Remarque: Pour maintenir des conditions de culture stérile, toutes les procédures dans ce protocole sont réalisées en utilisant des pratiques de laboratoire stérile et réalisée sous hotte à flux laminaire. Avant de commencer, s&#39;assurer que tout média est équilibré à 37 ° C et de manière appropriée gazés. Préparation des boîtes de culture Geltrex enduits Note: voir l&#39;annexe pour l&#39;utilisation de boîtes de culture CELLstart couché. <ol><li> Dégeler un tube de Geltrex (1 ml) lentement à 2-8 ° C et diluer 1:100 c&#39;est dans 99 ml de Knockout D-MEM/F-12. Mélanger délicatement la solution. Remarque: Certaines des lignées de cellules iPS peuvent nécessiter une dilution différente Geltrex pour une croissance optimale. Voir l&#39;annexe pour des dilutions alternatives. </li><li> Couvrir toute la surface de chaque boîte de culture avec la solution Geltrex (1 ml pour un plat de 35 mm, 1,5 ml pour un plat de 60 mm). </li><li> Sceau chaque plat avec du Parafilm pour éviter le dessèchement et incuber les plats pendant 1 heure à 37 ° C. Remarque: À ce stade, vous pouvez stocker les boîtes de culture Geltrex enduits à 4 ° C jusqu&#39;à un mois. Sceau chaque plat avec du Parafilm pour empêcher la Geltrex de se dessécher. </li><li> Avant d&#39;utiliser, de transférer les plats Geltrex enduits d&#39;une hotte à flux laminaire et de leur permettre de s&#39;équilibrer à température ambiante (environ 1 heure). </li></ol> Préparation complète d&#39;alimentation sans SR KnockOut Medium <ol><li> Pour préparer 1 mL de 10 ug / ml solution FGF basique, de façon aseptique mélanger les composants énumérés ci-dessous. Aliquoter la solution et conserver à -20 ° C jusqu&#39;à 6 mois. <table border="0"><tbody><tr><td> FGF basique </td><td> 10 ug </td></tr><tr><td> D-PBS </td><td> 990μL </td></tr><tr><td> 10% de BSA </td><td> 10 uL </td></tr></tbody></table> Remarque: BSA peut être substitué avec HSA ou SR Knockout à la même concentration. </li><li> Pour préparer 50 ml de solution 2 Dispase mg / mL, de façon aseptique mélanger les composants énumérés ci-dessous. Filtre stériliser la solution et conserver à 4 ° C pendant 2 semaines. <table border="0"><tbody><tr><td> Dispase </td><td> 100 mg </td></tr><tr><td> D-PBS </td><td> 50 ml </td></tr></tbody></table></li><li> Pour préparer 100 ml de SR KnockOut complète Feeder-Free (KSR-FF) aseptiquement combiner les composants répertoriés dans le tableau ci-dessous. <table border="1"><tbody><tr><td> Composante </td><td> Concentration Stock </td><td> Concentration finale </td><td> Volume </td></tr><tr><td> Knockout DMEM/F12 (Cat. no. 12660-012) </td><td> - </td><td> 1X </td><td> 76,8 ml </td></tr><tr><td> Glutamax-I (cat. n ° 35050-061) </td><td> 200 mM </td><td> 2 mM </td><td> 1 ml </td></tr><tr><td> KnockOut SR (Cat. no. 10828-028) </td><td> - </td><td> 20% </td><td> 20 ml </td></tr><tr><td> KnockOut SR-GFC (Cat. no. A10580-01) </td><td> 50X </td><td> 1X </td><td> 2 ml </td></tr><tr><td> bFGF (Cat. no. PHG0024). </td><td> 10 ug / ml </td><td> 20 ng / mL </td><td> 200 uL </td></tr></tbody></table> Vous pouvez ranger le support KSR-FF à 2-8 ° C jusqu&#39;à une semaine. </li><li> Juste avant la pré-équilibration du milieu complet à la température et de gaz, ajouter aseptiquement le volume requis de 2 mercaptoéthanol (55 mM de concentration des stocks) pour une concentration de 0,1 mM final. Par exemple, pour préparer 100 ml de KSR-FMoyenne F ajoutez 182 uL de 55 mM de 2-mercaptoéthanol (1:550 dilution) Alternativement, le 2-mercaptoéthanol peuvent être ajoutés au milieu de 1X terminé et stocké à 2-8 ° C jusqu&#39;à une semaine. </li></ol> Préparation de congélation / Cryoconservation Medium Le milieu de cryopréservation se compose de deux médias; milieu de congélation A et B. milieu de congélation Ils sont ajoutés aux cellules à différents moments et doivent rester séparés. Ils sont chacun 50% du volume total des moyennes Cryoconservation nécessaire. Le volume total des moyennes Cryoconservation nécessaire est de 1 mL pour chaque flacon d&#39;être gelés. Un plat de 90% confluentes 60mm de CSEh peuvent être utilisés pour la banque 2 flacons de CSEh dans les médias cryoconservation. Préparer une quantité suffisante de chaque milieu de congélation avec un supplément de 2 à 5 ml pour assurer excédent. <table border="1"><tbody><tr><td> Un milieu de congélation (50% du volume final): </td><td> 50% DMEM/F12 </td><td> KSR 50% </td></tr><tr><td> Milieu de congélation B (50% du volume final): </td><td> 80% DMEM/F12 </td><td> 20% de DMSO </td></tr></tbody></table> Exemple: Si vous êtes de congélation 20 flacons de cellules, vous aurez besoin de 20 ml de Medium cryoconservation. Ajouter 4 ml d&#39;excédent pour un total de cryoconservation moyen 24ml. Cela signifie que vous aurez besoin 12 mL de milieu de congélation A. (50% de 24ml) et 12 mL de milieu B de congélation (50% de 24ml) <table border="1"><tbody><tr><td> Un milieu de congélation (12 ml): </td><td> 6 ml DMEM/F12 </td><td> 6 ml KSR </td></tr><tr><td> Milieu de congélation B (12 ml): </td><td> 9,6 ml DMEM/F12 </td><td> 2,4 ml de DMSO </td></tr></tbody></table> La composition finale du milieu cryoconservation est de 65% DMEM/F12, KSR 25%, et 10% de DMSO. Cryoconservation iPSCs <ol><li> Pré-chauffer le volume requis de Dispase dans un bain d&#39;eau à 37 ° C. Se reporter au Tableau 1 ci-dessous pour plus de détails sur les volumes requis. </li><li> Pré-équilibrer le volume requis de KSR-FF dans un bain à 37 ° C pendant 15 min de l&#39;eau. Reportez-vous au tableau 1below pour des détails sur les volumes requis. </li><li> Aspirer le milieu épuisé du récipient de culture à l&#39;aide d&#39;une pipette, et rincer les cellules deux fois avec du D-PBS. </li><li> Ajouter délicatement solution de dispase préchauffée à la cuve de culture (par exemple, 1 ml de solution de dispase par boîte de culture de 60 mm). Agiter le récipient de culture pour enrober la surface des cellules entières. </li><li> Incuber le récipient de culture à 37 ° C pendant 3 