We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

NOTA: Questo metodo è stato utilizzato nella ricerca riportata nel Awe et al 10.. 1. Riprogrammazione umano adulto Dermal fibroblasti con STEMCCA <ol><li> Fibroblasti dermici Thaw adulti umani (HUFS), di passaggio a 4 o inferiore, in un bagno d&#39;acqua a 37 ° per 2 minuti, avendo cura di non immergere la parte superiore del flaconcino con l&#39;acqua. </li><li> Posizionare la poltiglia 1 ml di fibroblasti umani in una fiala conica 15 ml e posizionare lentamente 4 ml di supporti HUF standard preriscaldata a 37 ° C, in un modo goccia a goccia, per diluire le dimetil solfossido (DMSO) e risospendere le cellule. </li><li> Centrifugare la fiala a 200 xg per 10 min a temperatura ambiente. Aspirare il supernatante e risospendere le cellule in un volume adeguato di media per creare una sospensione di 100.000 cellule / ml e aggiungere 2 ml in un pozzetto di una piastra a sei pozzetti, pre-rivestito per 20 minuti con 0,2% di gelatina. NOTA: Una sorella bene con un numero uguale di cellule per linea e condizione deve essere seminato for il conteggio delle cellule / molteplicità di infezione (MOI) di calcolo. </li><li> Oscillare la piastra di lato all&#39;altro e avanti e indietro per distribuire uniformemente le cellule. Incubare per una notte a 37 ° C, 5% CO 2 in un incubatore umidificato. </li><li> Il giorno di trasduzione, ottenere conta delle cellule (s) dalla sorella ben (s) circa 24 ore dopo la placcatura HUF iniziale. </li><li> Scongelare 2 x 10 8 TU, o, concentrato lentiviral equivalente (STEMCCA) e portare a 1 x 10 8 TU / ml in HUF media. </li><li> Trasdurre cellule con un rapporto di 10-MOI in HUF multimediale integrato con 8 mg / ml agente trasfezione. Mescolare pozzi trasdotte uniformemente e incubare una notte a 37 ° C, 5% CO 2 in un incubatore umidificato. </li><li> Circa 24 ore dopo la trasduzione lentivirale, passare i mezzi di HUF di cellule staminali pluripotenti umane (PSC) medie standard. </li><li> Nei giorni 2 a 6, cambiare i media quotidiana di media PSC umana. </li><li> Il giorno 6, piatto due 0,2% di gelatina rivestite piatti da 10 cm di1,5-1,75 x 10 6 MEF derivati ​​da CF1 topi in HUF supporti 12. </li><li> Il giorno 7, facendo attenzione a non centrifugare più di 100 grammi, l&#39;uso della temperatura ambiente tripsina per sollevare la giornata 7 fibroblasti riprogrammate da un pozzetto della piastra sei bene e il passaggio ad un rapporto di 01:16, placcando tutte le cellule in modo uniforme tra due piastre 10 cm, con i media PSC umano fresco, che sono stati placcati con MEF il giorno prima. Distribuire aggregati di cellule in un movimento a spirale in senso orario e posto a 37 ° C, 5% CO 2 incubatore umidificato notte. </li><li> Circa 48 ore dopo la semina le cellule riprogrammate sul MEF e ogni giorno dopo, cambiare fuori trascorsi supporti PSC umani con mezzi freschi. </li><li> Dopo 3 o 4 settimane, prendere le singole colonie con tipico ESC come la morfologia con un ago 21-gauge e subclone fuori in un 24-pozzetti stipulando circa 8 a 10 pezzi singoli di colonie specifiche nei singoli pozzetti in un 0,2% di gelatina rivestita 24 pozzetti pre-seeded con MEF derivate da topi CF1 a medio PSC umana. NOTA: Dopo l&#39;espansione delle colonie raccolte, le colonie devono essere caratterizzato come pluripotenti dalla formazione corpo embrioide e analisi di espressione di marcatori pluripotenza a livello proteico. </li></ol> 2. Vector Integrazione analisi in loco e STEMCCA Excision <ol><li> Analizzare il sito di integrazione STEMCCA da amplificazione lineare non restrittiva PCR come descritto 10. </li><li> Dopo saggiare per garantire una integrazione in una zona desiderata del genoma, STEMCCA accise aspirando il