Vorremmo ringraziare i membri del laboratorio Lee per le discussioni importanti sul manoscritto. Il lavoro in laboratorio Lee è stato sostenuto da sovvenzioni dal Robertson Investigator Award of Foundation di New York Stem Cell e dal Maryland Stem Cell Research Fund (TEDCO).

1. Preparazione di cellule e dei media (1 ° giorno) <ol><li> Cultura ed espandere fibroblasti umani con i media DMEM contenente 10% FBS. </li><li> Piastre fibroblasti umani (Figura 1) su una piastra da 24 pozzetti alla densità appropriata per pozzetto il giorno prima trasduzione. NOTA: Le seguenti diluizioni seriali sono raccomandati (200K, 100K, 50K, 25K, 12.5K e 6.25K), perché diversi tipi di cellule hanno diverse capacità di attacco. </li><li> Incubare le cellule per un giorno a 37 ° C, 5% CO 2 incubatore, assicurando le cellule hanno pienamente aderito ed esteso. </li></ol> 2. Eseguire trasduzione (Day 2) <ol><li> Il giorno di trasduzione, controllare la densità cellulare e scelto pozzetti densità più efficienti (Figura 2). E &#39;meglio scegliere tre diverse densità: alta (80 ~ 90%), medio (50 ~ 70%) e bassa (20 ~ 40%). </li><li> Almeno 1 ora prima di trasduzione, aspirare il fibroblsupporti AST dalle cellule e modificare nuovi 300 ml di mezzo fibroblasti. </li><li> Rimuovere un set di 4 diversi tubi di virus Sendai dal -80 ° C di stoccaggio. Scongelare ogni provetta allo stesso tempo in un bagno d&#39;acqua a 37 ° per alcuni secondi, e poi le valvole fuori dal bagno di acqua. Dopo di che, scongelare a temperatura ambiente; provette da centrifuga a 6000 xg per 10 sec e disporli sul ghiaccio fino all&#39;uso. Non ricongelare e scongelare il virus dal momento che i titoli non manterrà. </li><li> Aggiungere i volumi di ciascuna delle quattro tubi virus Sendai (OCT-4, Klf-4, c-Myc e Sox-2) indicati al tubo micro-centrifuga. Assicurarsi che la soluzione viene mescolata ben pipettando delicatamente. Ad esempio, se 50K cellule / pozzetto di una 24-pozzetti lookwell per trasduzione: (Titer sulla base del certificato di analisi di Life Technologies, può essere diverso da lotto a lotto) Tubo hOct-4 = 6,0 x 10 7 CIU, 3 MOI = 5 ml Tubo hSox-2 = 6,5 x 10 7 CIU, 3 MOI = 4.6 μ; L Tubo hKlf-4 = 6,3 x 10 7 CIU, 3 MOI = 4.8 ml Tubo hc-Myc = 7.8 x 10 7 CIU, 3 MOI = 3,8 microlitri Totale = 18.2 ml di quattro fattori virali miscela / uno così (50K celle) della piastra da 24 pozzetti </li><li> A seconda densità cellulari; aggiungere 2X, 1X, 0.5X e volume della miscela virus nel pozzo. Agitare delicatamente la piastra anteriore alla parte posteriore, a sinistra ea destra (2X: 36.4 ml di miscela di virus, 1X: 18,2 microlitri e 0.5X: 9.1 ml). </li><li> Mettere le cellule a 37 ° C, 5% CO 2 incubatore e incubare una notte. </li></ol> 3. Sostituzione della Cultura Media (Day 3 &amp; Day 5) <ol><li> 24 ore dopo la trasduzione, sostituire il mezzo con 500 microlitri media fibroblasti fresco. </li><li> Il giorno 5, cambiare il Piano con i media fibroblasti fresca. </li></ol> 4. Preparare piatti MEF per il Co-cultura (Giorno 8) <ol><li> Preparare cellule MEF in 60 millimetri capsule di Petri con il 10% FBS conteneva DMEM uno day prima passaging fibroblasti trasdotte sul feeder cellule MEF. Nota: Stiamo usando 5 x 10 5 del MEF in ogni piatto 60 millimetri. </li></ol> 5. Avvio co-coltura con cellule trasdotte Feeder MEF (Giorno 9) <ol><li> Prima di placcatura cellule trasdotte, aspirare il 10% FBS conteneva DMEM supporto dai piatti alimentatore MEF e aggiungere fresco del 10% FBS conteneva DMEM. </li><li> Rimuovere il terreno dai fibroblasti trasdotti che sono 24 pozzetti e lavare le cellule una volta con D-PBS. Aggiungere 200 ml di 0,25% tripsina / EDTA nelle cellule di fibroblasti inthe 24-pozzetti. Dopo meno di 5 minuti di incubazione con tripsina / EDTA, quando le cellule iniziano a staccarsi dalla piastra, raccogliere le cellule con mezzo di coltura in tubo da 15 ml. Poi si centrifuga a 6000 g per 4 min. NOTA: Pooling 2X, 1X, e il virus 0.5X è raccomandato fibroblasti trasdotte. </li><li> Rimuovere il mezzo surnatante e lavare per risospendereil pellet cellulare con 4-5 ml di terreno fresco fibroblasti di neutralizzazione da tripsina. Poi centrifugare a 135 g per 4 min. </li><li> Cellule Piastra come densità diluizione seriale e iniziare a co-coltura