Les auteurs tiennent à remercier les subventions suivantes du NIH pour soutenir cette recherche: K08DK082783 (AR), P30DK56465 (BTS), U01HL099993 (BTS), T32HL00715036 (SKS), et K12HL0806406 (SKS); et la Fondation McCarthy JP (AR).

Le consentement éclairé, tel qu&#39;approuvé par les Institutional Review Board de la Cancer Research Center Fred Hutchinson et de l&#39;hôpital de Philadelphie s enfants », a été obtenu à partir de patients avant de recueillir des échantillons de sang périphérique. Toutes les lignes directrices institutionnelles ont été observées. Toutes les expériences animales, y compris la génération MEF et formation de tératome ont été approuvés par le Comité de protection des animaux et institutionnel. 1. Ficoll séparation des PBMC et Expansion des érythroblastes - Jour 0 <ol><li> Diluer le sang périphérique de 1: 1 avec une solution saline tamponnée au phosphate de Dulbecco (DPBS). Dans un tuyau à fond rond en polystyrène de 15 ml, 7 ml soigneusement la couche de sang dilué sur 3 ml de la température ambiante de Ficoll-Hypaque. </li><li> Centrifuger l&#39;échantillon à 400 g pendant 30 min. </li><li> Les PBMC aura réglé à l&#39;interface entre le plasma et le Ficoll-Hypaque, vu comme une couche blanche trouble. L&#39;utilisation d&#39;un transfert pipette stérile, recueillir le PBMC dans un tube de 15 ml conique. Porter le volume à 10 ml à l&#39;aide de DPBS. </li><li> En utilisant un hématimètre, compter les PBMC. </li><li> Pipette 2 × 10 6 PBMC dans un tube de 15 ml conique séparé et centrifuger à 300 g pendant 5 min. Centrifuger les cellules restantes et le gel à une concentration de 2 x 10 6 CMSP / ml dans 90% de sérum bovin fœtal (FBS) / 10% de diméthylsulfoxyde (DMSO). </li><li> Préparer Expansion: milieu libre QBSF-60 sérum (abri de la lumière) + pénicilline / streptomycine (1%) + acide ascorbique (50 ng / ml) + facteur des cellules souches (SCF, 50 ng / ml) + interleukine 3 (IL 3, 10 ng / ml) + érythropoïétine (EPO, 2 U / ml) + insuline-like growth factor 1 (IGF-1, 40 ng / ml) + dexaméthasone (1 uM; abri de la lumière; dégeler une partie aliquote de chaque média changer). </li><li> Remettre en suspension 2 × 10 6 PBMC dans 2 ml de fraîchement préparé milieu d&#39;expansion et le placer dans un puits d&#39;une plaque de culture de 12 tissu puits et incuber dans un incubateur humidifié à 37 ° avec une atmosphère de 5% O 2, 5% CO 2 et 90% N 2. </li><li> Le jour 3 et le jour 6, remplacer médias. <ol><li> Recueillir les cellules et laver le puits avec 2 ml de QBSF-60 pour recueillir toutes les cellules restantes. </li><li> Centrifuger la suspension de cellules à 300 xg pendant 5 min et le surnageant aspiré. </li><li> Reprendre les cellules dans 2 ml de la nouvelle extension moyenne et les retourner à la même plaque 12 puits. </li></ol></li></ol> 2. reprogrammation des cellules par nucléofection - Jour 9 <ol><li> Pipeter les plasmides de reprogrammation dans un tube Eppendorf de 1,5 ml stérile (tableau 1). </li><li> Mélanger la solution de nucléofection avec supplément (fourni par le fabricant) et la pipette dans un tube Eppendorf de 1,5 ml stérile. </li><li> Introduire à la pipette 2 ml de moyen d&#39;extension dans un puits d&#39;une plaque 12 puits et la place dans une humidifié à 37 ° C incubateur (5% de O 2, 5% CO 2 et 90% N 2) pour atteindre l&#39;équilibre avant l&#39;ajout de cellules. </li><li> Recueillir leLavage des cellules cultivées et le puits avec 2 ml de QBSF-60 pour recueillir les cellules restantes. </li><li> Centrifuger les cellules à 300 g pendant 5 min et aspirer le surnageant. </li><li> Laver les cellules par remise en suspension dans 5 ml de DPBS et centrifugation à 300 xg pendant 5 min. </li><li> Aspirer le surnageant. </li><li> Reprendre le culot cellulaire dans 100 pi de solution de nucléofection + Supplément, en prenant soin d&#39;éviter les bulles de production. </li><li> Introduire à la pipette la suspension cellulaire dans le tube contenant les plasmides de reprogrammation, pipette de haut en bas une fois, et la pipette l&#39;échantillon dans le fond de la cuvette de nucléofection. </li><li> Placez la cuvette dans la machine de nucléofection et exécuter le programme T-019. </li><li> Transférer 100 ul de l&#39;expansion préchauffé moyen (à partir de la plaque d&#39;équilibrage à l&#39;étape 2.3) dans la cuvette de nucléofection. </li><li> Recueillir