minutes. </li><li> Retirez le récipient de l&#39;incubateur, aspirer la solution Dispase, et laver délicatement les cellules avec du D-PBS. </li><li> Gratter délicatement les cellules de la surface de la boîte de culture cellulaire à l&#39;aide d&#39;un grattoir, et le transfert des cellules dans un tube de centrifugeuse de 15 ml stérile. </li><li> Rincer la boîte de culture à deux reprises avec KSR-FF, doucement &quot;pulvériser off&quot; toutes les cellules qui n&#39;ont pas détaché. Piscine de rinçage moyenne avec des cellules dans le tube de 15ml. </li><li> Centrifuger le tube à 200 xg pendant 5 minutes à température ambiante pour un culot cellulaire. </li><li> Aspirer délicatement le surnageant sans perturber le culot cellulaire et le jeter. </li><li> Préparer une quantité suffisante de cryotubes. Une fois que les cellules sont en contact avec le DMSO, ils devraient être aliquotés et congelés dans les 2-3 minutes. </li><li> Tapotez doucement le tube, pour bien déloger le culot cellulaire du fond du tube et remettre en suspension les cellules dans un milieu de congélation A. [par un léger pipetage de haut en bas en utilisant un 5-mL pipette sérologique]. Suite à la suspension uniforme de touffes, ajouter un volume égal de milieu de congélation B à elle, d&#39;une manière goutte à goutte, tout en douceur tourbillonnant la suspension cellulaire à se mélanger. Remarque: À ce stade, les cellules sont en contact avec le DMSO, et les travaux doivent être effectués de manière efficace. Une fois que les cellules sont en contact avec le DMSO, ils devraient être aliquotés et congelés dans les 2-3 minutes. </li><li> Aliquote de 1 ml de la suspension cellulaire dans chaque cryovial. </li><li> Placer rapidement les flacons dans un Mr. Frosty [rapidement geler] et de le transférer à -80 ° C la nuit. </li><li> Après une nuit à -80 ° C, le transfert des cellules à un réservoir d&#39;azote liquide pour le stockage à long terme. </li></ol> dégel du flacon IPSC et la récupération des cellules Remarque: KSR-FF peuvent être remplacés par KSR contenant MEF milieu conditionné ou moyennes KSR pour une utilisation avec les mangeoires. Voir l&#39;annexe pour les instructions pour la préparation des supports. Préchauffer 10mL de KSR-FF moyennes dans un tube de 50 ml. <ol><li> Equilibrer la quantité appropriée de Geltrex enduit de plats à la température ambiante et aspirer le liquide de la vaisselle juste avant plaquager cellules. </li><li> Retirez délicatement la CSEh (ou COPSi) flacon de réservoir d&#39;azote liquide de stockage. </li><li> Rapidement dégel flacon congelé des cellules dans un bain à 37 ° C l&#39;eau, jusqu&#39;à ce que juste un petit morceau congelé reste dans le flacon. </li><li> Vaporiser flacon avec 70% d&#39;isopropanol de décontaminer et de le transférer à la hotte de culture de tissus stériles. </li><li> Transférer de façon aseptique le contenu entier du flacon dans un tube de 15 ml conique à l&#39;aide d&#39;une pipette 5 ml. </li><li> Rincer la fiole avec 1 ml de KSR-FF et ajoute à cela le tube de 15 ml centrifugeuse. </li><li> Lentement, ajouter 4 ml de KSR-FF goutte à goutte à des cellules dans le tube de 15 ml conique. Tout en ajoutant le support, déplacez doucement le tube avant et en arrière de mélanger les cellules. (Ce qui réduit le choc osmotique dans les cellules). </li><li> Centrifuger le tube 15 ml avec la suspension cellulaire à 1000rpm (200 x g) pendant 2 minutes à température ambiante. </li><li> Soigneusement aspirer et jeter le surnageant sans perturber le culot cellulaire. </li><li> Re-suspendre le culot cellulaire dans la pré-chauffé KSR-FF (p.ex. plats mm 4mL/60) en utilisant une pipette 5 ml et doucement la pipette les cellules de haut en bas jusqu&#39;à ce culot cellulaire est complètement dispersée. A la viabilité supérieure à 70% est attendue, cependant, si la viabilité est inférieure à 70%, il peut prendre plus de temps pour les cellules pour atteindre la confluence. </li><li> En utilisant la même pipette, transférer la suspension cellulaire goutte à goutte à boîte de culture Geltrex enduits pré-équilibrée à la température ambiante. </li><li> Placez la boîte de culture dans un incubateur à 37 ° C, avec une atmosphère humidifiée de 4 à 6% de CO 2 dans l&#39;air. Navire dans un tourbillon soigneusement nord au sud, d&#39;est en ouest, afin de répartir uniformément les cellules. </li><li> Doucement fluides changer la boîte de culture de 24 heures après décongélation et quotidiennement par la suite pour éliminer les débris cellulaires et de fournir des nutriments frais jusqu&#39;à ce que le plat est d&#39;environ 70-80% de confluence. Pour la culture continue et repiquage de vos cellules iPS, reportez-vous à notre protocole intitulé «Feeder-Free Culture et repiquage des cellules iPS humaines utilisant remplacement complet KnockOut Sérum Feeder milieu exempt&quot; </li></ol> Résultats attendus Les cellules doivent être à peu près 70-80% confluentes avant cryoconservation. Dispase résultats de digestion dans le curling et pliage des colonies le long des marges colonie. Après la récolte des cellules, ils sont gelés tant que petites touffes dans les médias cryoconservation. Une fois le flacon est décongelé, les cellules de récupérer et de joindre à l&#39;antenne de pré-revêtu comme de petites touffes. Ils grandissent rapidement conduit à un plat confluentes en 5-6 jours. Tableau 1 - Volumes recommandée <table border="1"><tbody><tr><td> Composante </td><td> Plat 35mm </td><td> Plat 60mm </td><td> Plat 100mm </td></tr><tr><td> Remplissez KnockOut SR moyennes </td><td> 2 ml </td><td> 4 ml </td><td> 10 ml </td></tr><tr><td> Solution Geltrex </td><td> 1 ml </td><td> 1,5 ml </td><td> 4-5 ml </td></tr><tr><td> Dispase </td><td> 0,5 ml </td><td> 1 ml </td><td> 3-4 ml </td></tr><tr><td> D-PBS pour le rinçage </td><td> 2 ml </td><td> 4 ml </td><td> 10 ml </td></tr></tbody></table> S&#39;il vous plaît <a href="http://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">cliquez ici pour voir l&#39;appendice</a> .

