Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC Differenziazione di cellule neurali progenitrici <ol><li> Crescere ed espandere hiPSCs in cellule staminali embrionali umane (HES) supporto (Tabella 1) co-coltura di un mouse fibroblasti embrionali (MEF) feeder layer fino grandi (ma subconfluenti) le colonie sono pronti per la differenziazione neuronale attraverso un corpo embrioide (EB) intermedio (Figura 2). Routine condizioni di coltura hiPSC sono ben descritte altrove 16,17; brevemente, crescere hiPSCs nei media HES su uno strato alimentatore mEF fino confluenti, passaggio poi enzimaticamente con collagenasi (1 mg / ml di DMEM) ed espandere di circa 1: 3 ogni 7 giorni. </li><li> Per preparare piastre rivestite Poly-L-ornitina / laminina, piastre cappotto con 10 g / ml Poly-L-ornitina in acqua sterile per superfici plastiche (50 g / ml per superfici di vetro) e incubare a temperatura ambiente per 3-24 ore. Lavare almeno una volta con acqua sterile e poi cappotto con 5g / ml laminina in PBS sterile a temperatura ambiente per 3-24 ore. NOTA: Se avvolto in plastica, piastre possono essereimmagazzinato per fino a sei mesi a -20 ° C. </li><li> Il giorno 1, enzimaticamente sollevare hiPSCs subconfluenti (generalmente coltivate su MEF, o hiPSCs coltivate in mezzi definiti quali TeSR 18 su Matrigel) dalla piastra come grandi colonie utilizzando 1mg / ml Collagenase IV in DMEM / F12. NOTA: Dopo 1-2 ore di incubazione a 37 ° C, le colonie sarà fluttuante nel piatto. </li><li> Lavare delicatamente le colonie hiPSC (da stabilirsi, non centrifugazione) 1-2x con DMEM / F12, risospendere in 2 ml / pozzetto di N2 media / B27 (Tabella 1) e il trasferimento a piatti non aderente 6 pozzetti (combinare 3-pozzetti in 1 -Beh) 19. NOTA: Durante la notte, le colonie si formano ammassi sferici fluttuanti definito &quot;corpi embrionali&quot; (EbS). Aspettatevi morte cellulare sostanziale. </li><li> Il giorno 2, piastre di inclinazione e permettere EBs di stabilirsi. Rimuovere con attenzione dei media e lavare una volta con DMEM / F12 per rimuovere i detriti. Gli alimenti con N2 / B27 multimediale integrato con inibitori SMAD doppio (0.1M LDN193189 e 10M SB431542) 12 </sup&gt;. </li><li> Nei giorni 3-7, neuralization avverrà nel contesto di una doppia inibizione SMAD; controllare che EBs sono rotondi ma non cistica. Concime EBs ogni secondo giorno con mezzi N2 / B27 integrati con 0.1M LDN193189 e 10M SB431542. </li><li> Nei giorni 7-14, dopo placcatura di EB per Poli-L-Ornitina / laminina piastre rivestite, controllare utilizzando un microscopio in campo chiaro che rosette neurali cominciano ad apparire nel giro di pochi giorni (caratterizzata da grappoli di cellule rotonde neuroepiteliali con apico-basale polarità; Figura 1). Continuare ad alimentare EB aderenti ogni secondo giorno con N2 / B27 multimediale integrato con 0,1 M LDN193189, 10M SB431542 e 1 g / ml laminina. </li></ol> 2. Raccolta di Neural Rosette NOTA: Si consiglia di rosette neurali siano enzimatica raccolti utilizzando Neural Rosette Selection reagente 20 o reagente simile selezione. Anche se rosette neurali possono essere raccolti manualmente in 6 pozzetti rivestiti Poli-L-Ornitina / laminina, thimetodologia s prende una formazione da padroneggiare, e, a seconda abilità dell&#39;utente, può richiedere una seconda fase di raccolta a 20 giorni per arricchire ulteriormente per i PNG e riducono tipi di cellule non neuronali. <ol><li> Per la selezione enzimatica di rosette neurali al giorno 14, i media aspirato da EBs aderito e aggiungere 1 ml di neurale reattivo selezione rosetta per bene per un 6-pozzetti. Incubare a 37 ° C per 1 ora. </li><li> Con un Pipetman P1000, rimuovere delicatamente enzima da ciascun pozzetto. Aggiungere 1 ml di DMEM / F12 per pozzetto per lavare. </li><li> Con un P1000, raccogliere i 1 ml di DMEM / F12 e rapidamente espellere il DMEM / F12 di nuovo nel pozzo, staccando così le rosette dalla piastra. Raccogliere le rosette in un tubo falco. </li><li> Dispensare un altro 1 ml di DMEM / F12 e rapidamente espellere nello stesso bene a staccare restanti rosette. (Non triturare. Cerca di non rompere gli aggregati rosetta). Raccogliere le rosette neurali nella stessa provetta falco. </li><li> Ripetere il punto 2.4, se necessario. Se rosette non si staccano facilmente, aggiungere mori enzima e ripetere il processo (punti 2.1-2.4) </li><li> Spin cellule a 300 xg per 3 min. Aspirare il lavaggio, e ri-sospendere rosette a 2 ml di NPC supporti (Tabella 1). Trasferire le cellule (1: 1) in un poli-L-Ornitina / laminina rivestite 6 pozzetti. </li><li> Da giorni 15-21, le rosette neurali si espanderanno; rosette di alimentazione ogni secondo giorno con NPC media. </li><li> Valutare la qualità delle rosette neurali e garantire che fibroblasti, come piatti, non si stanno espandendo all&#39;interno della cultura. NOTA: Se non rosette persistono, la cultura NPC a volte può essere salvato dalla raccolta manuale, selezionando solo il meglio delle rosette raccolte in Accutase. Dissociarsi 15 min a 37 ° C. Pellet, lavare e piatto in NPC mezzi su 24 pozzetti rivestiti Poli-L-Ornitina / laminina. </li></ol> 3. espansione delle cellule progenitrici neurali NOTA: hiPSC NPC può essere coltivata su entrambi Matrigel- o Poli-L-Ornitina / laminina piastre rivestite. Usiamo tipicamente Matrigel-piastre quanto possono essere preparati in modo più rapido ed a costi inferiori. <ol><li> Per preparare piastre Matrigel rivestite, scongelare rapidamente e risospendere un&#39;aliquota congelate 1mg di Matrigel in 24 ml freddo DMEM / F12 e distribuire immediatamente 2 ml per ciascun pozzetto di due piastre a sei pozzetti. Incubare per almeno 1 ora a 37 ° C. NOTA: Matrigel deve solo essere aspirato, non lavato, immediatamente prima dell&#39;uso. </li><li> Concime NPC ogni secondo giorno e mantenere ad altissima densità o saranno spontaneamente differenziarsi a neuroni. NOTA: PNG se più consolidati sono in genere divisi 1: 4 ogni settimana, NPC molto basse di passaggio possono essere suddivisi 1: 2 come di rado come una volta ogni due settimane. </li><li> Per dividere, supporti primo aspirare e aggiungere 1 ml di caldo Accutase (1X) per pozzetto di 6-pozzetti. Incubare a 37 ° C per 10-15 minuti. </li><li> Trasferire delicatamente le cellule staccate in una provetta da 15 ml contenente DMEM / F12 con il minor stress meccanico possibile. Non titolare cellule mentre in enzima. Cellule pellet di Spinning a 1000 xg per 5 min. </li><li> Aspirare il lavaggio, e risospendere NPC in ~ 1 ml NPC supporto per pozzetto originale di 6-bene. NOTA: Aspettatevi che un pozzo confluente di un piatto 6-bene ha 5-10.000.000 cellule; questo può essere confermato contando un&#39;aliquota 10l di cellule usando un hematocytometer prima di ri-placcatura. </li><li> Dopo ri-sospensione, NPC ri-piastra come NPC, neuroni o neurosfere. Per mantenere NPC, piatto circa 1-2 milioni di cellule per pozzetto di un 6-pozzetti. L&#39;efficienza della formazione neurosfere può variare tra esperimenti e linee cellulari, ma generalmente si verifica meglio se tra 200.000 e 1.000.000 cellule vengono seminate per pozzetto di una piastra da 6 pozzetti non aderente; se si verifica aggregazione delle neurosfere, ridurre il numero delle cellule seminate. Per differenziare a neuroni, piastra circa 200.000 cellule per pozzetto di un 6-pozzetti, sostituendo i media NPC con i media neurone. </li><li> Immunoistochimica validare ogni linea NPC stabilito. Una volta che il materiale cellulare sufficiente è stata EXPANded, PNG etichette con anticorpi per nestina e SOX2. Linee Validated NPC dovrebbero essere differenziate in neuroni per 4-6 settimane, ed etichettati con anticorpi per III-TUBULINA e MAP2AB. <ol><li> Fissare cellule in 4% paraformaldeide in PBS a 4 ° C per 10 min. Permeabilize NPC a temperatura ambiente per 15 min in 1,0% Triton in PBS, e quindi bloccare nel siero asino 5% con 0,1% Triton a temperatura ambiente per 30 min. </li><li> Incubare con anticorpo primario nel siero asino 5% con 0,1% Triton, durante la notte a 4C. NOTA: Si consiglia i seguenti anticorpi: capra anti-Sox2, 1: 200; Nestin topo anti-umana, 1: 200; coniglio anti-βIII-tubulina, 1: 500; topo anti-βIII-tubulina, 1: 500; topo anti-MAP2AB, 1: 200. (Figura 2). </li><li> A seguito di un lavaggio con PBS, incubare cellule anticorpi secondari con l&#39;appropriato anticorpo secondario coniugato alla capra, il mouse o il coniglio a 1: 300, in in 5% di siero asino con 0.1% Triton per 1-2 ore a temperatura ambiente. Per visualizzare i nuclei, cellule macchia con 0,5 mg / ml DAPI (4 &#39;, 6-diamidino-2-fenilindolo) e poi montare con Vectashield. </li></ol></li></ol> 4. NPC trasduzione <ol><li> Utilizzare spinfection (centrifugazione di piastre di coltura di cellule in presenza del virus a 1.000 xg per 1 ora) per aumentare la percentuale di cellule trasfettate 21. Aspirare i media e sostituirlo con la sovraespressione pertinente (o controllo) lentivirus (o