This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

Basato Single-cell di tipo non-colonia monostrato (NCM) cultura della hPSCs. 1. Preparativi <ol><li> Rendere 500 ml di terreno per la cultura di fibroblasti embrionali di topo (MEF): medie DMEM supplementato con 10% FBS, 2 mM L-glutammina, e 0,1 mm aminoacidi non essenziali (NEAA). </li><li> Isolare fibroblasti embrionali di topo (MEF), le cellule derivate dal ceppo CF1 a seguito di un protocollo di routine 16 e cultura MEF sul 0,1% di gelatina rivestite 6-pozzetti di coltura cellulare in DMEM. In alternativa, l&#39;acquisto di azioni del MEF al passaggio 3 da risorse commerciali. </li><li> Preparare piatti Matrigel. <ol><li> Diluire 5 ml di hESC qualificati Matrigel magazzino con 5 ml di DMEM/F12 medio (raffreddato a ~ 4 ° C) e conservare il 50% aliquote in una -20 ° C freezer. Scongelare ghiacciato Matrigel magazzino in frigorifero (~ 4 ° C) O / N e diluire ulteriormente la Matrigel in mezzo DMEM/F12 freddo (per dare luogo ad una concentrazione di lavoro 2,5%). </li><li&gt; Coat 6 pozzetti piastre di coltura cellulare con 1,5 ml di 2,5% Matrigel per bene, conservare le piastre rivestite in frigorifero O / N (Nota: utilizzare la piastra Matrigel rivestite entro 2 settimane). </li><li> Rimuovere la piastra Matrigel dal frigorifero, lasciare che la piastra si riscaldi a temperatura ambiente nella cappa di coltura cellulare per 10-30 min (Nota: non scaldare la piastra in un incubatore coltura cellulare), e aspirare DMEM prima della placcatura hPSCs. </li></ol></li><li> Rendere 500 ml mezzo hESC: 80% medio DMEM/F12, 20% KSR, 2 mM L-glutammina, 0,1 mM aminoacidi non essenziali, 0,1 mM β-mercaptoetanolo, e 4 ng / ml di FGF-2. </li><li> Preparare 10 ml di 2X HPSC medio di congelamento: 60% FBS, 20% DMSO e 20 mM Y-27632 in mTeSR1 mezzo, sterilizzare per filtrazione, e utilizzare il mezzo entro 1 settimana. </li></ol> . 2 Protocollo 1 (Elementare): Grow HPSC Colonie su Alimentatori <ol><li> Utilizzare numeri passaggio MEF a 5 o 6 (designato come p5 e p6) per cultura HPSC per vedere Resul coerentets. Mitoticamente inattivare MEF trattando le cellule con 10 pg / ml mitomicina C per 3 ore a 37 ° C, lavare le cellule 3x con di 1X Dulbecco Phosphate-Buffered Saline (D-PBS), e poi si dissociano MEF con 0,05% tripsina in 0,53 mM EDTA. In alternativa, irradiare MEF alla dose di 8.000 rad considerando un irradiatore a raggi X. </li><li> Contare il numero di cellule utilizzando il metodo di esclusione del Trypan Blue macchia sotto microscopio. In alternativa, utilizzare un contatore automatico cella. </li><li> Piastre MEF irradiati su lastre di polistirene 6 pozzetti rivestiti con 0,1% di gelatina ad una densità di 1,88 x 10 5 cellule per pozzetto (cioè, 1,96 x 10 4 cellule / cm 2). Incubare le cellule a 37 ° C e 5% di CO 2 per 24 ore. </li><li> Rimuovere il terreno di coltura MEF per aspirazione con una pipetta Pasteur (Nota: nessun fasi di lavaggio necessari in questo momento). </li><li> Triturare HPSC colonie dalla cultura precedente in piccoli ciuffi, esaminano i grumi sotto il microscopio per assicurare loro dimensioni variano from 50 a 100 micron di diametro, e piastra ciuffi HPSC sulla cima MEF strati feeder in un pozzetto contenente 2 ml di HPSC medie. </li><li> Cambiare hESC mezzo al giorno per 3 a 5 giorni, record di crescita colonia