immédiatement l&#39;ensemble de l&#39;échantillon et le dépôt dans le puits de préchauffé Moyen Foisonnement. Éviter de recueillir le, des débris flottants de cellules mortes blanc. </li&gt; <li> Placer la plaque dans une humidifié à 37 ° C incubateur (5% de O 2, 5% CO 2 et 90% N 2) pour la croissance. </li></ol> 3. Placage Feeder cellules - Jour 10 ou 11 <ol><li> Au jour 10, les cellules plaque d&#39;alimentation. <ol><li> Revêtez un 6 et plaque de culture de tissu avec de la gélatine. <ol><li> Introduire à la pipette 1 m de gélatine à 0,1% en solution saline tamponnée de Hank (HBSS) dans chaque puits de la plaque. </li><li> Placer la plaque dans une humidifié à 37 ° C incubateur pendant au moins 5 min. </li><li> Immédiatement avant utilisation, aspirer la solution de gélatine à partir de la plaque. </li></ol></li><li> Décongeler une fiole de 2 x 10 6 fibroblastes embryonnaires de souris irradiées à 3000 cGy dans 10 ml d&#39;préchauffé MEF Media (milieu de Eagle modifié par Dulbecco (DMEM) + FBS (10%) + pénicilline / streptomycine (1%)). </li><li> Centrifuger les cellules à 300 g pendant 5 min et aspirer le surnageant. Reprendre les cellules dans 12 ml de MEF médias et pipette 2 ml inPour chaque puits de la plaque 6 puits revêtu de gélatine. Placer la plaque dans une humidifié à 37 ° C incubateur (5% de O 2, 5% CO 2 et 90% N 2) jusqu&#39;au jour 12. </li></ol></li></ol> 4. étalement des cellules sur des cellules nourricières et changement de support - Jour 12 <ol><li> Recueillir les cellules dans une nucleofected 15 ml tube conique et collecter toutes les cellules restantes par un lavage des puits avec 2 ml de QBSF-60. Centrifuger l&#39;échantillon à 300 g pendant 5 min. </li><li> Préparer reprogrammation moyenne: Iscove modification de Dulbecco (IMDM) + FBS (10%) + non animale L-glutamine (1 mM) + non-acides aminés essentiels (NEAA, 1%) + pénicilline / streptomycine (1%) + β mercaptoéthanol (0,1 mM) + le facteur de croissance des fibroblastes (bFGF, ajoutées à l&#39;alimentation fraîche, 10 ng / ml) + acide ascorbique (frais ajoutés à l&#39;alimentation, 50 pg / ml). </li><li> Aspirer le surnageant et remettre en suspension les cellules dans 12 ml de reprogrammation moyenne. Introduire à la pipette 2 ml par puits de la suspension de cellules dansla plaque à 6 puits de cellules nourricières (tel que préparé dans la section 3). Centrifuger la plaque à 50 xg pendant 30 min à température ambiante. </li><li> Placer la plaque dans une humidifié à 37 ° C incubateur (5% de O 2, 5% CO 2 et 90% N 2) pour la croissance. Changer la reprogrammation moyenne tous les deux jours jusqu&#39;à ce que les colonies iPSC commencent à apparaître (1-2 semaines). </li><li> Préparer hESC moyenne: DMEM / F12 1: 1 + Knockout sérum de remplacement (20%) + pénicilline / streptomycine (1%) + NEAA (1%) + non animale L-glutamine (1 mM) + pyruvate de sodium (1%) + bicarbonate de sodium (0,12%) + β-mercaptoéthanol (0,1 mM) + bFGF (ajoutés à l&#39;alimentation fraîche, 10 ng / ml) </li><li> Préparer les inhibiteurs de HDAC: butyrate de sodium (ajouté à l&#39;alimentation fraîche, 100 M), de l&#39;acide suberanilohydroxamic (SAHA, a ajouté à l&#39;alimentation fraîche, 200 nM). Une fois colonies iPSC apparaissent, commencent à se nourrir les cellules avec des inhibiteurs de CSEh moyen + HDAC, le changement moyen chaque jour. REMARQUE: le butyrate de sodium est une petite molécule qui est adsorbé sur des tubes en plastique, therefore stocker dans un tube en verre de borosilicate à 4 ° C. </li></ol> 5. Les clones isolation iPSC <ol><li> Une fois colonies iPSC sont assez grands pour être isolé (20-25 cellules), les prendre avec une pipette P20. </li><li> Préparer les inhibiteurs de HDAC: butyrate de sodium (ajouté à l&#39;alimentation fraîche, 100 M), SAHA (ajoutée à l&#39;alimentation fraîche, 200 nM) </li><li> Placez les colonies isolées sur iMEFs dans les différents boîtes de 35 mm contenant des inhibiteurs d&#39;HDAC CSEh moyen + + Supplément Clonage &amp; Recovery. </li><li> Le jour après la cueillette des colonies, changer de support de CSEh inhibiteurs HDAC moyen +. </li><li> Changer le support tous les jours jusqu&#39;à ce que les colonies iPSC sont prêts pour des passages. </li><li> Retirer les inhibiteurs d&#39;HDAC dans le milieu une fois colonies se stabilisent à passage 2-3. </li></ol>