retrovirus), titolato alla molteplicità desiderato di infezione (MOI) (in genere 1-10), diluito nei media NPC. Utilizzare 1,5 ml di volume per pozzetto di una piastra da 6 pozzetti (o un volume opportunamente scalati per altri tipi di piastre di coltura tissutale). Spin a 1.000 xg e 37 ° C per 1 ora in una centrifuga piatto. NOTA: Idealmente, la trasduzione NPC con lentiviral o vettori retrovirali entro 1-2 giorni di scissione. Sia che si utilizzi commerciali o virus in laboratorio preparati, può essere utile per poi titolare adeguatamente lotti di virus in anticipo per diluizione seriale. (Figura 3). </li><li> Per ridurre celmorte lular, sostituire il supporto entro 8 ore di spinfection. NOTA: l&#39;integrazione virale e l&#39;espressione del transgene dovrebbero essere rilevabili entro 24 ore dal gene reporter fluorescenza. (Se il virus non ha un reporter fluorescente, questo è più difficile da valutare; trasfezione di successo può essere meglio convalidato da immunoblot, immunoistochimica o qPCR per i prodotti iperespressione.) Espressione completa può richiedere 3-7 giorni; spinfections ripetuti con virus possono essere richieste ulteriori per aumentare la percentuale di cellule transfettate. Spinfections ricorrenti possono verificarsi ogni giorno, ma non deve essere così frequenti. Idealmente, se si utilizza un reporter fluorescente,&gt; 80% delle cellule deve essere etichettato. </li></ol> 5. neurosfere Migration Assay NOTA: Neurosfere forma spontaneamente, seguendo la dissociazione enzimatica di NPC (da un modo identico a quello utilizzato per l&#39;espansione NPC - passaggi 3,3-3,6), se le cellule sono coltivate in sospensione in NPC media. <ol><li> In breve, crescere dissociarsid NPC nei media NPC per 48 ore in piastre non aderenti al fine di generare neurosfere. (Figura 4). </li><li> Per la migrazione neurosfere, preparare piatti Matrigel freschi utilizzando supporti NPC freddo, piuttosto che DMEM, in una piastra a 96 pozzetti, 1-2 ore prima di neurosfere raccolta. Non aspirare Matrigel dai pozzi. </li><li> Come originariamente pubblicato per cellule staminali embrionali derivate da neurosfere 22, selezionare manualmente neurosfere NPC-derivati ​​al microscopio, dopo il lavaggio una volta nei media NPC per rimuovere i detriti cellulari. Trasferire una neurosfere a ciascun pozzetto di una piastra a 96 pozzetti Matrigel rivestite. </li><li> Aggiungere un ulteriore 0,5 mg Matrigel, diluito nei media NPC freddi, le neurosfere in ciascuna piastra da 96 pozzetti. </li><li> Centrare manualmente ogni singolo neurosfere nel mezzo del pozzo con un puntale. NOTA: È importante scegliere neurosfere di dimensioni simili, al fine di ridurre la variabilità nei risultati. </li><li> Lasciare la migrazione a verificarsi per 48 ore e poi fissare le cellule wesima 4% paraformaldeide e macchia con marcatori immunoistochimici desiderati. </li><li> Se la migrazione radiale è determinato, neurosfere fotografia nel loro interamente utilizzando un obiettivo 4x microscopio. Escludere qualsiasi contatto neurosfere bordo del pozzo o una seconda neurosfere dall&#39;analisi. NOTA: Se si verifica la migrazione per più di 48 ore, la migrazione radiale risultante sarà probabilmente troppo grande per immagine utilizzando un obiettivo 4x. </li><li> Misurare la migrazione media da ogni neurosfere utilizzando il software NIH ImageJ (Figura 5). Questo può essere fatto utilizzando uno dei metodi di analisi descritti di seguito: <ol><li> Migrazione Radial: <ol><li> Tracciare manualmente il bordo delle più lontane migrazione delle cellule con lo strumento di selezione a mano libera quindi utilizzare la funzione (Ctrl + M) Misura per calcolare l&#39;area della forma risultante. Prima di misurare l&#39;area assicurarsi che Area sia selezionata nella finestra Imposta Misurazioni trovato nella scheda Analizza. Utilizzare ilvalore della misura dell&#39;area e l&#39;equazione per l&#39;area di un cerchio (A = πr 2) per determinare il raggio (esterno). </li><li> Tracciare il bordo del neurosfere originale, misurare l&#39;area e calcolare il raggio (interna) nello stesso modo. Calcolare la migrazione totale radiale come differenza fra il raggio esterno ed interno. </li></ol></li><li> Greatest migrazione cellulare: <ol><li> Misurare la distanza percorsa delle cinque celle lontane dal bordo del neurosfere interna utilizzando lo strumento di selezione lineare seguita dalla funzione (Ctrl + M) Misura. </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