fotografia, contrassegnare tutte le colonie morfologicamente alterati o differenziati (~ 5%), e rimuovere manualmente le colonie contrassegnati con delicatezza aspirazione con una pipetta Pasteur. </li><li> Risciacquare le restanti colonie MEFs due volte (2 min ciascuno con D-PBS), e incubare le colonie con 2 ml di 1 mg / ml di collagenasi IV in HPSC medio per 10 a 30 minuti), ed esaminare il distacco delle colonie HPSC dal MEF superficie rivestita (Nota: utilizzare un raschietto per aiutare il processo di distacco solo se le colonie sono strettamente collegati alla piastra). </li><li> Aggiungere 5 ml di HPSC mezzo a ciascun pozzetto per minimizzare le reazioni enzimatiche, trasferimento colonie ad una provetta da 15 ml, permettere colonie HPSC sedimentare per 3 a 5 minuti a temperatura ambiente, e garantire la sedimentazione delle colonie tramite visualizzazione diretta del cells nella parte inferiore del tubo (Nota: non centrifugare la provetta in questa fase). </li><li> Rimuovere il surnatante contenente MEF residue e dissociato singole cellule, risospendere le colonie con 5 ml di hESC medie, e ripetere l&#39;operazione di sedimentazione due volte. </li><li> Rimuovere le medie, triturare colonie HPSC in piccoli ciuffi, negozio in 1X HPSC congelamento medio (o CryoStore CS10 mezzo congelamento) per la crioconservazione (1 confluente bene per fiala congelata) o piastra su un 6-pozzetti per passaging cellulare. </li></ol> 3 Protocollo 2:. Convertire HPSC colonie da alimentatori a NCM <ol><li> Sciacquare pellets HPSC in D-PBS una volta, incubare il pellet con 1 ml di 1X Accutase per 10 minuti, ed esaminare la reazione enzimatica sotto un microscopio per assicurare dissociazione singola cellula. </li><li> Terminate le reazioni enzimatiche da risospendere delicatamente le cellule in 5 ml di mTeSR1 mezzo contenente 10 mM Y-27632 seguito da centrifugazione a 200 xg a temperatura ambiente per 5 min(Nota, non utilizzare eccessive forze di centrifugazione che causano danni alle cellule). </li><li> Aggiungere 5 ml di mTeSR1 mezzo al pellet, risospendere il pellet come singole cellule, e filtro dissociato cellule attraverso un colino cella 40 pm (per eliminare eventuali aggregati di cellule residue). </li><li> Seed 1,3-2 x 10 6 hPSCs in un pozzetto (1,4-2,1 x 10 5 cellule / cm 2) di un 6-pozzetti rivestiti con 2,5% hESC qualificato Matrigel, aggiungere mTeSR1 medio fino a 2,5 ml, e comprendono 10 micron Y-27632 nel mezzo (per facilitare il 24 hr placcatura singola cellula iniziale). In alternativa, utilizzare le seguenti piccole molecole di sostituire 10 micron Y-27632 per migliorare la placcatura unicellulare: 1 micron Y-39983 (ROCK I inibitore), 1 mM phenylbenzodioxane (inibitore ROCK II), 1 micron thiazovivin (un inibitore ROCK romanzo) , e inibitore JAK 2 mM 1. </li><li> Sostituire il mezzo con drug-free medio mTeSR1 il giorno successivo (entro 24 ore), dissociare le cellule da uno in piùbene, e contare il numero di cellule per determinare singola cellula efficienza placcatura (Nota: approssimativamente, dal 50 al 90% della singola cellula placcatura di efficienza che può essere raggiunto in questa fase). </li><li> Permettono alle cellule di crescere come una cellula formata singolo monostrato per alcuni giorni con 3 ml di mTeSR1 medie. Cambiare medio giornaliero. </li><li> Determinare