di fibroblasti con i media sul MEF: come 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 e 1/128 a 60 mm piatto. A seconda del tasso di morte delle cellule durante la prima settimana di trasduzione, non si può avere bisogno di diluire a 1/2 o 1/128 densità. Mettere da parte cellule rimanenti per estrarre l&#39;RNA totale come controllo positivo per Transgene RT-PCR. </li><li> Porre 60 millimetri piatti 37 ° C, 5% CO 2 incubatore durante la notte. </li></ol> 6. Feed Cellule Staminali embrionali umane a medio e Controllo delle Cellule (Giornata 10) <ol><li> Il giorno dopo, cambiare i media fibroblasti ai media ES umana con 10 pM Y-27632. Dopo di che, cambiare medio giornaliero con terreno ES umana: 800 ml DMEM/F12 + 200 ml Knockout siero sostituzione + 10 ml di L-Glutammina + 10 ml di 10 mM MEM aminoacidi non essenziali minimi+ 1 ml di 2-mercaptoetanolo + 10 ng / ml di fattore base di crescita dei fibroblasti. </li><li> Fare un cambiamento dei media ogni giorno con mezzi freschi umane ES. Dopo una settimana, controllare i piatti una volta ogni 2 ~ 3 giorni sotto un microscopio per la formazione di grumi di cellule ES colonia-like. A seconda del tipo di cellula, la cultura vorrà più o meno tempo prima colonie si trovano. </li></ol> 7. Picking staminali pluripotenti indotte colonie di cellule ed espandere la Cells (giorno 20 ~) <ol><li> Tre settimane dopo la trasduzione, le colonie dovrebbero apparire cresciuto correttamente per la raccolta. </li><li> Il giorno prima di prendere le colonie, preparare alimentatore MEF a 24 pozzetti. NOTA: Stiamo usando 12,5 x 10 5 del MEF in una piastra da 24 pozzetti. </li><li> Il giorno dopo, cambiare mezzo di piatti MEF dai media fibroblasti a supporto ES umana con 10 pM Y-27632. Scegliere manualmente una colonia in ogni tempo sotto il microscopio alla raccolta-cappuccio e macchie più piccole pipettando e trasferirlisulla nuova preparati MEF dishes.Try di scegliere diversi cloni. </li><li> Passaggio ed espandere l&#39;ciascun pozzetto di una piastra da 24 pozzetti di una piastra inizialmente 6 bene. Poi da un 6-pozzetti a 60 mm piatti prima di ulteriore moltiplicazione. </li></ol> 8. Caratterizzazione di iPSCs umano (dopo 10 passaggi) <ol><li> Immunofluorescenza <ol><li> Piatto iPSCs umani in lamiera e della cultura 24 pozzetti durante 4-5 giorni con cambio giornaliero dei media. </li><li> Per avviare test immunofluorescenza, lavare la piastra con D-PBS volta. Poi fissare le cellule immediatamente nello 0,4% paraformaldeide per 30 min. Dopo che lavare tre volte in D-PBS per 5 min. </li><li> Trattare cellule fissate con 0.1% Triton X-100 in PBS soluzione per 15 minuti a temperatura ambiente. </li><li> Aspirare la soluzione permeablization (0,1% triton X-100 soluzione), poi aggiungere BSA soluzione bloccante 0,5% per 1 ora a temperatura ambiente. </li><li> Eseguire una rilevazione di Nanog, Oct-4, SSE (antig embrionale stadio-specificait) e TRA-1-81 (tumore rifiuto antigene) utilizzando un anticorpo anti-umano in soluzione BSA allo 0,5% (controllare la tabella materiale per tasso di diluizione di anticorpi). Incubare anticorpo primario per 2 ore a temperatura ambiente. Quindi lavare tre volte in D-PBS per 5 minuti ciascuno. </li><li> Verificare usando la microscopia a fluorescenza dopo 1 ora di incubazione con Alexa Fluor 488 di capra anti-topo IgG come 2 ° anticorpi, che vengono diluiti in soluzione 1:2.000 BSA 0,5% a temperatura ambiente. Colorare tutti i nuclei di ogni MEF cellule feeder e hiPSCs con DAPI. </li></ol></li><li> RT-PCR di Transgene Conferma <ol><li> Estrarre mRNA totale di pellet cella utilizzando reagenti di estrazione di RNA. </li><li> Fare una trascrizione inversa su aliquote (1 mg) di RNA totale e il cDNA risultante per l&#39;amplificazione PCR da trascrittasi inversa. </li><li> Preparare le miscele PCR per contenere 2 ml di cDNA, 10 ml di 2X PCR master mix, 2 ml di 2 pmol di primer(Avanti: 1 ml e reverse: 1 ml) e 6 ml di DW. Per rilevare l&#39;espressione genica, apportare la RT-PCR con i primer elencati nella Tabella 1. </li><li> Eseguire la RT-PCR con gene GAPDH come controllo per l&#39;efficienza delle reazioni di amplificazione. Visualizzare e analizzare prodotto di PCR dell&#39;1% elettroforesi su agarosio-gel. </li></ol></li></ol>

