BS è stato sostenuto da Università degli Studi di Milano-Bicocca Summer Student Programma di formazione alla ricerca; ricerca è stata sostenuta da fondi del Ministero della Salute italiano e dal Ministero italiano dell&#39;Istruzione, dell&#39;Università e della Ricerca e la Fondazione Humanitas per GC, si ringraziano Michael VG Latronico per criticamente la lettura del manoscritto.

1. Manutenzione alimentatore-Free e Passaging delle iPS umane Cell Lines <ol><li> Preparare i piatti Matrigel rivestite. Scongelare una fiala di Matrix ESC-umano qualificato in ghiaccio per un&#39;ora e diluirlo in 25 ml di DMEM-F12 medium. Aggiungere 1 ml di ciascuna piastra 35 mm (o importi equivalenti per superficie, se sono utilizzati altri piatti), mantenendo tutto il ghiaccio e la garanzia che tutte le lastre e tubi sono pre-raffreddata. Coprire le piastre con un foglio di alluminio. </li></ol> Nota: le concentrazioni di magazzino Matrigel variano a seconda del lotto. Istruzioni per la diluizione sono indicati sulla scheda tecnica. <ol start="2"><li> Mantenere le piastre in ghiaccio durante la notte e rimuovere dal ghiaccio il giorno successivo. Le piastre possono essere conservate in frigorifero fino ad una settimana prima di utilizzare. </li><li> Preparare il terreno completo mTESR1 (o scongelare una fiala di Nutristem media) e l&#39;H-ESM (medio ESC umane) in base alla tabella sottostante. <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> H-ESM </td><td> FINALE CONC </td><td> Per 500 ml </td></tr><tr><td> Knockout Serum Replacement (KSR) </td><td> 20% </td><td> 100 ml </td></tr><tr><td> Glutamax 100X </td><td> 2 mM </td><td> 5 ml </td></tr><tr><td> MEM non essenziali aminoacidi </td><td> 0,1 mM </td><td> 5 ml </td></tr><tr><td> Penicillina-streptomicina </td><td> 100 U / ml - 0,1 mg / ml </td><td> 5 ml </td></tr><tr><td> 2-mercaptoetanolo </td><td> 0,1 mM </td><td> 900 microlitri </td></tr><tr><td> B27 Supplemento - senza Vitamina </td><td> 1% </td><td> 10 ml </td></tr><tr><td> N2 Supplement </td><td> 1% </td><td> 5 ml </td></tr><tr><td> Knockout DMEM </td><td> - </td><td> 370 ml </td></tr></tbody></table> Archivio mezzo può essere conservato a 4 ° C per non più di 2 settimane. Per preparare il terreno di coltura completo, FGF di base viene aggiunto solo before l&#39;alimentazione ad una concentrazione finale di 20 ng / ml. </li></ol> Nota: Complete H-ESM condizionato su fibroblasti embrionali di topo può essere utilizzato al posto di mTESR1 o Nutristem per le cellule iPS. <ol start="4"><li> Posizionare le piastre rivestite in un incubatore a 37 ° C per 30 min a 2 ore prima della passaging. </li><li> Le cellule devono essere diversi passaggi circa ogni 5 giorni, anche se non hanno raggiunto la confluenza. Le colonie non dovrebbero toccare. </li><li> Sostituire mezzo di crescita con 1 ml di dispasi (1 mg / ml, sciogliere da 10 mg / ml di brodo in PBS). </li><li> Rimuovere differenziare le aree con tirato vetro pipette Pasteur. Le aree che dovrebbero essere rimossi comprendono strutture a cratere o cistica nel centro di colonie, e le aree in cui le cellule si sono separati dalle colonie e appiattiti e sembrano essere la migrazione verso l&#39;esterno. </li><li> Incubare a 37 ° C sotto una cappa di coltura fino al confine colonie diventano più luminose e cominciano a staccarsi (di solito circa 5-7 min). Discard dispasi. Lavare con 1 ml di H-ESM e scartare. </li><li> In 1 ml di H-ESM, utilizzare un sollevatore di cellulare (spatola-come raschietto) per colonie raccolto e raccogliere in una provetta Eppendorf 15. Risciacquare con 1 ml di H-ESM e aggiungere al tubo. Spin a 1.000 rcf per 4 minuti e rimuovere il surnatante. </li><li> Risospendere il pellet in 1 ml di terreno di crescita pre-riscaldato (sia mTESR1 o Nutristem). Evitare di rompere i grumi troppo - pipetta su e giù per meno di 6-8x. Determinare quante cellule devono essere placcato e rimuovere le cellule non necessarie (di solito 1:04 o 1:5 di diluizione), ad esempio per 2 piatti