empiricamente la pianificazione per passaging adattato NCM seconda della densità cellulare. Passage quando la crescita delle cellule raggiunge confluenza al giorno 3 o 4 ° giorno, o al punto di tempo 4 ore dopo la scomparsa dei confini cellula-cellula. Dissociare le cellule in 1 ml di Accutase, utilizzare un 1 a 3 rapporto di splittaggio (cioè, 1 confluenti ben di cellule piastrate in 3 pozzetti della piastra da 6 pozzetti) per passaging cella, e stabilizzare adattato NCM da 5 passaggi. </li><li> Il congelamento delle cellule <ol><li> Utilizzare un pozzetto confluenti (~ 5-6 x10 6 cellule) per una fiala congelata: dissociare celle da una confluenti bene con 1 ml di Accutase per 10 min. </li><li> Diluire wesima 5 ml di mTeSR1 medie e centrifugare le cellule come descritto sopra. </li><li> Risospendere il pellet in 500 ml di mTeSR1 medio e poi aggiungere lentamente 500 ml di 2X HPSC medio di congelamento (contenenti 20 micron Y-27632) in un flaconcino di crioconservazione. In alternativa, risospendere le cellule in 1X HPSC medio congelamento contenenti inibitore JAK 2 mM 1 o 1X CryoStor CS10 medio in presenza di 10 pM o Y-27632 o 2 inibitore JAK 1 mM. </li><li> Mettere fiale congelate in un cryocontainer ghiaccio freddo (bottom-riempita con isopranol) e trasferire il cryocontainer ad un -80 ° C freezer immediatamente. </li><li> Trasferire le cellule dal -80 ° C congelatore ad un serbatoio di azoto liquido (il giorno successivo) per la crioconservazione a lungo termine. </li></ol></li><li> Scongelamento delle cellule <ol><li> Utilizzare un flaconcino crioconservazione per la placcatura 1 pozzetto di una piastra da 6 pozzetti. </li><li> Pre-riscaldare medio mTeSR1 in un bagno d&#39;acqua a 37 ° C per 10 min, cellule disgelo nella stessa bagnomaria per 2 min,goccia a goccia aggiungere celle a 5 ml di pre-riscaldato medio mTeSR1 contenenti 10 mM Y-27632, e centrifuga come sopra descritto. </li><li> Risospendere delicatamente i pellet cellulari con 2 ml di mTeSR1 mezzo contenente 10 mM Y-27632 e lentamente trasferire cellule ad una ben pre-rivestito con Matrigel (Nota: evitare di produrre bolle d&#39;aria). </li><li> Cambia medio giornaliero e si aspettano una crescita HPSC raggiungere confluenza al giorno 3 o 4. </li></ol></li></ol> 4 Protocollo 3:. Convertire HPSC colonie su Matrigel per NCM Cultura <ol><li> Rimuovere un piatto Matrigel rivestite da frigorifero, posizionare la piastra nella cappa coltura tissutale da 10 a 30 minuti, rimuovere il terreno da ciascun pozzetto della piastra, e aggiungere 2 ml di pre-riscaldato medio mTeSR1 a ciascun pozzetto. </li><li> Trasferimento triturato ciuffi HPSC dalla cultura alimentatore (Passo 2.10 del protocollo di base) alla piastra sopra Matrigel. Aggiungi mTeSR1 medio fino a 2,5 ml di volume finale in ogni pozzetto. </li><li> Cambiare mezzo al giorno per 3 a 4 giornis fino colonie raggiungono l&#39;80-90% di confluenza. </li><li> Per passaging cella: sciacquare le colonie con D-PBS due volte, trattare le cellule con 1 ml di 2 mg / ml dispasi a 37 ° C per 15 a 20 minuti, e sedimenti e passaggio cellule come grumi. </li><li> Per la cultura NCM: ripetere i passaggi 3,1-3,9 descritto nel protocollo 2. </li></ol> 5 Protocollo 4:. NCM Cultura della hPSCs su LN-521 <ol><li> Scongelare il ricombinante soluzione LN-521 a 4 ° C prima del giorno di