<ol>
	<li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Koche, R. P., 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=Reprogramming+factor+expression+initiates+widespread+targeted+chromatin+remodeling.">Reprogramming factor expression initiates widespread targeted chromatin remodeling.</a> Cell Stem Cell. 8 (1), 96-105 (2011).</li><li>Baum, C., Kustikova, O., Modlich, U., Li, Z., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+and+oncogenesis+by+chromosomal+insertion+of+gene+transfer+vectors.">Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors.</a> Human Gene Therapy. 17 (3), 253-263 (2006).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448 (7151), 313-317 (2007).</li><li>Yu, J., 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=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324 (5928), 797-801 (2009).</li><li>Hong, H., 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=Suppression+of+induced+pluripotent+stem+cell+generation+by+the+p53-p21+pathway.">Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.</a> Nature. 460 (7259), 1132-1135 (2009).</li><li>Okita, K., 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8 (5), 409-412 (2011).</li><li>Yates, J. L., Warren, N., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+replication+of+plasmids+derived+from+Epstein-Barr+virus+in+various+mammalian+cells.">Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells.</a> Nature. 313 (6005), 812-815 (1985).</li><li>Ware, C. B., 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=Histone+deacetylase+inhibition+elicits+an+evolutionarily+conserved+self-renewal+program+in+embryonic+stem+cells.">Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells.</a> Cell Stem Cell. 4 (4), 359-369 (2009).</li><li>Mali, P., 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=Butyrate+greatly+enhances+derivation+of+human+induced+pluripotent+stem+cells+by+promoting+epigenetic+remodeling+and+the+expression+of+pluripotency-associated+genes.">Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.</a> Stem Cells. 28 (4), 713-720 (2010).</li><li>Azuara, V., 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=Chromatin+signatures+of+pluripotent+cell+lines.">Chromatin signatures of pluripotent cell lines.</a> Nature Cell Biology. 8 (5), 532-538 (2006).</li><li>Sommer, A. G., 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=Generation+of+human+induced+pluripotent+stem+cells+from+peripheral+blood+using+the+STEMCCA+lentiviral+vector.">Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector.</a> J. Vis. Exp. (68), e4327 (2012).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5, 237-241 (2009).</li><li>Abbondanzo, S. J., Gadi, I., Stewart, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+Embryonic+Stem+Cell+Lines.">Derivation of Embryonic Stem Cell Lines.</a> Methods in Enzymology. 225, 803-823 (1993).</li><li>Kim, H. J., Bae, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors:+molecular+mechanisms+of+action+and+clinical+trials+as+anti-cancer+drugs.">Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs.</a> American Journal of Translational Research. 3 (2), 166-179 (2011).</li></ol>

Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrents.