Abbiamo descritto i metodi con cui differenziare hiPSCs in PNG, un tipo di cellule neurali in cui una frazione significativa della firma genica dei neuroni hiPSC derivati ​​si conserva e che possono servire come proxy per i percorsi di sviluppo potenzialmente contribuiscono alla patogenesi della malattia 8, 11. Inoltre, come abbiamo descritto, gli NPC sono una popolazione neuronale robusto replicativa e facilmente trasdotta, che riteniamo possa essere adatto per studi molecolari e biochimici di predisposi...

Studi di espressione genica di neuroni differenziati in vitro da umani cellule staminali pluripotenti indotte (hiPSCs) da noi 1 e altri 2,3 indicano che i neuroni hiPSC assomigliano fetale piuttosto che adulti tessuto cerebrale. Allo stato attuale, i modelli basati hiPSC può essere più appropriato per lo studio della predisposizione, piuttosto che di fine &#39;di, malattia neurologica. Abbiamo precedentemente riportato che una frazione significativa della firma gene della schizofrenia neuroni hiPSC-derivati ​​è conservata in celle schizofrenia hiPSC di derivazione progenitrici neurali (NPC), che indica che i PNG può essere un tipo di cellule utili per lo studio dei meccanismi molecolari che contribuiscono alla schizofrenia 1 . Noi e altri hanno segnalato la migrazione aberrante, aumento dello stress ossidativo e le specie reattive dell&#39;ossigeno, la sensibilità al di sotto della soglia stress ambientali e alterata funzione mitocondriale nella schizofrenia hiPSC NPC 1,4-6, così come è diminuito neuronale connectivity e funzione sinaptica nei neuroni schizofrenia hiPSC 5,7-10. Se i fattori molecolari che contribuiscono alla migrazione aberranti e / o stress ossidativo nella schizofrenia hiPSC NPC alla base anche la connettività neuronale ridotta in schizofrenia neuroni hiPSC-derivati, PNG potrebbero essere una popolazione neurale robusto e altamente replicativa con cui studiare i meccanismi responsabili della malattia. Inoltre, perché si può rapidamente generare un gran numero di cellule e hanno bisogno di non aspettare settimane o mesi per la maturazione neuronale, test a base di NPC sono adatti per lo studio di coorti di pazienti più grandi e sono più suscettibili di screening ad alto rendimento. Crediamo che hiPSC NPC può servire come proxy per i percorsi di sviluppo potenzialmente contribuiscono alla patogenesi della malattia, come è già stato dimostrato in disturbi diversi come la schizofrenia 1 e Huntington della malattia 11. Per differenziare NPC da hiPSCs, neurale inizialeproduzione si ottiene l&#39;inibizione dual-SMAD (0,1 mM LDN193189 e 10mM SB431542) 12. Antagonizzando BMP e TGF segnalazione con queste piccole molecole, endoderma e mesoderma specifica è bloccato, accelerando la differenziazione neuronale e che porta alla formazione di rosette neurali visibili entro una settimana dalla placcatura. Patterning neurale si verifica all&#39;inizio di questo processo, presumibilmente durante il periodo di formazione di rosette neurale e subito dopo. In assenza di altre indicazioni, queste cellule neurali primitive assumono una anterior proencefalo simile destino 13. Subito dopo la formazione di rosette neurale, e continua per tutta l&#39;espansione NPC, NPC prosencefalo sono coltivate con FGF2 8,14. Hanno dual potenziale lignaggio e possono essere differenziati a popolazioni neurali del 70-80% neuroni III-TUBULINA-positivi e 20-30% proteina acidica fibrillare gliale (GFAP) astrociti -positive (Figura 1). La maggior parte dei neuroni del prosencefalo hiPSC sono VGLUT1-positivi, E così sono presumibilmente glutamatergica, anche se circa il 30% dei neuroni sono GAD67-positivi (GABAergica) 8. NPC sono regolarmente diversi passaggi più di dieci volte in vitro, pur mantenendo i profili di differenziazione coerenti, e senza accumulare anormalità del cariotipo. Gruppi hanno riportato PNG Passaging più di 40 volte 15, però, scopriamo che oltre dieci passaggi, PNG mostrano maggiore propensione per la differenziazione astrociti. NPC ben tollera più freeze-disgelo e può essere la transizione a crescere come neurosfere semplicemente coltura in piastre non aderenti. NPC sono efficacemente trasdotti da vettori virali, consentendo una rapida valutazione delle conseguenze molecolari e cellulari di perturbazioni genetiche, e facilmente espandibile per produrre materiale sufficiente per studi biochimici. Inoltre, poiché i vettori virali consentono robusto over-espressione e / o atterramento di geni-malattia rilevanti, sia di controllo o paziente derivato neurcellule di Al, si può utilizzare questa piattaforma per testare l&#39;effetto di background genetico in queste manipolazioni. Anche se non è adatto per synaptic o saggi di attività a base richiedono neuroni maturi, PNG può essere una pratica alternativa per molte analisi molecolari o biochimici semplici di cellule neuronali derivate dal paziente.

