Nous tenons à remercier les membres du laboratoire Lee pour des discussions utiles sur le manuscrit. Le travail dans le laboratoire Lee a été soutenue par des subventions de Robertson Investigator Award de New York Fondation des cellules souches et du Maryland souches Fonds de recherche sur les cellules (TEDCO).

1. Préparation de la cellule et des médias (Jour 1) <ol><li> Culture et élargir fibroblastes humains avec du milieu DMEM contenant 10% de FBS. </li><li> Plate fibroblastes humains (figure 1) sur une plaque de 24 puits à la densité appropriée pour bien le jour avant la transduction. REMARQUE: Les dilutions en série suivantes sont recommandées (200K, 100K, 50K, 25K, 12.5K et 6.25K) parce que différents types de cellules ont la capacité de fixation différents. </li><li> Incuber les cellules pour un jour de plus dans un 37 ° C, 5% de CO 2 incubateur, assurant que les cellules ont pleinement adhéré et étendu. </li></ol> 2. Effectuer transduction (Jour 2) <ol><li> Le jour de la transduction, vérifier la densité cellulaire et choisir les plus efficaces puits de la densité (figure 2). Il est préférable de choisir trois densités différentes: haute (80 ~ 90%), moyen (50 ~ 70%) et faible (20 ~ 40%). </li><li> Au moins 1 heure avant la transduction, aspirer le fibroblmédias ast des cellules et modifier nouveaux 300 pi de milieu de fibroblastes. </li><li> Supprimer un jeu de 4 différents tubes de virus Sendai de -80 ° C stockage. Décongeler chaque tube en même temps dans un bain-marie à 37 ° pendant quelques secondes, puis prendre les tubes à partir du bain d&#39;eau. Après cela, les décongeler à la température ambiante; tubes de centrifugation à 6000 g pendant 10 secondes et les placer sur la glace jusqu&#39;à utilisation. Ne pas réutiliser gel et de dégel du virus depuis les titres ne seront pas maintenir. </li><li> Ajouter les volumes de chacun des quatre tubes de virus Sendai (Oct-4, Klf-4, c-Myc et Sox-2) indiquées au tube de micro-centrifugeuse. Assurez-vous que la solution est bien mélangée par pipetage. Par exemple, si 50K cellules / un puits de 24 puits plaque lookwell pour la transduction: (Titre basé sur le certificat d&#39;analyse de Life Technologies, peut être différent d&#39;un lot à) Tube hOct-4 = 6,0 x 10 7 SDI, 3 MOI = 5 pi Tube hSox-2 = 6,5 x 10 7 SDI, 3 MOI = 4,6 μ; L Tube hKlf-4 = 6,3 x 10 7 SDI, 3 MOI = 4,8 pi Tube hc-Myc = 7,8 x 10 7 SDI, 3 MOI = 3,8 pi Total = 18.2 pi de quatre facteurs de virus mélange / un puits (50K cellules) de la plaque de 24 puits </li><li> Selon les densités cellulaires; ajouter 2X, 1X, 0,5 X et le volume du mélange de virus dans le puits. Secouer doucement le devant de la plaque à l&#39;arrière, à gauche et à droite (2X: 36,4 pi de mélange de virus, 1X: 18,2 pi et 0.5X: 9,1 pi). </li><li> Placer les cellules dans un 37 ° C, 5% CO 2 incubateur et incuber pendant une nuit. </li></ol> 3. Remplacement du milieu de culture (Jour 3 &amp; Jour 5) <ol><li> 24 heures après la transduction, remplacer le milieu avec 500 moyen de fibroblastes ul frais. </li><li> Le jour 5, changement de milieu avec les médias des fibroblastes frais. </li></ol> 4. Préparer des plats du MEF pour la co-culture (Jour 8) <ol><li> Préparer les cellules MEF dans 60 boîtes de culture mm avec 10% de FBS contenait un milieu DMEM day repiquer avant le fibroblaste transduit sur cellules nourricières du MEF. Remarque: Nous utilisons 5 x 10 5 MEF dans chaque boîte de 60 mm. </li></ol> 5. Démarrer co-culture de cellules transduites avec des cellules nourricières du MEF (Jour 9) <ol><li> Avant étalement des cellules transduites, aspirer 10% de FBS contenait milieu DMEM de plats d&#39;alimentation du MEF et ajouter frais 10% de FBS contenait milieu DMEM. </li><li> Supprimer le milieu à partir des fibroblastes qui sont transduites dans la plaque à 24 puits et laver les cellules une fois avec du D-PBS. Ajouter 200 ul de 0,25% de trypsine / EDTA dans les cellules de fibroblastes inThe plaque de 24 puits. Après moins de 5 minutes d&#39;incubation avec de la trypsine / EDTA, lorsque les cellules commencent à se détacher de la plaque, de recueillir les cellules avec un milieu de croissance dans 15 ml de tube conique. Puis centrifuger les dans 6000 g pendant 4 min. REMARQUE: La mise en commun 2X, 1X, 0.5X et virus fibroblastes transduction est recommandé. </li><li> Retirez le milieu surnageant et laver à remettre en suspensionle culot cellulaire avec 5.4 ml de milieu de fibroblastes frais pour la neutralisation de la trypsine. Puis centrifuger à 135 xg les pendant 4 min. </li><li> Cellules de la plaque que la densité de dilution en série et commencer