utilizzo e rendere la soluzione di rivestimento laminina (LCS) diluendo la laminina scongelato con 1X D-PBS (contenente Ca 2 + / Mg 2 +) ad una concentrazione finale di 10 ug / ml. </li><li> Aggiungere 1 ml di LCS da un pozzetto in 6 pozzetti (nota: evitare a secco durante il processo di rivestimento). </li><li> Sigillare la piastra ricoperta con Parafilm per evitare l&#39;evaporazione e conservare il piatto in frigorifero (4 ° C) O / N (nota: utilizzare la piastra entro 1 settimana). </li><li> Rimuovere delicatamente le LCS con una pipetta Pasteur, senza peruching la superficie rivestita e aggiungere 2 ml di mTeSR1 mezzo ad un pozzetto. </li><li> Riscaldare tutte le soluzioni di coltura (inclusi D-PBS) in un bagno d&#39;acqua a 37 ° C per 25 min. </li><li> Convertire colonie HPSC a NCM <ol><li> Dissociare aggregati cellulari alle celle singole con Accutase come descritto nel protocollo 2. </li><li> Seme circa 1,3-2 x 10 6 hPSCs in uno LN-521 rivestita di (1,4-2,1 x 10 5 cellule / cm 2) senza la presenza di inibitori ROCK. </li><li> Cambiare mezzo al giorno per 3-4 giorni. </li><li> Dissociare le cellule in 1 ml di Accutase al giorno 3 o 4 per il prossimo passaging cellulare utilizzando un 1 a 3 rapporto di splittaggio. </li></ol></li></ol> . 6 Protocollo 5: NCM cultura per il plasmide DNA Transfezione <ol><li> Adattare le colonie HPSC alla cultura NCM come descritto nei protocolli 2-4. </li><li> Piatto dissociata hPSCs a 12-pozzetti con una densità cellulare di 7,5 x 10 5 cellule / pozzetto in mTeSR1 medio in presenza di 10 mM Y-27632. </li><li> Sostituire mTeSR1 media con drug-free mTeSR1 medio tra 4-8 ore dopo la placcatura le cellule. </li><li> Per ogni trasfezione, diluire 2,5 mg di plasmidi di espressione (ad esempio, pmaxGFP) e 5 ml di Lipofectamine 2000 in provette Eppendorf separati in 125 ml ciascuna di Opti-MEM Ridotto Serum Medium. </li><li> Dopo 5 min, miscelare i reagenti diluito e incubare per 20 min a temperatura ambiente (per formare complessi trasfezione). </li><li> Aggiungere 250 microlitri complessi trasfezione per ciascun pozzetto contenente hPSCs in 1 ml mTeSR1 medio e incubare le cellule a 37 ° C in un incubatore CO 2 per 24 ore. </li><li> Esaminare l&#39;efficienza di trasfezione sotto un microscopio a fluorescenza e fotografia casualmente per il calcolo della effettiva efficienza di trasfezione. </li></ol> . 7 Protocollo 6: NCM cultura per trasfezione di microRNA <ol><li> Dissociare hPSCs semi-confluenti al giorno 2 in condizioni NCM con l&#39;aggiunta di 1 ml di Accutase per pozzetto in 6<html> -Pozzetti. </li><li> Aggiungere 10 ml di mTeSR1 mezzo per diluire le reazioni enzimatiche e centrifugare a 200 xg per 5 min. </li><li> Risospendere il pellet cellulare con mTeSR1 medio, seminare le cellule ad una densità di 7,5 x 10 5 cellule per pozzetto in una piastra 12 pozzetti in mTeSR1 terreno contenente 10 mM Y-27632, e lasciare che le cellule attaccano per 4 ore. </li><li> Sostituire il mezzo con Y27632-libera mTeSR1 medie. </li><li> Utilizzare disponibili in commercio microRNA (ad esempio, un non-targeting miRIDIAN miRNA Transfezione controllo etichettato con Dy547). Titolare concentrazioni che vanno 0-160 nM utilizzando i reagenti forniti e seguire le istruzioni del produttore. </li><li> Ottimizzare l&#39;efficienza di trasfezione per ciascuna concentrazione