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			<td>for HES media</td>
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			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
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			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">Glutamax</td>
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			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
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			<td style="width:216px;">&#160;#21985-023</td>
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			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">FGF2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
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			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0074</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">BDNF</td>
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			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
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			<td style="width:500px;"></td>
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			<td height="42" style="height:42px;width:288px;">Accutase</td>
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			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
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			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
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			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Rosette neurali possono essere identificati morfologicamente, utilizzando un microscopio campo chiaro, per l&#39;aspetto caratteristico come cluster rotonde di cellule neuroepiteliali con apico-basale polarità (Figura 1). Anche se gli NPC sono tipicamente coltivate a densità molto elevata delle cellule, subito dopo passaging, leggermente a forma di piramide soma e bipolare struttura neurite è visibile (Figura 1D). NPC convalidate esprimono nestina e SOX2 nella maggior parte delle cel...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Una guida per generazione e l&#39;uso hiPSC NPC derivati ​​per lo Studio delle Malattie Neurologiche

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

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Medicine

18.1K Views.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Video: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Metodo per Novel Anti-Cancer Drug Development utilizzando Espianti Tumore di campioni chirurgici


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Ricerca

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizzando la Stimolazione Magnetica Transcranica per studiare il sistema neuromuscolare umano

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Utilizzando Saccadometry con stimolazione cerebrale profonda per studiare normali e patologiche funzione cerebrale

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Un regime Fenotipizzazione per topi geneticamente modificati utilizzati per studiare geni implicati in malattie umane di invecchiamento

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Impostazione di una Stroke algoritmo squadra e svolgono la formazione basata sulla simulazione nel Dipartimento di Emergenza - Una guida pratica

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Sintesi radiochimica automatizzata di [<sup&gt; 18</sup&gt; F] 3F4AP: un nuovo tracciante PET per le malattie Demyelinating di imaging

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Usando la formazione immagine retinica per studiare la demenza

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Impianto di hiPSC-derivato del muscolo cardiaco patch dopo lesione del miocardio in un modello della cavia

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Una guida per generazione e l'uso hiPSC NPC derivati ​​per lo Studio delle Malattie Neurologiche

Una guida per generazione e l'uso hiPSC NPC derivati per lo Studio delle Malattie Neurologiche