vecchio supporto ESC umana. Poi, unire 45 ml di virus adeno-Cre-PuroR concentrato con 8 mg / ml agente trasfezione in 3 ml di media PSC standard per la 24 ore. </li><li> Dopo il periodo di incubazione di 24 ore, aspirare il surnatante virale mista e lavare le cellule due volte con i media PSC umani. Inserire supporti PSC umani freschi insieme con 2 ug / ml puromicina per un periodo di 5 giorni, cambiando i media ingegno giornalierah mezzi freschi e antibiotici. </li><li> Dopo 5 giorni, subclone rimanendo colonie con un ago 21-gauge su 0,2% pozzi gelatina rivestite, in un 12-pozzetti pre-rivestito con MEF derivati ​​da CF1mice ed espandere come sopra. NOTA: Dopo l&#39;espansione sufficiente per ottenere un titolo congelato, è necessaria la conversione in condizioni di assenza di alimentazione. Aspettatevi almeno il 95% la morte colonia di cellule staminali, come la maggior parte delle cellule non saranno trasdotte con successo e si soccombere antibiotico nel periodo di 5 giorni di incubazione. Sebbene adeno-Cre-PuroR integra in un efficienza 0,001-1% in cromosomi di accoglienza delle cellule infette, reexposing un campione di colonie senza fattore di puromicina per assicurare la morte cellulare 100% confermerà nessuna integrazione è verificato 13. </li><li> Scongelare una fiala di pre-aliquotati matrice membrana basale su ghiaccio, per un periodo di circa 2 ore (raccomandazioni del produttore) finché la matrice è un liquido e diluire ad una concentrazione finale di 1:30 fredda mezzi basale. Coal numero desiderato di pozzetti in una piastra a sei pozzetti di 1 ml per pozzetto. Lasciate riposare a temperatura ambiente per 1 ora. </li><li> Cross-botola (con un ago calibro 18, o un bicchiere di plastica o &quot;punta&quot;), almeno 20 o 30 colonie da un pozzo in precedenza rivestito con MEF. Fare attenzione a ridurre MEF edificante e trasferire dalla piastra. <ol><li> Generare il dispositivo tratteggio attraverso un certo numero di approcci differenti, dal riscaldamento più complicato, tirando, e la finitura di una pipetta Pasteur di vetro su una fiamma libera, per il semplice utilizzo di una punta di plastica pipetta sterile o, come nel nostro caso, 21- e / o calibro 18 aghi. </li></ol></li><li> Aspirare la matrice dal rivestito ben dopo incubazione. </li><li> Togliere i pezzi delle colonie delicatamente raschiando dal vecchio piatto con una pipetta 200 microlitri e posizionare con cura in 3 ml di mezzi freschi combinate 1 nel piatto di matrice rivestita. Incubare per una notte a 37 ° C, 5% CO 2 incubatore umidificato. Cambia i media 48 ore dopo passaging. </li><li> Estratto DNA genomico da hiPSCs convertiti post- asportato e senza alimentatore, a più del 80% di confluenza, con kit di estrazione del DNA commerciale. </li><li> Analizzare per una corretta asportazione con primer specifici per le integrazioni esogeni del lentivirus STEMCCA: gDNA-Hendo-MycS-forward, 5&#39;-acgagcacaagctcacctct-3 &#39;; gDNA-hWPRE-reverse, 5&#39;-tcagcaaacacagtgcacacc-3 &#39;. Ricostituire primer liofilizzati con acqua PCR-grade ad una soluzione stock di 10 micron. </li><li> Eseguire una PCR con il seguente protocollo cinque fasi: denaturazione iniziale a 95 ° C per 3 min; 35 cicli di ciascuno dei seguenti: denaturazione a 98 ° C per 20 sec, ricottura di primer a 62 ° C per 15 sec, ed estensione a 72 ° C per 15 sec; seguito da un singolo ciclo di estensione finale a 72 ° C per 3 min. </li><li> Effettuare un gel di agarosio al 3% in peso su 1,5 g di agarosio e l&#39;immissione in 50 ml di tampone Tris-acetato 1x-EDTA. Forno a microonde fino agarosio è sciolto e posto in gel macchia DNA a pdiluizione Roper, mescolare e dispensare il volume corretto in cassette gel a solidificare per 1 ora a temperatura