a 1:05, togliere 600 ml, aggiungere 1,6 ml di mezzo fresco. </li><li> Rimuovere Matrigel dalle piastre. Cellule posto goccia a goccia, distribuendo in modo uniforme sul piatto. Evitare di scuotere il tubo di troppo, in modo da indurre le cellule a muoversi verso il centro. </li><li> Lavare tubo con 1 ml di mezzo e di divisione tra le piastre. Volume finale in ogni piastra deve essere 1,5 -2 ml. </li><li> Cambiare medio di tutti i giorni, tranne il giorno dopo passaging. Sebbene siaideale per cambiare il mezzo quotidiana, puo essere cambiata occasionalmente ad una frequenza di una volta ogni due giorni se sono passati più di 2-3 giorni dall&#39;ultimo passaggio senza influenzare potenziale pluripotenza o differenziazione. </li><li> Se la linea cellulare deve essere congelato, risospendere invece in 1-2 ml mFreSR congelamento media utilizzando una pipetta ml 2 e posto 1 ml / esageratamente. Posto flaconi in un CRYOBOX a -80 ° C e trasferimento azoto liquido in 3-4 giorni. </li></ol> Nota: microdissezione manuale delle cellule iPS deve essere effettuata allo stereomicroscopio. Una fecondazione in vitro (IVF) workstation (ad esempio una workstation IVF da KSystem) integrato con uno stereomicroscopio permette la manipolazione delle cellule in condizioni sterili mentre sono tenuti su una superficie riscaldata. Se questo non è disponibile, uno stereomicroscopio può essere spostata sotto una cappa a flusso laminare regolare. 2. Corpi embrionali Aggregazione e Cardiac Induction <ol><li> Preparare EBM-20 medio secondo la tabella sottostante. <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> EBM-20 </td><td> FINALE CONC </td><td> Per 500 ml </td></tr><tr><td> Di siero fetale bovino (provenienza Sud America) </td><td> 20% </td><td> 100 ml </td></tr><tr><td> MEM aminoacidi non essenziali </td><td> 0,1 mM </td><td> 5 ml </td></tr><tr><td> Penicillina - streptomicina </td><td> 100U/ml - 0,1 mg / ml </td><td> 5 ml </td></tr><tr><td> Glutamax 100X </td><td> 0,1 mM </td><td> 5 ml </td></tr><tr><td> 2-mercaptoetanolo </td><td> 50um </td><td> 450 microlitri </td></tr><tr><td> DMEM/F12 </td><td> - </td><td> 385 ml </td></tr></tbody></table> Uso di FBS di origine Sud America è fondamentale per determinare l&#39;efficienza di differenziazione: l&#39;occupazione di altri tipi di sieri possono influireil processo di differenziazione. Per preparare EBM-20, acido ascorbico lista completa (AA) ad una concentrazione finale di 50 pg / ml. Terreno di crescita completo può essere conservato a 4 ° C per non più di una settimana. </li><li> Harvest iPS colonie come descritto sopra (passi 1,6-1,9). </li><li> Risospendere delicatamente in 1 ml EBM-20 con una pipetta ml 2. Evitare di rompere i grumi troppo - pipetta su e giù per non più di 2-3x, controllando la dimensione delle colonie allo stereomicroscopio. Piastra su piastre di fissaggio ultra-bassi in rapporto 1:1 (per esempio per un piatto di 35 mm, utilizzare 1 pozzetto di un 6-pozzetti). Sciacquare provetta con 1 ml di EBM-20 e di aggiungere al piatto. </li></ol> Nota: La dimensione delle colonie influenzano il processo di differenziazione, controllando l&#39;efficienza e la tempistica di induzione cardiaco. Aggregazione di omogeneamente dimensioni EBs hanno dimostrato di ridurre la variabilità osservata durante la differenziazione. <ol start="4"><li> Il giorno 3, cambiare EBM-20 una volta utilizzando un microscopio per evitare di ririmozione di EBS. Mantenere pipetta vicino a bordo del piatto, e togliere medie. </li><li> Il giorno 7, interruttore EBs alle piastre rivestite con 0,1% di gelatina e precedentemente posizionato a 37 ° C per 30 min. Se necessario, la gelatina può essere rimosso e piastre lasciato sotto una cappa di coltura cellulare O / N. Aggiungere 35-40 EBs per piastra da 35 mm. </li><li> Controllare EBs per battere le zone e cambiamento EBM-20 due volte a settimana. Delimitare le zone dove battono sono situati sulla parte superiore del coperchio piatto, per facilitare la loro