in linee HPSC dal imaging di fluorescenza Dy547 delle cellule vive sotto un microscopio a 24 ore dopo la trasfezione. </li><li> Calcolare le efficienze di trasfezione in base alla percentuale di cellule positive Dy547 su tutte le cellule impressi. </li></ol>. tle &quot;&gt; 8 Protocollo 7: NCM cultura per la trasduzione di vettori lentivirali <ol><li> Ripetere i passaggi 7,1-7,4 del protocollo 6. </li><li> Calcolare la quantità di particelle virali da utilizzare per la trasduzione: Utilizzare la seguente formula: TU = (MOI x CN) / VT, mentre TU indica il numero totale di unità di trasformazione, MOI è uguale alla molteplicità desiderato di infezione nel pozzo (MOI o TU / cell), CN specifica il numero di celle in ogni bene, e VT indica archivi titoli virali (TU / ml). Ad esempio, dato che i titoli azionari virali per SMART-shRNA vettore è uguale a 1 x 10 9 TU / ml (unità di trasformazione per ml), 7,5 x 10 5 cellule per pozzetto al momento della trasduzione e un MOI previsto di 20: utilizzare 15 microlitri delle particelle virali di archivi per ogni bene (nota: questa condizione è raccomandato per l&#39;induzione lentivirali transitorio). </li><li> Pre-riscaldare 300 ml di mTeSR1 medio con 10 mg / ml di polibrene per 30 min. </li><li> Aggiungere 15 ml di archivi particelle virali inil mezzo mTeSR1 pre-riscaldato contenente polibrene e mescolare delicatamente la soluzione. </li><li> Sostituire il mezzo di coltura cellulare con 300 ml di mezzo preriscaldata contenenti particelle virali. </li><li> Dopo 4 ore di incubazione, esaminare turboGFP fluorescenza (un creatore di espressione shRNA) per determinare l&#39;efficienza di trasduzione. </li><li> Aggiungere 300 ml di ulteriore mezzo mTeSR1 alla trasduzione bene quando le cellule iniziano a esprimere turboGFP. </li><li> Dopo 12-16 ore di incubazione, riesaminare l&#39;espressione turbo-GFP. </li><li> Effettuare esperimenti di follow-up desiderati con queste cellule trasdotte entro 72 ore. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenicity+as+a+clinical+hurdle+for+pluripotent+stem+cell+therapies.">Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies.</a> Nat Med. 19, 998-1004 (2013).</li><li>Domogatskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin-511+but+not+-332,+-111,+or+-411+enables+mouse+embryonic+stem+cell+self-renewal+in+vitro.">Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro.</a> Stem Cells. 26, 2800-2809 (2008).</li><li>Domogatskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+laminins.">Functional diversity of laminins.</a> Annu Rev Cell Dev Biol. 28, 523-553 (2012).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nat Biotechnol. 28, 611-615 (2010).</li><li>Liew, C. 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The authors declare that they have no competing financial interests.

Ci sono due modi principali per hPSCs coltura in vitro: coltura convenzionale colonia-tipo (di celle alimentatori o matrici extracellulari) e cultura sospensione delle hPSCs gli aggregati senza alimentatori 6. Le limitazioni di entrambi colonia-tipo e sospensione metodi di coltura comprendono eterogeneità accumulati e cambiamenti epigenetici ereditabili. Cultura NCM, sulla base sia passaging singola cellula e cellula placcatura ad alta densità, rappresenta un nuovo metodo di coltura per la crescita...