ambiente. </li><li> Carico 12 ml di prodotto di PCR mescolato con 3 ml di colorante di caricamento in un gel di agarosio al 3%. Eseguire il gel per 30 minuti a 80 V. </li><li> Visualizza con luce ultravioletta per l&#39;assenza di una banda che indica il corretto escissione. </li></ol> 3. La quantificazione di differenze di espressione genica utilizzando PCR quantitativa di pre e post-hiPSCs asportati <ol><li> Estratto RNA totale da hiPSCs convertiti post- asportato e senza alimentatore utilizzando kit di estrazione RNA commerciali. </li><li> Reverse-trascrivere fino a 1 mg di RNA totale utilizzando kit commerciali in conformità con le istruzioni del produttore. Utilizzare ancorato-oligo (dT) 18 e primer esameriche casuali. NOTA: Assicurarsi che il protocollo PCR è su misura per le trascrizioni gene di più o di meno di 4 kb. Dovranno essere fatti per i qualsiasi gene STEMCCA originariamente integrato primer specificinto. </li><li> Ricostituire il primer liofilizzato con acqua PCR-grade ad una soluzione stock di 20 micron. </li><li> Utilizzare 5 ng di campione per 20 microlitri di reazione che consiste di sonda UPL 10 micron, 2x LightCycler 480 Sonde Maestro, e 20 micron avanti e indietro primer. </li></ol> 4. Conversione in condizioni GMP-grade <ol><li> Il primo giorno di transizione i hiPSCs post- asportato verso condizioni di GMP-grade, fare una miscela di mezzi composto da PSC umana combinata multimediali 1 (i media combinata 1) e di grado clinico buone pratiche di fabbricazione (GMP) di media compatibile (supporti combinate 2 ) ad un rapporto di 80:20. </li><li> Aspirare off i vecchi media combinato 1 e collocare 3 ml di nuovi media combinati 1 e 2, pre-riscaldato, al rapporto di 80:20. Cambiare i media al giorno per 3 giorni con questo rapporto specifico. Cellule posto di nuovo in un 37 ° C, 5% CO 2 incubatore. </li><li> Il giorno 4, rendere il supporto combinato 1 e 2 con un rapporto 50:50. Aspirare il vecchio media e soste con i media pre-riscaldato, con i media combinata 1 e 2 al rapporto di 50:50. Cambiare i media per 3 giorni con questo rapporto specifico. Cellule posto di nuovo in un 37 ° C, 5% CO 2 incubatore. </li><li> Il giorno 7, rendere i media combinata 1 e 2 con un rapporto 20:80. Aspirare il vecchio media e sostituirli con supporti pre-riscaldato, con i media combinati 1 e 2 al rapporto di 20:80. Cambiare i media per 3 giorni con questo rapporto specifico. Cellule posto di nuovo in un 37 ° C, 5% CO 2 incubatore. </li><li> Il giorno 10, effettuare i media combinata 1 e 2 a 0: rapporto di 100. Aspirare il vecchio media e sostituirli con supporti pre-riscaldato, con i media combinata 1 e 2 a 0: rapporto di 100. Cambiare media ogni giorno a questo rapporto specifico. Le cellule sono ora in media liberi-xeno completamente definito. Cellule posto di nuovo in un 37 ° C, 5% CO 2 incubatore. NOTA: Durante l&#39;intero processo di conversione, è necessario passaging routine con un ago calibro 18 per mantenere la giusta dimensione delle colonie e la densità. Piano su passaging cellule ogni 4 o 5 giorni sulla base della dimensione delle colonie, confluenza, e differenziazione. </li><li> Coat un pozzetto in una piastra a sei pozzetti con un substrato senza xeno definito come raccomandato dal produttore. </li><li> Passaggio meccanicamente un confluente bene, già convertito in-Xeno gratuita condizioni di media, da una piastra di sei bene con un ago 18-gauge. È importante sottolineare che, posizionare nuovi media (supporti combinati 2) sulle cellule con un inibitore ROCK 1x per 1 ora, prima passaging pre-condizione di media. </li><li> Aspirare la soluzione substrato sintetico largo della nuova piastra che le cellule vengono trasferiti. Rimuovere tutti i supporti contenenti i grumi di