localizzazione con lo stereomicroscopio. Aree contraenti dovrebbero comparire dopo circa 10-20 giorni. </li><li> Quando le aree con contrazione spontanea appaiono, passare medio EBM-2 (preparare come EBM-20, con il 2% di FBS, invece) e isolano come descritto di seguito nella sezione 3. </li><li> Continuare a controllare altre zone per aver picchiato e cambiare media due volte a settimana fino a quando sono necessarie aree battendo per ulteriori esperimenti. </li></ol> Nota: L&#39;efficienza del processo di differenziazione è fortemente dipendentediversi fattori, il più critico dei quali sono le linee cellulari specifici e il lotto del siero usati per indurre differenziazione cardiaca. Per ottenere la massima efficienza, linee cellulari di prova per potenziale cardiomyogenic e vari lotti di siero. 3. Battere aree di isolamento e di cultura <ol><li> Cappotto piastre da 12 pozzetti (4 cm 2 di area) e le piastre di interesse con 5 pg / cm 2 fibronectina, diluiti in PBS per fare un volume sufficiente a coprire completamente l&#39;intera superficie (totale 300 pl per pozzetto in piastre da 12 pozzetti). Tenere scoperto in cappuccio di coltura cellulare e lasciare asciugare. Le piastre possono essere confezionati e conservati a 4 ° CO / N. </li><li> Rimuovere area utilizzando un bicchiere battendo tirato pipetta Pasteur e bisturi, come necessario. Trasferimento alla fibronectina piastre rivestite con una pipetta da 1 ml. </li><li> Aggiungere 1 ml di EBM-2. </li><li> Attraversare fuori la piastra per indicare le aree dove sono stati rimossi e le cellule cambiare il mezzo. Le piastre possono essere conservate per un massimo di 1 mese, come altre aree possono iniziare bmangiare in seguito. </li></ol> Nota: Se sono necessari più piccoli ciuffi battendo, pipettare l&#39;area espiantato su e giù diverse volte con lo stereomicroscopio, finché non si rompe in piccoli pezzi. Questo si tradurrà in gruppi di poche cellule battente (vedi film 3). <ol start="5"><li> Cambiare media due volte a settimana fino al momento delle analisi. </li></ol> Nota: Cardiomyocytes ottenuti da questo protocollo possiedono caratteristiche morfologiche e funzionali fetale-simili; maturazione verso un fenotipo adulto-simile può essere ottenuto aumentando il tempo di coltura per differenziare ulteriormente queste cellule. 4. Singolo Battere celle di isolamento e di analisi <ol><li> Cappotto vetrini 2-camera (4 cm 2 area) o altri supporti appropriati con 5 pg / cm 2 fibronectina diluito in PBS sufficiente a coprire l&#39;intera superficie cultura. Se viene utilizzato un supporto di vetro, cappotto con laminina e fibronectina (5 mcg / cm &lt;sup&gt; 2 ciascuno). </li><li> Rimuovere zona battendo con una pipetta Pasteur / bisturi dalle tavole della sezione 3. </li><li> Usando una pipetta 1 ml, trasferire le cellule su una provetta 15 ml contenente 500 microlitri collagenasi II (480 U / ml in PBS con Mg e Ca) e incubare 15 minuti a 37 ° C, agitando il tubo una volta durante l&#39;incubazione. </li><li> Pipetta su e giù con una pipetta 200 microlitri. Se eventuali grumi rimangono, trasferimento a fresco collagenasi II e ripetere il punto 4.3. </li><li> Ripetere il punto 4.4 fino a quando non ha grandi ciuffi sono lasciati. </li><li> Inattivare collagenasi con 3 ml di EBM-2 (no AA). Il tubo può quindi essere lasciato sotto il cofano in un rack riscaldata finché altri tubi sono pronti. Centrifugare a 1000 rcf per 4 min. </li><li> Risospendere pellet in 1 ml di 0,25% tripsina / EDTA (diluito da uno stock di 0,5% in 1X PBS) e incubare a 37 ° C per 5 min. Combina frazioni di digestioni dallo stesso campione iniziale. </li><li> Inattivare la tripsina con 4 ml EBM-2 (no AA). Passare cellule attraverso un ago 18G in una siringa da 2,5 ml non più than 7x. Centrifugare a 1000 rcf per 4 min. </li><li> Risospendere il pellet di cellule in 1 ml di EBM-2 per pozzetto e la piastra. Cellule possono essere utilizzati per analisi funzionali e morfologiche 2-3 giorni dopo la placcatura. </li></ol>