La capacità di hPSCs di differenziare verso tessuti adulti multilineage ha aperto nuove strade alla cura dei pazienti che soffrono di malattie gravi che coinvolgono cardiovascolare, epatico, pancreatico e sistemi neurologici 1-4. Vari tipi di cellule derivate da hPSCs sarebbe anche fornire piattaforme cellulari robuste per la modellazione della malattia, l&#39;ingegneria genetica, screening di farmaci e test tossicologici 1,4. La questione chiave che assicura loro applicazioni cliniche e farmacologiche futuri è la generazione di un gran numero di hPSCs clinico-grade in coltura cellulare in vitro. Tuttavia, i sistemi di coltura attuali sono insufficienti o intrinsecamente variabile, che coinvolge vari alimentazione e senza alimentatore culture del hPSCs come colonie 5,6. Colony-tipo crescita delle quote hPSCs molte caratteristiche strutturali della massa cellulare interna (ICM) di embrioni di mammiferi. L&#39;ICM è incline a differenziarsi in tre strati germinaliin un ambiente multicellulare causa dell&#39;esistenza di gradienti di segnalazione eterogenei. Pertanto, l&#39;acquisizione di eterogeneità in sviluppo embrionale precoce è considerata come un processo necessario per la differenziazione, ma una caratteristica indesiderata della cultura HPSC. L&#39;eterogeneità nella cultura HPSC è spesso indotto da eccessivi segnali apoptotici e differenziazione spontanea a causa di condizioni di crescita ottimali. Così, in colonia-tipo coltura, le cellule eterogenee si osservano spesso nella periferia delle colonie 7,8. È stato anche dimostrato che le cellule in cellule staminali embrionali umane (hESC) Colonie risposte differenziali Esporre a molecole di segnalazione come BMP-4 9. Inoltre, metodi di coltura colonia producono produzioni di cellule basse così come i tassi di recupero molto bassi cellulare da crioconservazione a causa di tassi di crescita incontrollabili e segnale apoptotico Percorsi di 6,9. Negli ultimi anni, diverse culture sospensioni sono state sviluppate per hPSCs coltura, soprattarly per l&#39;espansione di grandi quantità di hPSCs in-e alimentatore condizioni prive di matrice 6,10-13. Ovviamente, diversi sistemi di coltura hanno i loro vantaggi e svantaggi. In generale, l&#39;eterogeneità hPSCs rappresenta uno dei principali inconvenienti di tipo colonia e cultura aggregato metodi, che sono ottimali per la consegna di DNA e RNA materiali in hPSCs per l&#39;ingegneria genetica 6. Chiaramente, vi è la necessità imperativa di sviluppare nuovi sistemi che aggirano alcune carenze delle attuali metodi di coltura. Le scoperte di piccoli inibitori della molecola (come l&#39;inibitore ROCK Y-27632 e JAK inibitore 1) che migliorano la sopravvivenza a cella singola spianare la strada per dissociato-HPSC cultura 14,15. Con l&#39;uso di queste piccole molecole, abbiamo recentemente sviluppato un metodo di coltura in base al tipo non-colonia (NCM) crescita di dissociate-hPSCs 9. Questo metodo di coltura romanzo combina passaging cella singola e ad alta densitàplaccatura metodi, che ci permette di produrre grandi quantità di hPSCs omogenee sotto cicli di crescita costante senza grandi anomalie cromosomiche 9. In alternativa, cultura NCM potrebbe essere implementato con diverse piccole molecole e matrici definite (quale laminine) al fine di ottimizzare il metodo di coltura per le applicazioni di larghezza. Qui vi presentiamo diversi protocolli dettagliati basato sulla cultura NCM e delineare le procedure dettagliate per l&#39;ingegneria genetica. Per dimostrare la versatilità dei protocolli di NCM, abbiamo provato anche la cultura NCM con inibitori ROCK diversi e con il singolo laminina isoforma 521 (vale a dire, LN-521).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Countess automated cell counter&#160;&#160;</td>
			<td style="width:220px;">Invitrogen Inc.&#160;</td>
			<td style="width:187px;">C10227</td>
			<td style="width:187px;">Automatic cell counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Faxitron Cabinet X-ray System</td>
			<td style="width:220px;">Faxitron X-ray Corporation, Wheeling, IL&#160;</td>
			<td style="width:187px;">Model RX-650</td>
			<td style="width:187px;">X-ray irradiation of MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">MULTIWELL six-well plates&#160;</td>
			<td style="width:220px;">Becton Dickinson Labware</td>
			<td style="width:187px;">353046</td>
			<td style="width:187px;">Polystyrene plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11965&#8211;092</td>
			<td style="width:187px;">For MEF medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">mitomycin C</td>
			<td style="width:220px;">Roche&#160;</td>
			<td style="width:187px;">107 409</td>
			<td style="width:187px;">Mitotic inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Trypsin</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">25300-054</td>
			<td style="width:187px;">For MEF dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM/F12&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11330&#8211;032</td>
			<td style="width:187px;">For hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Opti-MEM I Reduced Serum Medium&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">31985-062</td>