<ol>
	<li>Takahashi, K. <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+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K. <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, 861-872 (2007).</li><li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <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+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <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+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <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.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td>
</tr>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>17105-041</td>
<td>Note: see appendix for alternative passaging methods</td>
</tr>
<tr>
<td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>DMSO, 500mL</td>
<td>&nbsp;</td>
<td>JT Baker</td>
<td>JT9224-1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Liquid nitrogen storage tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol freezing containers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 mL Cryovials</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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cryopreserving and recovering of human ips cells using complete knockout serum replacement feeder-free medium

La découverte en 2006 que les fibroblastes humains et de souris pouvaient être reprogrammées pour produire des cellules iPS 1-3 avec des qualités remarquablement similaires aux cellules souches embryonnaires a créé une nouvelle source précieuse de cellules pluripotentes pour la découverte de médicaments, la thérapie cellulaire, et la recherche fondamentale. Les médias et les réactifs GIBCO ont été à la pointe de la recherche de cellules souches pluripotentes pendant des années. Knockout DMEM supplémenté avec remplacement Knockout sérum est le média de choix pour la croissance des cellules souches embryonnaires et maintenant la culture de cellules iPS 3-9. Cet or média standard du système peut maintenant être utilisé pour le chargeur sans la culture avec l&#39;ajout de SR Knockout facteur de croissance cocktail. Traditionnelle ES humaines et des méthodes de culture de cellules iPS nécessite l&#39;utilisation de la souris ou humains couches nourricières de fibroblastes, ou le chargeur milieu conditionné. Ces méthodes de culture sont une main-d&#39;œuvre, dur à l&#39;échelle et il est difficile de maintenir les cellules indifférenciées hanches à cause des conditions indéfini. Invitrogen a développé SR Knockout facteur de croissance cocktail pour vous permettre de passer facilement de vos hanches et des cultures de cellules hES d&#39;alimentation sans tout en conservant votre utilisation du SR Knockout.