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Cellule staminali pluripotenti hanno la potenzialità di differenziarsi spontaneamente in CMS, anche se con bassa efficienza ed elevata variabilità tra le linee. Lo sviluppo di nuovi metodi di induzione è quindi concentrata sul miglioramento dell&#39;efficienza del processo e muoversi verso protocolli più definiti. Tuttavia, la maggior parte di questi nuovi metodi di differenziazione sono piuttosto complessi, che richiedono condizioni elaborate cultura, controllo fine dei tempi e la concentrazione dei reagenti, e il ...

Storicamente, l&#39;indagine dei meccanismi genetici e molecolari che guida lo sviluppo umano e la malattia si è basata sulla generazione di modelli animali geneticamente modificati. Tuttavia, numerosi fenotipi umani non riescono ad essere replicato correttamente nei topi, soprattutto a causa delle differenze biologiche esistenti tra le due specie. D&#39;altra parte, l&#39;accesso ai tessuti umani può essere limitata e spesso non permettono abbastanza materiale da ottenere per studi sperimentali approfonditi. Il campo della biologia cardiovascolare soffre di entrambe queste limitazioni: la fisiologia del cuore umano è significativamente diversa da quella del topo, e una quantità significativa di tessuto cardiaco è accessibile solo post mortem o durante la chirurgia cardiaca. Trovare una fonte ottimale per la differenziazione delle funzionale umana CM è quindi diventato un tema centrale in biologia cardiovascolare, ed è stato fatto molto sforzo per risolvere questo problema. Sono stati proposti vari tipi di cellule, including mioblasti scheletrici, cellule locali cardiache staminali del midollo osseo, cellule mononucleari, progenitori endoteliali e cellule staminali mesenchimali. Tuttavia, i dati ottenuti utilizzando queste cellule non sia stato coerente 4. La derivazione di cellule staminali embrionali umane (ESC) prima 5 e la scoperta rivoluzionaria di staminali pluripotenti (iPS), cellule indotte da Yamanaka e Thomson 6,7 poi sembrava fornire soluzioni: queste cellule hanno la capacità di crescere indefinitamente e il potenziale per dare salire a tutti i derivati ​​delle cellule dei tre foglietti embrionali, tra cui il CMS. L&#39;uso di cellule iPS offre ulteriori vantaggi: essendo derivati ​​da cellule adulte autologhe, portano lo stesso genoma come l&#39;individuo o paziente da cui sono derivati. Essi consentono quindi lo sviluppo di modelli in vitro che facilitano l&#39;indagine di malattie genetiche umane e meccanismi di sviluppo. In virtù di questa caratteristica, le cellule iPS possono anche Oproblemi legati alla vercome rigetto immunitario e questioni etiche 8. Per queste ragioni, anche se i CES rappresentano ancora il gold standard nel campo della biologia delle cellule staminali, i ricercatori in tutto il mondo si stanno ora orientando più verso la tecnologia delle cellule iPS. cellule iPS vengono ora impiegati per modellare la malattia umana in vitro per le varie condizioni, tra cui disturbi cardiovascolari congenite 9,10. Recentemente, l&#39;applicazione di modelli di malattia delle cellule iPS è stata eseguita per i disturbi cardiaci monogeniche (cioè lunghe sindromi del QT, tachicardia ventricolare polimorfa catecolaminergica) e patologie nelle quali difetti cardiaci fanno parte di un fenotipo complesso (cioè Leopard e Timothy sindromi, cardiomiopatia dilatativa). Questi rapporti hanno confermato che le cellule iPS specifiche per ogni paziente che si differenziano in CM presentano caratteristiche fenotipiche e funzionali simili, come la malattia in vivo. Tuttavia, cisono ancora molte sfide per migliorare l&#39;efficacia di indurre le cellule iPS nella discendenza cardiaco. Spontanea generazione di CMS dalla CES umane attraverso la formazione di aggregati chiamati corpi embrionali (EBS) si sono dimostrati di successo 17. Dopo questa scoperta, sono stati proposti molti altri metodi. Molti gruppi hanno notevolmente migliorato l&#39;efficienza del protocollo di differenziazione e si sono spostati verso i reagenti di coltura chimicamente definite e animale-prodotto-libero (vedi Mummery, C., et al., Ricerca Circolazione (2012) per una rassegna completa di tutti i metodi esistenti 3 ). Tuttavia, il metodo classico basato su EB aggregazione rappresenta ancora il più comunemente impiegato per l&#39;esecuzione di studi funzionali e di indagare i meccanismi di malattia. La nostra proposta di protocollo si basa sull&#39;aggregazione di cellule iPS in EBs e cultura in presenza di acido ascorbico e di siero, che ha dimostrato di migliorare il processo di differenziazione cardiaca e di Positively influenzare la maturazione di queste cellule 18,19. In questo articolo andremo attraverso questa metodologia in dettaglio e vi mostreremo come utilizzare alimentatore-Free linee di cellule iPS per la generazione di paziente-specifici iPS derivate CMS.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media and Reagents</td>