			<td style="width:187px;">For hPSC transfection</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Heat-inactivated FBS</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">16000&#8211;044</td>
			<td style="width:187px;">Component of MEF medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Knockout Serum Replacer&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">10828&#8211;028</td>
			<td style="width:187px;">KSR, Component of hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">14190-144</td>
			<td style="width:187px;">D-PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Non-essential amino acids&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">11140&#8211;050</td>
			<td style="width:187px;">NEAA, component of hPSC medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">L-Glutamine&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">25030&#8211;081</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:203px;">mTeSR1 &#38; Supplements</td>
			<td rowspan="2" style="width:220px;">StemCell Technologies</td>
			<td rowspan="2" style="width:187px;">5850</td>
			<td style="width:187px;">Animal protein-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TeSR2 &#38; Supplements</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">5860</td>
			<td style="width:187px;">Xeno-free medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:220px;">Sigma&#160;</td>
			<td style="width:187px;">7522</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;"> 
			MEF (CF-1) ATCC</td>
			<td style="width:220px;">American Type Culture Collection (ATCC)&#160;</td>
			<td style="width:187px;">SCRC-1040</td>
			<td style="width:187px;">For feeder culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">hESC-qualified Matrigel</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">354277</td>
			<td style="width:187px;">For feeder-free culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Laminin-521</td>
			<td style="width:220px;">BioLamina</td>
			<td style="width:187px;">LN521-02</td>
			<td style="width:187px;">Human recombinant protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">FGF-2 (recombinant FGF, basic)</td>
			<td style="width:220px;">R&#38;D Systems, MN</td>
			<td style="width:187px;">223-FB</td>
			<td style="width:187px;">Growth factor in hPSC medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CryoStor CA10&#160;</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">7930</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Accutase</td>
			<td style="width:220px;">Innovative Cell Technologies</td>
			<td style="width:187px;">AT-104</td>
			<td style="width:187px;">1X mixed enzymatic solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">JAK inhibitor I</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">420099</td>
			<td style="width:187px;">An inhibitor of Janus kinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">688000</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0012</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-39983</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0029</td>
			<td style="width:187px;">ROCK I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Phenylbenzodioxane&#160;</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0030</td>
			<td style="width:187px;">ROCK II inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Thiazovivin</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0017</td>
			<td style="width:187px;">A novel ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">BD Falcon Cell Strainer&#160;</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">352340</td>
			<td style="width:187px;">40-&#181;m cell strainer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nalgene 5100-0001 Cryo 1&#176;C</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">C6516F-1</td>
			<td style="width:187px;">&#8220;Mr. Frosty&#8221; Freezing Container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Lipofectamine 2000&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11668-027</td>
			<td style="width:187px;">Transfection reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DharmaFECT Duo&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">T-2010-02</td>
			<td style="width:187px;">Transfection reagent</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Non-targeting miRIDIAN miRNA Transfection Control</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">IP-004500-01-05</td>
			<td style="width:187px;">Labeled with Dy547, to monitor the delivery of microRNAs&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">SMART-shRNA</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">To be determined</td>
			<td style="width:187px;">Lentiviral vector</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">pmaxGFP</td>
			<td>amaxa Inc (Lonza)</td>
			<td>Included in every transfection kit</td>
			<td style="width:187px;">Expression plasmid for transfection control</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">4-Oct</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-5279</td>