à co-culture avec des milieux de fibroblastes sur MEF: telle que 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 et 1/128 par 60 plat mm. Selon le taux de mort cellulaire au cours de la première semaine de la transduction, on ne peut pas besoin de diluer à 1/2 ou 1/128 densité. Mettez de côté les cellules restantes pour extraire l&#39;ARN total comme un contrôle positif pour Transgene RT-PCR. </li><li> Placer des boîtes de 60 mm à 37 ° C, 5% CO 2 incubateur pendant une nuit. </li></ol> 6. Flux cellules souches embryonnaires humaines à moyen et surveiller les cellules (Jour 10) <ol><li> Le lendemain, changer de support fibroblastes aux médias ES humain avec 10 uM Y-27632. Après cela, changer moyen quotidien avec le milieu ES humain: 800 ml de DMEM/F12 + 200 ml Sérum de remplacement Knockout + 10 ml de L-Glutamine + 10 ml de 10 mM de MEM acides aminés non essentiels minimaux+ 1 ml de 2-mercaptoéthanol + 10 ng / ml de facteur de croissance basique des fibroblastes. </li><li> Faire un changement quotidien des médias avec les médias de nouvelles souches embryonnaires humaines. Après une semaine, vérifiez les plats une fois par 2 ~ 3 jours sous un microscope pour la formation d&#39;amas de cellules ES colonies comme. Selon le type de cellule, la culture aura plus ou moins de temps avant colonies se trouvent. </li></ol> 7. Cueillette de la souches pluripotentes induites colonies cellulaires et Développer les cellules (Jour 20 ~) <ol><li> Trois semaines après la transduction, les colonies doivent apparaître grandi bien pour la cueillette. </li><li> La veille de la cueillette des colonies, préparer MEF alimentation en plaque de 24 puits. NOTE: Nous utilisons 12,5 x 10 5 MEF dans une plaque de 24 puits. </li><li> Le lendemain, changement de milieu de plats MEF de médias de fibroblastes de médias ES humain avec 10 uM Y-27632. Choisir manuellement une colonie à chaque fois sous le microscope dans la cueillette de capot et faire des petits bouquets de pipetage et de les transférersur fraîchement préparée MEF dishes.Try de choisir plusieurs clones. </li><li> Passage et développer chaque puits d&#39;une plaque de 24 puits à une plaque à 6 puits initialement. Ensuite, à partir d&#39;une plaque à 6 puits à une boîtes de 60 mm avant la propagation ultérieure. </li></ol> 8. Caractérisation des iPSCs humain (après 10 passages) <ol><li> Immunofluorescence <ol><li> Plaque CSPi humaines dans la plaque et de la culture de 24 puits pendant 4-5 jours avec changement de support quotidienne. </li><li> Pour commencer immunofluorescence, laver la plaque avec le D-PBS fois. Puis fixer les cellules immédiatement dans 0,4% de paraformaldéhyde pendant 30 min. Après que les laver trois fois dans du D-PBS pendant 5 min. </li><li> Traiter les cellules fixées avec 0,1% de Triton X-100 solution dans du PBS pendant 15 min à température ambiante. </li><li> Aspirer la solution de permeablization (0,1% triton X-100 solution), puis ajouter une solution à 0,5% de blocage de BSA pendant 1 heure à température ambiante. </li><li> Effectuer une détection de Nanog, Oct-4, l&#39;AESS (Antig embryonnaire spécifique au stadefr) et TRA-1-81 (antigène de rejet tumoral) en utilisant un anticorps anti-humain dans une solution de BSA à 0,5% (consulter le tableau matière de taux de dilution d&#39;anticorps). Incuber l&#39;anticorps primaire pendant 2 heures à température ambiante. Puis laver trois fois dans D-PBS pendant 5 min chacun. </li><li> Vérifier en utilisant la microscopie de fluorescence, après 1 h d&#39;incubation avec Alexa Fluor 488 anticorps IgG de chèvre anti-souris comme 2 ème anticorps, qui sont dilué 1:2000 dans une solution de BSA à 0,5% à la température ambiante. Colorer tous les noyaux de chacune des cellules nourricières du MEF et hiPSCs avec DAPI. </li></ol></li><li> RT-PCR de Transgene Confirmation <ol><li> Extrait ARNm total de culot cellulaire en utilisant des réactifs d&#39;extraction d&#39;ARN. </li><li> Faire une transcription inverse sur aliquotes (1 pg) de l&#39;ARN total et l&#39;ADNc résultant pour l&#39;amplification PCR par transcriptase inverse. </li><li> Préparer les mélanges de PCR pour contenir 2 pi de matrice d&#39;ADNc, 10 pi de 2X PCR Master Mix, 2 pi de 2 pmol d&#39;amorces(Avant: 1 pl et inverser: 1 pi) et 6 pi de DW. Pour détecter l&#39;expression des gènes, faire la RT-PCR avec les amorces énumérées dans le tableau 1. </li><li> Effectuer la RT-PCR avec le gène de la GAPDH comme contrôle pour l&#39;efficacité des réactions d&#39;amplification. Visualiser et analyser le produit de PCR par électrophorèse sur agarose-gel 1%. </li></ol></li></ol>