cellule staminali dal vecchio piatto e trasferimento a nuova piastra. </li><li> Cellule posto di nuovo in un 37 ° C, 5% CO 2 incubatore per 2 giorni con il minimo disturbo fisico (idealmente piatti, una volta all&#39;interno dell&#39;incubatrice, non sono direttamente toccati in alcun modo) per una massima attaccamento. NOTA: Le cellule richiederanno cambiamenti di media al giorno. Passagin continuag ogni 4 giorni saranno importanti. Utilizzando un ago 21-gauge, passaggio solo le parti di colonie con ottima morfologia ESC-like (elevato rapporto nucleocytoplasmic). Ciò garantirà la selezione per le colonie hiPSC corretto che crescerà bene e alla fine produrranno colonie omogenee. Tempo complessivo di derivazione del CES come colonie è tra 15-20 giorni dopo il cambio di supporto iniziale combinato. </li><li> Congelare le hiPSCs post- asportato GMP-grade per primo tratteggio incrociato tutte le colonie utilizzando un ago calibro 18. Sollevare tutte le parti fuori utilizzando una pipetta 200 microlitri e trasferire tutti i supporti contenenti le cellule ad una fiala conica 15 ml e centrifugare a 200 xg per 5 minuti a 4 ° C. </li><li> Mentre le cellule sono centrifugazione, fare un supporto congelamento costituito da volumi uguali di supporto combinato freddo 2 e congelamento media con una concentrazione finale di DMSO del 7,5%. </li><li> Dopo le cellule sono pellettizzati, aspirare off vecchia medio e goccia a goccia risospendere il pellet con i media di congelamento. Immediatamente transfer di fiale congelamento e mettere in un -80 ° C freezer. </li></ol> 5. hiPSCs Characterizing GMP-grade Post-asportati <ol><li> Per garantire una corretta espressione dei marcatori pluripotenza post-conversione, utilizzare QPCR per quantificare l&#39;espressione di marcatori pluripotenza chiave (SOX2, OCT4, e Nanog) utilizzando i primer elencati nella tabella 1 con la stessa procedura di cui al punto 3. metodologie QPCR seguire la stessi passaggi elencati al punto 3. </li><li> Utilizzare citometria a flusso per rilevare l&#39;acido non umano sialico Neu5Gc (N acido -glycolylneuraminic) in base a condizioni standard elencati nel kit come precedentemente pubblicato 10. </li><li> Lavare delicatamente le piastre di coltura cellulare con cellule utilizzando PBS 1x (PBS). </li><li> Dissociate colonie utilizzando 1x reagente di dissociazione per 5 min a temperatura ambiente. </li><li> Durante il tempo di incubazione di 5 minuti di passo 5.4, preparare la soluzione di blocco (disponibile inkit) facendo un 0,5% agente di blocco in ghiacciata PBS. </li><li> Etichettare la seguente serie di tubi per citometria a flusso di analisi: senza macchia; 4 &#39;, 6-Diamidino-2-fenilindolo (DAPI) DAPI; Controllo anticorpo 1: 200; Anticorpo primario 1: 200; Anticorpo secondario 1: 200 Donkey Anti-Pollo IgG (H + L); MEF campione; Cellule post-asportato esempio a matrice; e celle su supporto sintetico Campione post-asportato. </li><li> Scuotendo leggermente aggregati di cellule dal punto 5.4 con l&#39;aggiunta di 2 ml di soluzione bloccante e trasferire il contenuto di un tubo da 15 ml. Centrifugare a 80 xg per 5 minuti a 4 ° C. </li><li> Lavare le cellule due volte con soluzione bloccante e centrifugare come indicato al punto 5.7. </li><li> Delicatamente risospendere circa 1 x 10 6 cellule in 100 microlitri di soluzione bloccante con il corrispondente volume di diluizione di ciascun anticorpo. Incubare con dolce dondolo a 4 ° C per 1 ora. </li><li> Ripetere lava tre volte al punto 5.8. </li><li> Risospendere il pellet cellulare in 400 ml di diluente Buffer (dal kit) con 1: 100 di DAPI per tubi 2, 6, 7, e 8 come elencato passo 5.6. </li><li> Cellule filtrare con un colino 40 micron e attraversano citofluorimetro. </li></ol>