Pour la réussite génération iPSC lors de l&#39;utilisation de ce protocole, il ya plusieurs mises en garde importantes qui devraient être considérés. Au cours de la phase d&#39;expansion des érythroblastes, le calendrier de changement de support doit être strictement suivie, que les écarts peuvent conduire à la stimulation inefficace de la population de cellules progénitrices cible et un rendement plus faible de génération iPSC. Il est important de faire de nouveaux milieu d&#39;expansion avec la dexamétha...

iPSCs sont dérivées de tissus somatiques par l&#39;expression ectopique d&#39;un ensemble minimal de gènes de pluripotence. Cette technique a d&#39;abord été démontré par transduction rétrovirale des fibroblastes humains avec OCT4, SOX2, KLF4, et cmyc, qui sont fortement exprimés dans l&#39;état pluripotent 1. Ces exprimés de façon transitoire &quot;facteurs de reprogrammation épigénétique&quot; modifient le paysage et profil d&#39;expression génique de la cellule cible analogue aux cellules souches embryonnaires humaines 2. Une fois créé, iPSCs peut potentiellement être différenciées en tout type de tissu pour complément d&#39;enquête. Ainsi, ils sont prometteurs pour l&#39;utilisation dans la médecine régénérative, la modélisation des maladies, et des applications de thérapie génique. Cependant, ce qui perturbe le génome des virus intégrant a le potentiel de modifier l&#39;expression du gène endogène, influence le phénotype cellulaire, et en fin de compte de biaiser les résultats scientifiques. En outre, les intégrations virales aléatoires peuvent conduire à délétère e cellulaireffets, y compris la possibilité d&#39;une transformation maligne 3 ou ré-expression des transgènes oncogènes 4. Futures applications cliniques, il faudra intégrer les non-génération iPSC. Qu&#39;épisomes, qui sont chromosomiques circulaire molécules d&#39;ADN supplémentaires, offrent une stratégie visant à générer iPSCs rentables, sans intégration-5. La combinaison de vecteurs épisomiques indiqués dans le tableau 1 expriment la reprogrammation facteurs OCT4, SOX2, KLF4, MYCL, et LIN28A. Le plasmide pCXLE_hOCT3 / 4-shp53-F contient également une p53 shRNA pour la suppression temporaire de TP53 à améliorer la reprogrammation cellulaire 6. Le déficient vecteur pCXWB-EBNA1 réplication favorise l&#39;amplification des facteurs de reprogrammation et une efficacité accrue de reprogrammation en fournissant une augmentation transitoire de l&#39;expression de EBNA1 7. Le plasmide pCXLE_EGFP peut être ajouté au mélange de nucléofection dans le but de determining l&#39;efficacité de transfection ou pour des applications de tri des cellules. A l&#39;exception du p-EBNA1 CXWB, des plasmides épisomiques utilisées dans ce protocole contiennent l&#39;origine du virus d&#39;Epstein-Barr de la réplication du virus et le gène EBNA1, qui médient la réplication et la partition de l&#39;épisome au cours de la division de la cellule hôte 8. Les qu&#39;épisomes sont spontanément perdu avec extensions successives iPSC 7. Sous-clonage et la caractérisation des CSPi avec épisomaux perte de vecteur, qui peut être déduit de la perte d&#39;expression eGFP, peuvent conduire à l&#39;intégration complètement iPSCs gratuits pour futures applications cliniques. Inhérente au processus de génération iPSC est la suppression de gènes spécifiques de la lignée et la réactivation des gènes de pluripotence associée. Régulation de l&#39;expression des gènes se produit à plusieurs niveaux dans le noyau, y compris les modifications de l&#39;ADN et la chromatine pour permettre des facteurs de transcription, des éléments d&#39;ADN régulatrices, et l&#39;accès de l&#39;ARN polymérase à ciblergènes. Rénovation du paysage épigénétique via des modifications globales de la chromatine est un élément clé de ré-expression du programme génétique de la pluripotence. Une modification de la chromatine spécifique qui est importante dans la régulation de l&#39;expression génique est l&#39;acétylation des histones particulier à des résidus lysine, permettant l&#39;accès à cibler des gènes par le biais diminution de la tension de la bobine d&#39;histone-ADN. Les inhibiteurs de HDAC sont de petites molécules qui ont été montré pour améliorer la reprogrammation de cellules iPSC et CSEh auto-renouvellement 9,10, probablement en raison de l&#39;appui de l&#39;état acétylé 11. Le protocole décrit ci-dessous, adapté d&#39;une publication préalable en utilisant des lentivirus intégrant 12, fournit une méthode étape par étape pour la production de cellules iPSC optimisé de sang périphérique en utilisant qu&#39;épisomes et les inhibiteurs d&#39;HDAC. Les concentrations d&#39;inhibiteur d&#39;HDAC utilisés ici sont la moitié de ceux décrits par Ware, et al. 9, et ont systématiquement conduit à une augmentation de 2 fois dans les colonies iPSC entièrement reprogrammés plus de normeprotocoles de reprogrammation épisomaux sans inhibiteurs d&#39;HDAC. Ce niveau de reprogrammation va de pair avec l&#39;efficacité que nous observons avec les méthodes lentiviraux. En utilisant ce protocole, nous avons généré efficacement iPSC de personnes comme vieux de 87 ans.