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Les auteurs n&#39;ont rien à révéler.

Reprogrammation des cellules somatiques humaines à hiPSCs détient promesses sans précédent dans la biologie fondamentale, la médecine personnelle, et la transplantation 12. Auparavant, les générations iPSC humaines requises virus à ADN qui a un risque d&#39;intégration dans le génome de l&#39;hôte, ce qui peut créer des mutations génétiques indésirables qui limitent d&#39;autres applications cliniques telles que le développement de médicaments et de thérapies de transplantation 13.</sup...

Cellules souches embryonnaires humaines (CSEh) ont une capacité d&#39;auto-renouvellement in vitro et ont pluripotence, qui pourrait être potentiellement utile pour la modélisation de la maladie, pour le criblage de médicaments, et de développer des thérapies à base de cellules pour traiter les maladies et les blessures des tissus. Toutefois, les CSEh ont une limitation pour la thérapie de remplacement de la cellule en raison de barrières immunologiques, oncologiques et éthiques, et à étudier les gènes liés à la maladie, les CSEh spécifiques à la maladie ont pu être isolés par diagnostic pré-implantatoire génétique (DPI) des approches, mais il est encore techniquement difficile et les dons d&#39;embryons sont assez rares. Ces problèmes sont liés au progrès de la biologie des cellules souches, ce qui a conduit à la mise au point de cellules souches pluripotentes induites (hiPSCs). CSPi humaines sont génétiquement reprogrammées à partir de cellules somatiques humaines adultes et abritent souches caractéristiques de cellules pluripotentes comme similaires à CSEh, qui les rend une source utile pour la médecine régénérative, comme ddécouverte de tapis, la modélisation des maladies et de thérapie cellulaire chez un patient spécifique de manière 1,2. Jusqu&#39;à présent, il existe plusieurs méthodes pour générer des CSPi humaines, y compris le virus médiation (rétrovirus et adénovirus) 3, non-virus médiation (système de BAC et les vecteurs de transfection) 4 transductions de gènes et de protéines système de livraison 5-7. Même si une livraison de gènes du virus médiée peut assurer un certain niveau d&#39;efficacité, des vecteurs viraux peuvent laisser l&#39;empreinte génétique, car ils s&#39;intègrent dans les chromosomes de l&#39;hôte pour exprimer des gènes de reprogrammation d&#39;une manière incontrôlée. Même lorsque l&#39;intégration virale de facteurs de transcription peut activer ou inactiver les gènes de l&#39;hôte 8, il peut provoquer une aberration génétique inattendu et le risque de tumorigenèse 5,9. D&#39;autre part, l&#39;introduction directe de protéines ou d&#39;ARN dans les cellules somatiques ont été rapportés, mais ils ont quelques inconvénients tels que main-d&#39;œuvre, transf répétéeection, et le faible niveau de la reprogrammation de 7,10. Même adénovirus épisomique et sans intégration, virus adéno-associés, et les vecteurs plasmidiques sont encore relativement moins efficace 11. Pour ces raisons, il est plausible de choisir non-intégration des méthodes de reprogrammation avec une grande efficacité de la production iPSC et moins d&#39;anomalies génétiques. Dans cette étude, nous utilisons une reprogrammation base Sendai-virus. Ce procédé est connu pour être non-intégrés dans le génome de l&#39;hôte et produit constamment iPSCs humains sans intégration du transgène.

