenuto mediante simultanea retrovirus-mediata co-espressione di due fattori di trascrizione, Sox2 e ascl1. Questi PdiNs sono stati trovati per acquisire la capacità di azione ripetitiva potenziale di cottura e servire bersagli sinaptici per altri neuroni che indicano la loro capacità di integrare nelle reti neurali. Il nostro protocollo prevede una procedura semplice per l&#39;isolamento e lignaggio conversione di adulti pericytes cervello umano in Ins. </p&gt;

<table border="0" cellpadding="0" cellspacing="0" width="320">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">anti-CD140b-PE</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:80px;">558821</td>
			<td style="width:80px;">use 1:100</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:80px;">anti-CD146-FITC</td>
			<td style="width:80px;">AbD Serotec</td>
			<td style="width:80px;">MCA2141FT</td>
			<td style="width:80px;">use 1:100</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:80px;">anti-CD13-FITC</td>
			<td style="width:80px;">AbD Serotec</td>
			<td style="width:80px;">MVA1270A488T</td>
			<td style="width:80px;">use 1:100</td>
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			<td height="64" style="height:64px;width:80px;">anti-CD34-APC</td>
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			<td style="width:80px;">560940</td>
			<td style="width:80px;">use 1:100</td>
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			<td height="43" style="height:43px;width:80px;">anti-PDGFR&#946;</td>
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			<td style="width:80px;">3169S</td>
			<td style="width:80px;">use 1:200</td>
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			<td height="22" style="height:22px;width:80px;">anti-CD146</td>
			<td style="width:80px;">Abcam</td>
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			<td style="width:80px;">use 1:400</td>
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			<td style="width:80px;">Millipore</td>
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			<td style="width:80px;">use 1:400</td>
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			<td style="width:80px;">use 1:400</td>
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			<td style="width:80px;">T8660</td>
			<td style="width:80px;">use 1:400</td>
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			<td height="43" style="height:43px;width:80px;">anti-MAP2</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">M4403</td>
			<td style="width:80px;">use 1:200</td>
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			<td height="43" style="height:43px;width:80px;">anti-GFAP</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">G3893</td>
			<td style="width:80px;">use 1:600</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:80px;">anti-GFP</td>
			<td style="width:80px;">Aves Labs</td>
			<td style="width:80px;">GFP 10-20</td>
			<td style="width:80px;">use 1:1000</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:80px;">anti-RFP</td>
			<td style="width:80px;">Chromotek</td>
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			<td style="width:80px;">use 1:500</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:80px;">anti-S100&#946;</td>
			<td style="width:80px;">Sigma-Aldrich</td>
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			<td style="width:80px;">use 1:300</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:80px;">anti-NeuN</td>
			<td style="width:80px;">Millipore</td>
			<td style="width:80px;">MAB377</td>
			<td style="width:80px;">use 1:200</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:80px;">anti-GABA</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">A2052</td>
			<td style="width:80px;">use 1:500</td>
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			<td height="43" style="height:43px;width:80px;">anti-Calretinin</td>
			<td style="width:80px;">Millipore</td>
			<td style="width:80px;">AB5054</td>
			<td style="width:80px;">use 1:500</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:80px;">anti-vGluT1</td>
			<td style="width:80px;">SYnaptic SYstems</td>
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			<td style="width:80px;">use 1:1000</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:80px;">Rat IgG1 isotype control FITC</td>
			<td style="width:80px;">AbD Serotec</td>
			<td style="width:80px;">11-4301-81</td>
			<td style="width:80px;">use 1:100</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Rat IgG1 isotype control PE</td>
			<td style="width:80px;">AbD Serotec</td>
			<td style="width:80px;">12-4301-81</td>
			<td style="width:80px;">use 1:100</td>
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			<td height="85" style="height:85px;width:80px;">Rat IgG1 isotype control APC</td>
			<td style="width:80px;">AbD Serotec</td>
			<td style="width:80px;">17-4301-81</td>
			<td style="width:80px;">use 1:100</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:80px;">TrypLE</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">12605-010</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:80px;">DMEM, high glucose, GlutaMAX</td>
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			<td style="width:80px;">61965</td>
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			<td height="85" style="height:85px;width:80px;">B27 serum-free supplement</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">17504044</td>
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		<tr height="148">
			<td height="148" style="height:148px;width:80px;">fetal calf serum</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">10106-169</td>
			<td style="width:80px;">perform heat inactivation by incubation at 56 &#176;C for 30 min</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Penicillin-Streptomycin</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">15140122</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:80px;">HBSS (Hanks Balanced Salt Solution)</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">24020-091</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="198">
			<td height="198" style="height:198px;width:80px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS, 1&#215;; Invitrogen, cat. no. 14190)</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">14190</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:80px;">HEPES&#160;</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">H3375</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:80px;">Poly-D-lysine hydrobromide (PDL)</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">P0899</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">PFA (paraformaldehyde)</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">P1648</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Bovine serum albumin</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">A9418</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:80px;">Triton X-100</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">T9284</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:80px;">DAPI (4&#8242;,6-Diamidin-2-phenylindole) dilactate</td>
			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">042M4005</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Aqua-Poly/Mount</td>
			<td style="width:80px;">Polysciences</td>
			<td style="width:80px;">18606-20</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:80px;">Polypropylene round-bottom tubes&#160;</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:80px;">352063</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:80px;">Polystyrene round-bottom tubes with cell-strainer cap&#160;</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:80px;">352235</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:80px;">24-well plates</td>
			<td style="width:80px;">Orange Scientific</td>
			<td style="width:80px;">5530305</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:80px;">75 cm2 culture flasks</td>
			<td style="width:80px;">Greiner Bio-One</td>
			<td style="width:80px;">658175</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:80px;">25 cm2 culture flasks</td>
			<td style="width:80px;">Greiner Bio-One</td>
			<td style="width:80px;">690175</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Disposable pipettes 10 ml</td>
			<td style="width:80px;">Sarstedt</td>
			<td align="center" style="width:80px;">#########</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:80px;">Glass Pasteur pipettes (150 mm)</td>
			<td style="width:80px;">Fisherbrand</td>
			<td style="width:80px;">FB50251</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:80px;">Glass coverslips 12 mm diameter</td>
			<td style="width:80px;">Menzel</td>
			<td style="width:80px;">CB00120RA1</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Surgical disposable scalpels&#160;</td>
			<td style="width:80px;">B. Braun</td>
			<td style="width:80px;">5518083</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Tissue culture dishes&#160;</td>
			<td style="width:80px;">Greiner Bio-One</td>
			<td style="width:80px;">633180</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Conical tubes 15ml</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:80px;">352095</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:80px;">Laminar flow hood</td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:80px;">Centrifuge and swing-out rotor with adapters for 15 ml and 50 ml tubes</td>
			<td style="width:80px;">Hettich Lab Technology</td>
			<td style="width:80px;">1406</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:80px;">Flow cytometry cell sorter: FACSAria l with FACSDiva software&#160;</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Humified cell culture incubator</td>
			<td style="width:80px;">Eppendorf Galaxy 170R</td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Wather bath at 37 &#176;C</td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">FACSFlow sheath fluid&#160;</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:80px;">342003</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:80px;">Epifluorescence microscope BX61</td>
			<td style="width:80px;">Olympus</td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Confocal microscope LSM710</td>
			<td style="width:80px;">Zeiss</td>
			<td style="width:80px;"></td>
			<td style="width:80px;"></td>
		</tr>
	</tbody>
</table>

lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons

Diretto lignaggio riprogrammazione di cellule non neuronali nei neuroni indotti (INS) può fornire approfondimenti sui meccanismi molecolari alla base neurogenesi e attivare nuove strategie per la modellazione in vitro o la riparazione del cervello malato. Identificazione dei tipi di cellule non neuronali cerebrali residente suscettibili di conversione diretta in Ins potrebbe consentire per il lancio di un tale approccio in situ, cioè all&#39;interno del tessuto cerebrale danneggiato. Qui si descrive un protocollo sviluppato nel tentativo di identificare cellule derivate dal cervello umano adulto che soddisfano questa premessa. Questo protocollo prevede: (1) la coltura di cellule umane dalla corteccia cerebrale ottenuto da biopsie cerebrali adulte umane; (2) l&#39;espansione in vitro (approssimativamente richiede 2-4 settimane) e caratterizzazione di coltura mediante immunocitochimica e citofluorimetria; (3) l&#39;arricchimento da cellule fluorescenza-attivato (FACS) usando anti-recettore PDGF-β e anticorpi anti-CD146;(4) il retrovirus trasduzione mediata con la trascrizione neurogenica fattori Sox2 e ascl1; (5) e, infine, la caratterizzazione della risultante neuroni indotti periciti-derivato (PdiNs) mediante immunocitochimica (14 giorni a 8 settimane dopo trasduzione retrovirale). In questa fase, in possono essere sondato per le loro proprietà elettriche mediante registrazione patch-clamp. Questo protocollo prevede una procedura altamente riproducibile per la conversione in vitro stirpe di pericytes cervello residente in Ins umane funzionali.

Il primo risultato di questo protocollo dopo aver stabilito con successo una cultura da un esemplare di umano adulto corteccia cerebrale consiste nell&#39;identificazione della composizione cellulare della cultura. Immunocitochimica di proteine ​​specifiche di tipo cellulare rivela un notevole grado di eterogeneità tra culture derivati ​​da esemplari di diversi pazienti (Figura 1A). Quantificata mediante citometria di flusso vi è sempre una frazione sostanziale di cellule che esprimono PDGFRβ...

Watch this Scientific Journal Video about Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons at JoVE.com

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Targeting cellule cerebrali residente per discendenza diretta-riprogrammazione offre nuove prospettive per la riparazione del cervello. Qui si descrive un protocollo di come preparare le culture arricchito da pericytes cervello residente da adulto corteccia cerebrale umana e convertirli in neuroni indotti dall&#39;espressione retrovirus-mediata dei fattori di trascrizione Sox2 e Ascl1.

Lineage-riprogrammazione di cellule pericyte-derivate del cervello umano adulto in neuroni indotti

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

lineage-reprogramming-pericyte-derived-cells-adult-human-brain-into

Research

JoVE Journal

Neuroscience

12.2K Views.  Ludwig Maximilians University Munich.  Targeting cellule cerebrali residente per discendenza diretta-riprogrammazione offre nuove prospettive per la riparazione del cervello. Qui si descrive un protocollo di come preparare le culture arricchito da pericytes cervello residente da adulto corteccia cerebrale umana e convertirli in neuroni indotti dall'espressione retrovirus-mediata dei fattori di trascrizione Sox2 e Ascl1. 

Video: Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Studiare l&#39;integrazione degli adulti nati Neuroni

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

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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 ...

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

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

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

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

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

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 ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

A breve termine, le colture di fette libere dal cervello umano adulto

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migrazione, chemio-attrazione, e analisi co-cultura per le cellule endoteliali derivate da cellule staminali umane e neuroni GABAergic

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Ottenere Microglia Umana da Tessuto Cerebrale Umano Adulto

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

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 ...