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Riprogrammazione delle cellule somatiche umane per hiPSCs detiene promesse senza precedenti nella biologia di base, medicina personale, e il trapianto 12. In precedenza, le generazioni IPSC umane necessarie virus a DNA che ha il rischio di integrazione nel genoma dell&#39;ospite, che può creare mutazioni genetiche indesiderabili che limitano ulteriori applicazioni cliniche quali sviluppo di farmaci e terapie di trapianto 13. Con questa ragione, molti studi sono stati riportati per generare-vettore...

Le cellule umane staminali embrionali (hESC) hanno la capacità di auto-rinnovarsi in vitro e hanno pluripotenza, che potrebbe essere potenzialmente utile per la modellazione della malattia, per lo screening di stupefacenti, e di sviluppare le terapie a base di cellule per il trattamento di malattie e lesioni dei tessuti. Tuttavia, hESCs hanno una limitazione per la terapia di sostituzione cellulare a causa delle barriere immunologiche, oncologiche ed etiche, e di studiare i geni correlati alla malattia, hESCs malattie specifiche possono essere isolati attraverso pre-impianto diagnosi genetica preimpianto (PGD) si avvicina, ma è ancora tecnicamente impegnativo e le donazioni di embrioni sono piuttosto rari. Questi problemi sono legati al progresso della biologia delle cellule staminali, che ha portato allo sviluppo di cellule staminali pluripotenti indotte (hiPSCs). IPSCs umani sono geneticamente riprogrammate da cellule somatiche adulte umane e porto le caratteristiche di cellule simil-staminali pluripotenti simili a hESCs, che li rende una fonte utile per la medicina rigenerativa, come dscoperta tappeto, modelli di malattia e la terapia cellulare in paziente-specifici 1,2 maniera. Fino ad ora, ci sono diversi metodi per generare iPSCs umani, inclusi virus-mediata (retrovirus e adenovirus) 3, non virus mediata (sistema BAC e vettori di transfezione) 4 trasduzioni gene, e di sistema di consegna di proteine ​​5-7. Sebbene una consegna di geni del virus-mediata può garantire un certo livello di efficienza, vettori virali potevano lasciare impronta genetica, perché integrano nei cromosomi ospite per esprimere geni riprogrammazione in modo incontrollato. Anche quando integrazione virale di fattori di trascrizione può attivare o inattivare geni ospite 8, può causare un&#39;aberrazione genetica inattesa e il rischio di tumorigenesi 5,9. D&#39;altra parte, l&#39;introduzione diretta di proteine ​​o RNA in cellule somatiche sono stati segnalati, ma hanno alcuni svantaggi, come intensità di lavoro, trasf ripetutaezione, e basso livello di riprogrammazione 7,10. Anche episomale e non integranti adenovirus, virus adeno-associati, e vettori plasmidici sono ancora relativamente meno efficienti 11. Per queste ragioni, è plausibile scegliere non-integrazione metodi di riprogrammazione con elevata efficacia della generazione iPSC e un minor numero di anomalie genetiche. In questo studio, usiamo una riprogrammazione basata Sendai-virus. Questo metodo è noto per essere non integrata nel genoma dell&#39;ospite e produce costantemente iPSCs umani senza integrazioni transgene.