			<td style="width:187px;">Mouse IgG2b, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-1</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21702</td>
			<td style="width:187px;">Mouse IgM, differentiation marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-4</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21704</td>
			<td style="width:187px;">Mouse IgG3, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-60</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21705&#160;</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-81</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21706</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">CK8 (C51)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8020</td>
			<td style="width:187px;">Mouse IgG1, against cytokeratin 8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">&#945;-fetoprotein</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8399</td>
			<td style="width:187px;">AFP, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF-3&#946; (P-19)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-9187</td>
			<td style="width:187px;">FOXA2, goat polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Troponin T (Av-1)</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">MS-295-P0</td>
			<td style="width:187px;">Mouse IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Desmin&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">RB-9014-P1</td>
			<td style="width:187px;">Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Anti-NANOG</td>
			<td style="width:220px;">ReproCELL Inc, Japan</td>
			<td style="width:187px;">RCAB0004P-F</td>
			<td style="width:187px;">Polyclonal antibody&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Rat anti-GFAP</td>
			<td style="width:220px;">Zymed</td>
			<td style="width:187px;">13-0300</td>
			<td style="width:187px;">Glial fibrillary acidic protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Albumin (clone HSA1/25.1.3)</td>
			<td style="width:220px;">Cedarlane Laboratories Ltd. (</td>
			<td style="width:187px;">CL2513A</td>
			<td style="width:187px;">Mouse IgG1,</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Smooth muscle actin (clone 1A4)</td>
			<td style="width:220px;">DakoCytomation Inc</td>
			<td style="width:187px;">IR611/IS611</td>
			<td style="width:187px;">Mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nestin</td>
			<td style="width:220px;">Chemicon International</td>
			<td style="width:187px;">MAB5326</td>
			<td style="width:187px;">Rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TUBB3</td>
			<td style="width:220px;">Convance Inc</td>
			<td style="width:187px;">MMS-435P</td>
			<td style="width:187px;">Tuj1, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF4&#945; (C11F12)</td>
			<td style="width:220px;">Cell Signaling Technologies</td>
			<td style="width:187px;">3113</td>
			<td style="width:187px;">Rabbit monoclonal antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Paraformaldehyde (solution)</td>
			<td style="width:220px;">Electron Microscopy Sciences</td>
			<td style="width:187px;">15710</td>
			<td style="width:187px;">PFA, fixative, diluted in D-PBS</td>
		</tr>
	</tbody>
</table>

alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Uno schema generale della cultura NCM Figura 1 rappresenta un tipico schema cultura NCM indica le variazioni dinamiche di hPSCs dopo alta densità di placcatura unicellulare in presenza dell&#39;inibitore ROCK Y-27632. Questi cambiamenti morfologici includono connessioni intercellulari dopo la placcatura, la formazione di cluster cellulari, e la crescita cellulare esponenziale seguita da condensazione cella (Figura 1A). Un esperimento rappre...

Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis | Biology | Video

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Le colture per pluripotenti umane alternative staminali produzione di celle, manutenzione e analisi genetica

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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12.2K Views.  National Institutes of Health. Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Video: Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Topo Epidermal cresta neurale delle cellule staminali (EPI-NCSC) Culture

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e manutenzione di cellule staminali embrionali umane

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparazione del pool lisato piastrinico umani (pHPL) come supplemento efficiente per animali siero-Free Culture sulle cellule staminali umane

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

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 ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Produzione automatizzata di neuroni corticali e dopaminergici pluripotenti indotti dall'uomo con monitoraggio integrato delle cellule vive

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Una strategia in due fasi che combina modificazione epigenetica e segnali biomeccanici per generare cellule pluripotenti di mammifero

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

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.