<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>
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		<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>
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			<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>
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		<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>
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</table>

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

Il ya quelques années, la mise en place de cellules souches pluripotentes humaines induites (CISP) a inauguré une nouvelle ère dans le domaine biomédical. Les usages potentiels des CSPi humaines comprennent la modélisation pathogenèse des maladies génétiques humaines, la thérapie cellulaire autologue après la correction de gène, et le dépistage des drogues personnalisé en fournissant une source de cellules pertinentes et des symptômes spécifiques du patient. Cependant, il existe de nombreux obstacles à surmonter, par exemple en éliminant le facteur de reprogrammation expression transgène qui reste après la production de iPSCs humains. Plus important encore, l&#39;expression du transgène résiduelle dans CSPi humaines indifférenciées pourrait entraver différenciations propres et égarer l&#39;interprétation de la maladie pertinents dans les phénotypes in vitro. Avec cette raison, sans l&#39;intégration et / ou CSPi humaines libre transgènes ont été développés en utilisant plusieurs méthodes, telles que l&#39;adénovirus, le système de piggyBac, vecteur minicercle, vecteurs épisomiques, la livraison directe des protéines et la synthèse de l&#39;ARNm. Cependant, l&#39;efficacité de reprogramming utilisant des méthodes sans rapprochement est très faible dans la plupart des cas. Ici, nous présentons une méthode pour isoler CSPi humaines en utilisant Sendai-virus (virus à ARN) du système de reprogrammation base. Cette méthode de reprogrammation montre des résultats cohérents et de haute efficacité de manière rentable.

Habituellement fibroblastes infectés ne présentent pas de changements morphologiques dans plusieurs jours après la transduction du virus Sendai, mais cinq jours plus tard, ils commencent à avoir des formes différentes (Figure 1). Comme décrit dans un panneau de droite de la figure 1, les cellules n&#39;ont pas la morphologie des fibroblastes typique plus. Ils ont une forme ronde et grand noyau de cytoplasme. Même lorsque la transduction est réalisée dans 80% de confluence cellu...

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

Ici, nous vous présentons notre méthode établie pour reprogrammer des cellules somatiques humaines dans CSPi humaines sans transgène avec le virus de Sendai, qui montre des résultats cohérents et une efficacité accrues.

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec 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 ...

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Biology

22.2K vues.  Johns Hopkins University School of Medicine.  Ici, nous vous présentons notre méthode établie pour reprogrammer des cellules somatiques humaines dans CSPi humaines sans transgène avec le virus de Sendai, qui montre des résultats cohérents et une efficacité accrues. 

Vidéo: 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.

Génération de cellules souches pluripotentes induites par la reprogrammation des fibroblastes humains avec les lentivirus TF Stemgent Homme 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 ...

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Biologie

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.

Transfection et Nucleofecting homme cellules souches pluripotentes induites

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.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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.

Colonies sélectionner et isoler de l&#39;homme cellules souches pluripotentes induites à partir de fibroblastes adultes Reprogrammed

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.

Reprogrammation des cellules somatiques humaines dans cellules souches pluripotentes induites (CISP) Utilisation vecteur rétroviral avec la 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.

Génération de souris dérivées de cellules souches pluripotentes induites

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.

Génération de l&#39;homme cellules souches pluripotentes induites à partir de sang périphérique en utilisant le vecteur lentiviral STEMCCA

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.

Génération rapide et efficace des neurones à partir de cellules souches pluripotentes humaines dans un format plaque 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.

Génération de cardiomyocytes humains: Un protocole de différenciation des induits des cellules souches pluripotentes humaines chargeur sans

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.