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Reagent/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:503px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ficoll-Paque PLUS</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">45-001-750</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DPBS</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">RPMI 1640</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10437028</td>
			<td>Store at -20&#176;C until needed. Thaw, aliquot, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">154938</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">QBSF-60 serum-free medium</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">50-983-234</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">penicillin/streptomycin</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell Line Nucleofector Kit V</td>
			<td style="width:151px;">Lonza</td>
			<td style="width:119px;">VCA-1003</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2% gelatin solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1393</td>
			<td>Store at 4&#176;C, liquify in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Hank&#39;s Balanced Saline Solution (HBSS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14175-103</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:329px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">12440-061</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-glutamine, 200 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">non-essential amino acids (NEAA), 100X</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>Store at 4&#176;C, away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM/Ham&#39;s F12, 1:1</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">SH30023.02</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">KnockOut Serum Replacement</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium pyruvate, 100 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11360-070</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium bicarbonate, 7.5%</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25080094</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">PHG0263</td>
			<td>Make 10 mg/mL stock in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium butyrate&#160;</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887-1G</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">hES Cell Cloning &#38; Recovery Supplement</td>
			<td style="width:151px;">Stemgent</td>
			<td style="width:119px;">01-0014-500</td>
			<td>Store at -20&#176;C until needed. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ESC-qualified BD matrigel</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">35-4277</td>
			<td>Thaw overnight on ice at 4&#176;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20&#176;C until needed. Thaw at 4&#176;C, use immediately.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">StemSpan SFEM&#160;</td>
			<td style="width:151px;">STEMCELL Technologies</td>
			<td style="width:119px;">9650</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ascorbic acid, powdered</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403-100MG</td>
			<td>Make 5 mg/mL stock in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human stem cell factor (SCF)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">255-SC-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human interleukin 3 (IL-3)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">203-IL-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">erythropoietin (EPO)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">287-TC-500</td>
			<td>Make 1000 U/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">recombinant human insulin-like growth factor 1 (IGF-1)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">291-G1-200</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">&#946;-mercaptoethanol</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td>Make 1000X stock (100 mM) in DPBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dexamethasone</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902-25MG</td>
			<td>Make 50X stock (50 &#956;M) in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SAHA (vorinostat)</td>
			<td style="width:151px;">Cayman Chemical</td>
			<td style="width:119px;">149647-78-9</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">12 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">15 mL conical tube</td>
			<td style="width:151px;">Sarstedt</td>
			<td style="width:119px;">62553002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">1.5 mL Eppendorf tube</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">35 mm tisue culture plates</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">10 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">5 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">20 &#956;L pipette tips, barrier tips</td>
			<td style="width:151px;">Genessee</td>
			<td style="width:119px;">24-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">glass Pasteur pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pipette aid</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-681-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hOCT3/4-shp53-F</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hSK</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hUL</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-EGFP</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXWB-EBNA1</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">37624</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient ips cell generation from blood using episomes and hdac inhibitors