		</tr>
		<tr>
			<td>Human ESC-qualified Matrix</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Thaw the 5 ml bottle at 4 &deg;C on ice O/N, aliquot in pre-chilled cryo-tubes on ice, according to the data sheet dilution instructions and store at -20 &deg;C .</td>
		</tr>
		<tr>
			<td>Fibronectin, from human plasma, 0.1% solution</td>
			<td>Sigma</td>
			<td>F0896-2MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>PBS (no Mg and Ca), 10X</td>
			<td>Lonza</td>
			<td>BE17-515F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS (with Mg and Ca), 10X</td>
			<td>Bio Sera</td>
			<td>XC-S2067/500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase II, neutral protease, grade II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>Stock: 10 mg/ml in DMEM-F12 at -20 &deg;C, working solution: 1 mg/ml in PBS (no Mg/Ca)</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA, 0.5%</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase type 2</td>
			<td>Worthington</td>
			<td>4176</td>
			<td>Dilute to 480 U/ml (e.g. 1.6 mg/ml) in PBS with Mg and Ca</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>21331-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Thaw at 4 &deg;C, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (origin South America)</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>Batches should be tested for their cardiogenic potential. Thaw at 4 &deg;C, heat-inactivate at 56 &deg;C for 30 min, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>PenStrep, penicillin-Streptomycin, 100X</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GlutaMAX, 100X</td>
			<td>Life Technologies</td>
			<td>35050-038</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM NEAA, 100X</td>
			<td>Invitrogen</td>
			<td>11140-135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 supplement, 100X</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27 supplement, 50X</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>31350-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelatin, from porcine skin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td>Make stock at 1% in water, store at -20 &deg;C. Dilute to 0.1% in PBS and keep at 4 &deg;C</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td>Combine Supplement 5X with the basic medium, aliquot and store at -20 &deg;C. Do not keep complete medium at 4 &deg;C for more than 1 week</td>
		</tr>
		<tr>
			<td>Nutristem hESC XF</td>
			<td>Stemgent from Biological Industries</td>
			<td>05-100-1A</td>
			<td>Thaw at 4 &deg;C O/N, aliquot and store at -20 &deg;C until use. Aliquots may be kept at 4 &deg;C for no more than a week.</td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>Stem Cell Technologies</td>
			<td>5853</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-25G</td>
			<td>Prepare stocks at 10 mg/ml in water. Store aliquots at -20 &deg;C in the dark. Add to EBM at the final concentration (500X)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>35 mm culture dishes</td>
			<td>Becton Dickinson</td>
			<td>353001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Becton Dickinson</td>
			<td>353043</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permanox 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177437</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177380</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6-well plates, ultra-low-attachment surface</td>
			<td>Corning Incorporated</td>
			<td>3471</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Becton Dickinson</td>
			<td>304622</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes, 230 mm</td>
			<td>VWR International</td>
			<td>612-1702</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Becton Dickinson</td>
			<td>300188</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipments</td>
		</tr>
		<tr>
			<td>IVF Workstation</td>
			<td>KSystem, by Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nikon SMZ1500 Stereomicroscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