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Ce protocole décrit la procédure détaillée pour la cryoconservation de cellules iPS humaines dans un milieu de cryoconservation KnockOut SR et la récupération de ces cellules en complète KnockOut Feeder SR gratuit (KSR-FF) moyen ou le chargeur milieu à base de SR huitièmes de finale.

Cryoconservation et la récupération des cellules iPS humaines utilisant KnockOut complète Chargeur sans sérum remplacement moyen

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

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JoVE Journal

Biology

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Vidéo: Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Prédire l&#39;efficacité de la stratégie de remplacement de la population utilisant la modélisation mathématique

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

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Biologie

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Générer des cellules iPS de MEFS travers l&#39;expression forcée de Sox-2, Oct-4, c-Myc et Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolement et animale expansion sans sérum du cordon ombilical humain dérivé cellules stromales mésenchymateuses (CSM) et des cellules endothéliales progénitrices Colony Forming (CPEF)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

Chargeur sans adaptation, de la Culture et de repiquage des cellules iPS humaines à l&#39;aide complète d&#39;alimentation sans remplacement KnockOut Sérum Medium

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 ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolation des petits ARN non codant de sérum humain

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

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, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

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 ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Analyse comparative de l&#39;hormone de croissance dans le sérum en utilisant SPRi, Nano-SPRi et dosages ELISA

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Surveillance in Situ des assemblages de chaperon moléculaire s'est formé de façon transitoire dans les bactéries, les levures et les cellules humaines

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...