Ce manuscrit illustre un protocole pour créer efficacement des cellules gratuit intégration humains souches pluripotentes induites (CISP) du sang périphérique en utilisant des plasmides épisomiques et histone déacétylase (HDAC) inhibiteurs. Les avantages de cette approche sont les suivants: (1) l&#39;utilisation d&#39;une quantité minimale de sang périphérique en tant que matériau de source; (2) nonintegrating vecteurs de reprogrammation; (3) une méthode rentable pour générer des vecteurs iPSCs libres; (4) une seule transfection; et (5) l&#39;utilisation de petites molécules pour faciliter la reprogrammation épigénétique. En bref, des cellules mononucléaires du sang périphérique (PBMC) sont isolées à partir d&#39;échantillons de phlébotomie routine et ensuite mises en culture dans des facteurs de croissance définies pour obtenir une population de cellules progénitrices érythrocytaires hautement proliférative qui est remarquablement prêtent à une reprogrammation. Nonintegrating, plasmides épisomiques non transmissibles exprimant OCT4, SOX2, KLF4, MYCL, LIN28A, et un court en épingle à cheveux (sh) ARN p53 sont introduced dans les érythroblastes dérivés via une seule nucléofection. La co-transfection d&#39;un épisome qui exprime renforcée protéine fluorescente verte (eGFP) permet une identification facile des cellules transfectées. Un plasmide à réplication déficiente séparé d&#39;Epstein-Barr exprimant l&#39;antigène nucléaire 1 (EBNA1) est également ajouté au mélange réactionnel pour une expression accrue de protéines de épisomiques. Les cellules transfectées sont ensuite étalées sur une couche de fibroblastes embryonnaires de souris irradiées (les iMEFs) pour une reprogrammation continue. Dès que des colonies apparaissent comme iPSC à environ douze jours après nucléofection, les inhibiteurs de HDAC sont ajoutés au milieu pour faciliter le remodelage épigénétique. Nous avons trouvé que l&#39;inclusion d&#39;inhibiteurs HDAC augmente régulièrement la génération de colonies iPSC entièrement reprogrammé par 2 fois. Une fois colonies iPSC présentent typique cellules souches embryonnaires humaines (CSEh) morphologie, ils sont délicatement transférés à Imef tissus enduits des plaques de culture individuels pour la croissance et l&#39;expansion continue.

Trois jours après nucléofection et avant l&#39;étalement des cellules sur nucleofected iMEFs, l&#39;efficacité de nucléofection réussi doit être estimée par microscopie à fluorescence pour eGFP. La figure 1 représente une expérience typique de nucléofection à environ 5-10% de la population totale des cellules exprimant eGFP. Colonies reprogrammées iPSC vont commencer à apparaître environ deux semaines après nucléofection. Les colonies sont généralement ci...

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Nous décrivons ici un protocole pour générer des cellules souches pluripotentes induites humaines du sang périphérique en utilisant une stratégie de reprogrammation de episome base et les inhibiteurs d&#39;histone déacétylase.

Efficace sur les Cellules iPS génération de sang à l&#39;aide qu&#39;épisomes et les inhibiteurs de HDAC

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Research

JoVE Journal

Biology

12.7K vues.  Fred Hutchinson Cancer Research Center.  Nous décrivons ici un protocole pour générer des cellules souches pluripotentes induites humaines du sang périphérique en utilisant une stratégie de reprogrammation de episome base et les inhibiteurs d'histone déacétylase. 

Vidéo: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Collecte de sang du crabe américaine Horseshoe, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Recherche

Biologie

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Génération d&#39;une seule cellule suspensions de tissus de souris de neurones

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolement et culture de cellules endothéliales pulmonaires des souris néonatales

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Isométrique et excentrique évaluation de génération de forces des muscles squelettiques Isolé à partir des modèles murins de dystrophies musculaires

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Le poisson zèbre rapide et efficace génotypage utilisant la PCR avec haute résolution Melt analyse

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolement des vaisseaux sanguins dérivés multipotentes précurseurs du muscle squelettique humain

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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.

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 ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Profils MicroRNA d&#39;expression de cellules iPS humaines, l&#39;épithélium pigmentaire rétinien Derived From iPS, et fœtale épithélium pigmentaire rétinien

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...