generation of human cardiomyocytes: a differentiation protocol from feeder-free human induced pluripotent stem cells

Al fine di indagare gli eventi di guida lo sviluppo del cuore e per determinare i meccanismi molecolari responsabili delle malattie del miocardio negli esseri umani, è essenziale prima di generare cardiomiociti umani funzionali (CMS). L&#39;uso di queste cellule in scoperta di farmaci e studi tossicologici sarebbe anche molto vantaggioso, permettendo nuove molecole farmacologici per il trattamento dei disturbi cardiaci essere convalidate preclinicamente su cellule di origine umana. Delle possibili fonti di CMS, staminali pluripotenti (iPS) indotte sono tra i più promettenti, in quanto possono essere ricavati direttamente dal tessuto del paziente prontamente accessibile e possiedono una intrinseca capacità di dare origine a tutti i tipi di cellule del corpo 1. Sono stati proposti diversi metodi per differenziare le cellule iPS in CMS, che vanno dai corpi embrionali classiche (EBS) approccio aggregazione chimica definita protocolli 2,3. In questo articolo vi proponiamo un protocollo EB-based e mostriamo come questa metodologiad può essere impiegato per generare in modo efficiente le cellule CM-come funzionali di cellule iPS alimentatore-Free.

Nei nostri studi abbiamo generato CM da diverse linee di cellule iPS. Queste linee sono state inizialmente generate su uno strato di alimentatore MEF ma sono stati immediatamente immessi sul Matrigel usando un terreno specifico (sia mTESR1 o Nutristem). Vari metodi sono stati utilizzati per verificare mantenimento delle proprietà morfologiche, l&#39;espressione di marcatori tipici di cellule pluripotenti (OCT-4, SSEA4 e TRA1-60) e la loro stabilità genoma (Figura 1). Induz...

Watch this Scientific Journal Video about Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells at JoVE.com

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

Le cellule staminali pluripotenti, sia staminali (iPS), le cellule embrionali pluripotenti o indotto, costituiscono una preziosa fonte di cellule differenziate umani, tra cardiomiociti. Qui, ci si concentrerà sulla induzione cardiaco di cellule iPS, che mostra come usarli per ottenere cardiomiociti umani funzionali attraverso un protocollo embryoid corpi-based.

Generazione di cardiomiociti umani: Un protocollo di differenziazione da alimentatore-Free umani cellule staminali pluripotenti indotte

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

generation-human-cardiomyocytes-differentiation-protocol-from-feeder

Research

JoVE Journal

Biology

21.2K Views.  Humanitas Clinical and Research Center, Italy. Le cellule staminali pluripotenti, sia staminali (iPS), le cellule embrionali pluripotenti o indotto, costituiscono una preziosa fonte di cellule differenziate umani, tra cardiomiociti. Qui, ci si concentrerà sulla induzione cardiaco di cellule iPS, che mostra come usarli per ottenere cardiomiociti umani funzionali attraverso un protocollo embryoid corpi-based.

Video: Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Generazione di cellule staminali pluripotenti indotte da fibroblasti umani Riprogrammare con il Stemgent Lentivirus umani TF Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Ricerca

Biologia

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Trasfettando e Nucleofecting umani cellule staminali pluripotenti indotte

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Generazione di cellule pluripotenti indotte umane staminali da sangue periferico mediante il STEMCCA lentivirale Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Efficiente generazione indotta umane pluripotenti cellule staminali da cellule somatiche umane con Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Alta efficienza differenziazione delle cellule staminali pluripotenti umane di cardiomiociti e Caratterizzazione mediante citometria di flusso

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

Generazione e mantenimento di cellule staminali pluripotenti indotte da primati derivate dall'urina

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular ...

Caratterizzazione degli antigeni di superficie e capacità di proliferazione nelle MSC che guidano le hiPSC

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Autore in primo piano: Progressi nelle iPSC e nella ricerca sulle malattie genetiche 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Confronto di due metodi rappresentativi per la differenziazione di cellule staminali pluripotenti indotte umane in cellule stromali mesenchimali

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...