<ol>
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James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

Descriviamo una metodologia di derivazione hiPSCs senza fattore e renderli clinicamente rilevante convertendo queste cellule in condizioni GMP-grade per la differenziazione delle cellule a valle in future terapie umane. Sebbene questo protocollo è ampiamente applicabile ad una varietà di tipi cellulari, abbiamo scelto di riprogrammare fibroblasti dermici umani, a causa della facilità di estrazione dal paziente e la loro applicabilità alle terapie umane personalizzati. Una volta che le limitazioni sono state sanate, ...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:135px;">Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:521px;">Media and reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">DMEM/F12 (basal media)</td>
			<td style="width:111px;">Invitrogen (Carlsbad, CA, USA)</td>
			<td style="width:136px;">11330057</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Fetal bovine serum</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">16000044</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:140px;">Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">11140050</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Glutamax, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">PenStrep, penicillin-streptomycin, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Knockout serum replacement</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">10828028</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Trypsin/EDTA, 0.5%</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15400-054</td>
			<td style="width:135px;">Dilute stock out to 0.05% in 1x PBS</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Basic fibroblast growth factor</td>
			<td style="width:111px;">GlobalStem (Rockville, MD, USA)</td>
			<td style="width:136px;">GSR-2001</td>
			<td style="width:135px;">Reconstitute to 10 &#181;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &#8722;80 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">&#946;-mercaptoethanol</td>
			<td style="width:111px;">Millipore (Billerica, MA, USA)</td>
			<td style="width:136px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Matrigel (basement membrane matrix)</td>
			<td style="width:111px;">BD Biosciences (San Jose, CA, USA)</td>
			<td style="width:136px;">356231</td>
			<td style="width:135px;">Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">CELLstart (Synthetic Substrate)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1014201</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Stemmolecule Y27632</td>
			<td style="width:111px;">Stemgent (Cambridge, MA, USA)</td>
			<td style="width:136px;">04-0012-02</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Puromycin</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1113802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">LightCycler 480 Probes Master</td>
			<td style="width:111px;">Roche (Basel, Switzerland)</td>
			<td style="width:136px;">4707494001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">ProFreeze-CDM Medium/freezing medium</td>
			<td style="width:111px;">Lonza (Basel, Switzerland)</td>
			<td style="width:136px;">12-769E</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Dimethyl sulfoxide</td>
			<td style="width:111px;">Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td style="width:136px;">D8418</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">PBS</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">14190-250</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">100 BP DNA Ladder</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15628019</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">SYBR Safe DNA Gel Stain 10000x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">S33102</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:140px;">Agarose</td>
			<td style="width:111px;">Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td>
			<td style="width:136px;">161-3101</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Gelatin, from porcine skin</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">G1890-100G</td>
			<td style="width:135px;">Make stock at 0.2% in PBS, autoclave and store at room temperature</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">mTeSR1</td>
			<td style="width:111px;">StemCell Technologies (Vancouver, BC, Canada)</td>
			<td style="width:136px;">5850</td>
			<td style="width:135px;">Combine Supplement 5X with the basic medium, aliquot and store at 4 &#176;C for up to 2 weeks.&#160;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Stemedia NutriStem XF/FF Culture Medium</td>
			<td style="width:111px;">Stemgent</td>
			<td style="width:136px;">05-100-1A</td>
			<td style="width:135px;">Thaw at 4 &#176;C O/N, aliquot and store at 4 &#176;C for up to 2 weeks.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Primocin</td>
			<td style="width:111px;">InvivoGen (San Diego, CA, USA)</td>
			<td style="width:136px;">ant-pm-1</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Accutase (Dissociation Reagent)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1110501</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Donkey anti-Chicken IgG AlexaFluor 488</td>
			<td style="width:111px;">Jackson ImmunoResearch (West Grove, PA, USA)</td>
			<td style="width:136px;">703-546-155</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Polybrene/transfection agent</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:136px;">TR-1003-G</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Plasticware</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">12-well plates</td>
			<td style="width:111px;">VWR (West Chester, PA, USA)</td>
			<td style="width:136px;">29442-038</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">6-well plates</td>
			<td style="width:111px;">VWR</td>
			<td style="width:136px;">29442-042</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">10-cm plates</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z688819</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">18-gauge needle</td>
			<td style="width:111px;">Fisher Scientific (Pittsburgh, PA, USA)</td>
			<td style="width:136px;">148265D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">21-gauge needle</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">14-829-10D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Equipment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD LSRII Flow Cytometer</td>
			<td style="width:111px;">KSystem by Nikon (Tokyo, Japan)</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD FACSDiva Version 6.1.3 Software</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Kits</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">PureLink Genomic DNA Mini Kit</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">K182000</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">KAPA HiFi Hotstart ReadyMix PCR Kit</td>
			<td style="width:111px;">KAPA Biosystems (Wilmington, MA, USA)</td>
			<td style="width:136px;">KK2601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">High Pure RNA Isolation Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">11828665001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:140px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">4379012001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Sialix anti-Neu5Gc Basic Pack Kit</td>
			<td style="width:111px;">Sialix (Newton, MA, USA)</td>
			<td style="width:136px;">Basic Pack</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 1</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts mTeSR1 and Nutristem</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 2</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts TeSR2 and Nutristem</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:140px;">HUF Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Dulbecco&#8217;s modified Eagle&#8217;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:140px;">Human Pluripotent Stem Cell Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &#946;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;">DMEM/F12, Dulbecco&#8217;s modified Eagle&#8217;s medium/F12; PBS, phosphate-buffered saline.</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
	</tbody>
</table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

