Nota: Para mantener condiciones estériles cultura, todos los procedimientos descritos en este protocolo se realiza mediante prácticas de laboratorio y estériles a cabo bajo una campana de flujo laminar. Antes de comenzar, asegúrese de que cualquier medio es equilibrado a 37 ° C y adecuadamente el gas. Preparación Geltrex recubierto placas de cultivo Nota: véase el apéndice para el uso de CELLstart placas de cultivo revestidas. <ol><li> Descongelar un tubo de Geltrex (1 mL) lentamente a 2-8 º C y se diluye 1:100 en 99 ml de golpe de gracia D-MEM/F-12. Mezcle la solución suavemente. Nota: Algunas líneas de células iPS pueden requerir una dilución diferente Geltrex para un crecimiento óptimo. Véase el apéndice para las diluciones alternativa. </li><li> Cubrir toda la superficie de cada placa de cultivo con la solución Geltrex (1 ml por un plato de 35 mm, 1,5 ml de un plato de 60 mm). </li><li> Sello de cada plato con Parafilm para evitar la sequedad y se incuban las placas durante 1 hora a 37 ° C. Nota: En este punto usted puede almacenar las placas de cultivo Geltrex recubierto a 4 ° C durante un máximo de un mes. Sello de cada plato con Parafilm para evitar la Geltrex se seque. </li><li> Antes de usar, la transferencia de los platos Geltrex recubierto con una campana de flujo laminar y permitir que alcancen la temperatura ambiente (alrededor de 1 hora). </li></ol> Preparación del alimentador de Libre Completo SR golpe de gracia Media <ol><li> Para preparar 1 ml de 10 mg / ml de solución FGF básico, asépticamente mezclar los componentes enumerados a continuación. Alícuota de la solución y almacenar a -20 ° C hasta por 6 meses. <table border="0"><tbody><tr><td> FGF básico </td><td> 10 mg </td></tr><tr><td> D-PBS </td><td> 990μL </td></tr><tr><td> 10% de BSA </td><td> 10 l </td></tr></tbody></table> Nota: BSA se puede sustituir con HSA o SR golpe de gracia a la misma concentración. </li><li> Para preparar 50 ml de solución de 2 Dispasa mg / ml, asépticamente mezclar los componentes enumerados a continuación. Filtro de esterilización de la solución y se almacenan a 4 ° C durante un máximo de 2 semanas. <table border="0"><tbody><tr><td> Dispasa </td><td> 100 mg </td></tr><tr><td> D-PBS </td><td> 50 ml </td></tr></tbody></table></li><li> Para preparar 100 mL de SR anulación completa del alimentador-Free (KSR-FF) asépticamente combinar los componentes que figuran en la tabla de abajo. <table border="1"><tbody><tr><td> Componente </td><td> Concentración de la </td><td> La concentración final </td><td> Volumen </td></tr><tr><td> Nocaut DMEM/F12 (cat. n °. 12660-012) </td><td> - </td><td> 1X </td><td> 76.8 mL </td></tr><tr><td> Glutamax-I (Cat. N º 35.050-061) </td><td> 200 mM </td><td> 2 mM </td><td> 1 mL </td></tr><tr><td> SR golpe de gracia (art. no. 10.828-028) </td><td> - </td><td> 20% </td><td> 20 ml </td></tr><tr><td> Golpe de gracia SR-GFC (cat. n °. A10580-01) </td><td> 50 veces </td><td> 1X </td><td> 2 mL </td></tr><tr><td> bFGF (cat. n °. PHG0024). </td><td> 10 mg / ml </td><td> 20 ng / mL </td><td> 200 l </td></tr></tbody></table> Puede almacenar el medio KSR-FF a 2-8 º C durante un máximo de una semana. </li><li> Justo antes de pre-equilibrar el medio completo a la temperatura y gases, añadir asépticamente el volumen requerido de 2-mercaptoetanol (55 mM de concentración) para una concentración de 0,1 mM final. Por ejemplo, para preparar 100 ml de KSR-FMedio F añadir 182 l de 55 mM 2-mercaptoetanol (1:550 dilución) Por otra parte, el 2-mercaptoetanol puede ser añadido al medio 1X completaron y se almacenaron a 2-8 ° C durante un máximo de una semana. </li></ol> Preparación de congelación / Criopreservación Media El medio de criopreservación se compone de dos medios de comunicación, medio de congelación A y B. medio de congelación Se añaden a las células en diferentes momentos y deben permanecer separados. Son cada uno el 50% del volumen total del medio de criopreservación es necesario. El volumen total del medio de criopreservación se necesita 