<table border="0" cellpadding="0" cellspacing="0" width="1109">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">CytoTune-iPS Reprogramming Kit</td>
			<td style="width:195px;">invitrogen</td>
			<td style="width:119px;">A1378002</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CF-6,MEFs, neomycin-resistant, mitomycin C treated&#160;</td>
			<td>Global stem</td>
			<td>GSC-6105M&#160;</td>
			<td>5X105/6cm or 12.5X105/24 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td>
			<td style="width:195px;">invitrogen</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Cell Culture Plate, flat-bottom with lid</td>
			<td style="width:195px;">BD</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Y-27632</td>
			<td style="width:195px;">TOCRIS</td>
			<td style="width:119px;">1254</td>
			<td>10 uM (Stock: 10 mM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">basic fibroblast growth factor</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>PHG0263</td>
			<td>10 ng (Stock : 100 ug)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Knock-out serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fetal bovine serum</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine-200 mM (100X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>25030-081</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>11140050</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>21985023</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Phase Contrast Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EmbryoMax 0.1% Gelatin Solution</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:119px;">ES-006-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
			<td style="width:195px;">DSHB</td>
			<td>MC-813-70</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Tra-1-81</td>
			<td style="width:195px;">cell signaling</td>
			<td>4745S</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal Oct4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nanog</td>
			<td>R&#38;D</td>
			<td>AF1997</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Alexa Flouor 488 goat anti-mouse</td>
			<td>invitrogen</td>
			<td>948492</td>
			<td>1/2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
			<td style="width:195px;">cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
			<td></td>
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		<tr height="23">
			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">K0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:195px;">invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

Alcuni anni fa, la creazione di cellule staminali umane pluripotenti indotte (iPSCs) ha inaugurato una nuova era nel campo della biomedicina. Possibili usi di iPSCs umani comprendono la modellazione patogenesi delle malattie genetiche umane, la terapia cellulare autologa dopo la correzione genica e screening di farmaci personalizzati, fornendo una fonte di cellule rilevanti e sintomi specifici del paziente. Tuttavia, ci sono diversi ostacoli da superare, come eliminare l&#39;espressione del transgene fattore riprogrammazione restante dopo la produzione iPSCs umani. Ancora più importante, l&#39;espressione del transgene residuo in iPSCs umane indifferenziate potrebbe ostacolare differenziazioni corretta e fuorviare l&#39;interpretazione della malattia in questione in fenotipi vitro. Con questo motivo, privo di integrazione e / o iPSCs umani privi di transgene sono state sviluppate utilizzando diversi metodi, quali adenovirus, il sistema piggyBac, minicircle vettore, vettori episomale, la consegna diretta delle proteine ​​e sintetizzato mRNA. Tuttavia, l&#39;efficienza di reprograllo utilizzando metodi libero-integrazione è piuttosto basso nella maggior parte dei casi. Qui, vi presentiamo un metodo per isolare iPSCs umani utilizzando Sendai-virus (virus RNA) sistema di riprogrammazione based. Questo metodo di riprogrammazione mostra i risultati coerenti e di alta efficienza in modo economicamente efficiente.

Fibroblasti solito infette non mostrano cambiamenti morfologici in diversi giorni dopo la trasduzione del virus Sendai, ma cinque giorni dopo, cominciano ad avere forme diverse (Figura 1). Come descritto in un pannello di destra della figura 1, le cellule non hanno tipica morfologia fibroblasto più. Hanno una forma rotonda e più grande nucleo di citoplasma. Anche quando trasduzione viene eseguita nel 80% di confluenza cellulare, sembra che siano meno confluenti nel pozzo dopo comincia...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

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

Qui vi presentiamo il nostro metodo stabilito per riprogrammare cellule somatiche umane in libera-transgene iPSCs umane con virus di Sendai, che mostra esito coerente e una maggiore efficienza.

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 ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Biology

22.2K Views.  Johns Hopkins University School of Medicine.  Qui vi presentiamo il nostro metodo stabilito per riprogrammare cellule somatiche umane in libera-transgene iPSCs umane con virus di Sendai, che mostra esito coerente e una maggiore efficienza. 

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

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

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

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

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

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

Ricerca

Biologia

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

Trasfettando e Nucleofecting umani cellule staminali pluripotenti indotte

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

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

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

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

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

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

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 Mice Derived from Induced Pluripotent Stem Cells

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

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

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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

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

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

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

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

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Generazione rapida e efficiente di neuroni da cellule staminali umane pluripotenti in un formato piatto Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.