Umane pluripotenti indotte (cellule staminali hiPSCs) possono essere generati con le metodologie di riprogrammazione basata lentivirali-. Tuttavia, tracce di geni potenzialmente oncogeni rimanenti nelle regioni attivamente trascritti del genoma, limitano il loro potenziale per l&#39;uso in applicazioni terapeutiche umani 1. Inoltre, antigeni non umani derivanti dalla riprogrammazione di cellule staminali o di differenziazione in derivati ​​terapeuticamente rilevanti escludono questi hiPSCs vengano utilizzati in un contesto clinico umano 2. In questo video, vi presentiamo una procedura per la riprogrammazione e l&#39;analisi hiPSCs senza fattore gratuitamente transgeni esogeni. Questi hiPSCs poi possono essere analizzati per alterazioni di espressione genica nel introne specifica l&#39;lentivirus. Questa analisi può essere condotta utilizzando sensibile reazione a catena della polimerasi quantitativa (PCR), che ha un vantaggio rispetto meno sensibili tecniche precedentemente utilizzato per rilevare le differenze di espressione genica 3. La conversione completa inbuone prassi di fabbricazione clinica-grade (GMP) condizioni, permette di rilevanza clinica umana. Il nostro protocollo offre un altro metodo, a condizione che i criteri esenti attuali espandere e comprendono caratterizzati derivati ​​basati hiPSC senza fattore di applicazioni, ad terapeutiche umane derivanti hiPSCs GMP-grade, che dovrebbero eliminare qualsiasi rischio di immunogenicità a causa di antigeni non-umani. Questo protocollo è ampiamente applicabile per lentivirali riprogrammato cellule di qualsiasi tipo e fornisce un metodo riproducibile per la conversione di cellule riprogrammate in condizioni di GMP-grade.

Vi presentiamo un protocollo per derivare clinico-grade hiPSCs senza fattore utilizzando l&#39;approccio basato riprogrammazione-lentivirali STEMCCA. La figura 1A mostra un quadro rappresentativo di tre diverse linee di pre-asportato hiPSC, dopo la riprogrammazione con l&#39;approccio STEMCCA su uno strato di MEF. Il vantaggio principale del metodo riprogrammazione STEMCCA risiede nel successo riprogrammazione coerente ottenuto da più scienziati, in diversi gruppi di ricerca e posizioni. Figura...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Derivazione e caratterizzazione di un free-Transgene umana indotta pluripotenti staminali Cell Line e Conversione in definite condizioni clinico-grade

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K Views.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

Video: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivazione di cellule staminali embrionali umane da Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Ricerca

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selezione e isolamento di colonie di cellule pluripotenti indotte umane staminali riprogrammato da fibroblasti adulti

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

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 ...

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 ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

Estrazione, etichettatura e purificazione di cellule specifiche del lignaggio da follicoli antrali umani

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...