1 ml por cada vial a congelar. Un 90% confluentes plato de 60 mm de células madre se pueden utilizar para el banco 2 viales de células madre en los medios de criopreservación. Preparar un volumen suficiente de cada medio de congelación con un extra de 2 5 ml para asegurar exceso. <table border="1"><tbody><tr><td> Congelación Medio A (50% del volumen final): </td><td> 50% DMEM/F12 </td><td> 50% KSR </td></tr><tr><td> Congelación medio B (50% del volumen final): </td><td> 80% DMEM/F12 </td><td> 20% DMSO </td></tr></tbody></table> Ejemplo: Si se congelan 20 viales de células, tendrá 20 ml de medio de criopreservación. Agregar 4 ml por exceso por un total de medio Criopreservación 24ml. Eso significa que usted necesitará 12 ml de un medio de congelación. (50% de 24ml) y 12 ml de B medio de congelación (50% de 24ml) <table border="1"><tbody><tr><td> Congelación Medio A (12 ml): </td><td> 6 mL DMEM/F12 </td><td> 6 mL KSR </td></tr><tr><td> Congelación medio B (12 ml): </td><td> 9,6 ml de DMEM/F12 </td><td> 2,4 ml de DMSO </td></tr></tbody></table> La composición final de la media La crioconservación es un 65% DMEM/F12, el 25% KSR, y el 10% de DMSO. Criopreservar CMPi <ol><li> Pre-caliente el volumen necesario de Dispasa en un baño de agua a 37 ° C. Consulte la Tabla 1 a continuación para obtener información sobre los volúmenes requeridos. </li><li> Pre-equilibrar el volumen requerido de KSR-FF en un baño de agua 37 ° C for15 min. Consulte la tabla 1below para obtener información sobre los volúmenes requeridos. </li><li> Aspirar el medio transcurrido desde el recipiente de cultivo con una pipeta, y aclarar las células dos veces con D-PBS. </li><li> Suavemente agregar precalentado Dispasa solución al recipiente de cultivo (por ejemplo, 1 ml de solución Dispasa por placa de cultivo de 60 mm). Agitar el recipiente de cultivo para cubrir la superficie entera de la célula. </li><li> Incubar el recipiente de cultivo a 37 ° C durante 3 minutos. </li><li> Retire el vaso de la incubadora, se aspira la solución Dispasa, y lavar suavemente las células con D-PBS. </li><li> Raspe suavemente las células de la superficie de la placa de cultivo con un rascador de células, y la transferencia de las células a un tubo de centrífuga de 15 ml estéril. </li><li> Enjuague la placa de cultivo en dos ocasiones con KSR-FF, con suavidad &quot;pulverización de&quot; cualquier célula que no se han desprendido. La piscina del enjuague medio con las células en el tubo de 15 ml. </li><li> Centrifugar el tubo a 200 xg durante 5 minutos a temperatura ambiente para precipitar las células. </li><li> Con cuidado, aspirar el sobrenadante sin perturbar el sedimento celular y lo descarta. </li><li> Preparar una cantidad suficiente de crioviales. Una vez que las células están en contacto con DMSO, que debe ser en alícuotas y congelado a los 2-3 minutos. </li><li> Agitar suavemente el tubo, para desalojar completamente el pellet de células de la parte inferior del tubo y volver a suspender las células en medio de congelación A. [pipeteando suavemente hacia arriba y hacia abajo con un niño de 5 ml pipeta serológica]. Tras la suspensión uniforme de los grumos, agregar el mismo volumen de medio de congelación B a la misma, de manera gota a gota, mientras se agita suavemente la suspensión celular para mezclar. Nota: En este punto, las células están en contacto con DMSO, y el trabajo debe realizarse de manera eficiente. Una vez que las células están en contacto con DMSO, que debe ser en alícuotas y congelado a los 2-3 minutos. </li><li> Alícuota de 1 ml de la suspensión celular en cada criovial. </li><li> Rápidamente coloque los tubos en un Frosty el Sr. [para congelar rápidamente] y su transferencia a -80 ° C durante la noche. </li><li> Después de almacenamiento durante la noche a -80 ° C, la transferencia de las células a un tanque de nitrógeno líquido para su almacenamiento a largo plazo. </li></ol> IPSC deshielo vial y recuperación de las células Nota: KSR-FF puede ser reemplazado por KSR-MEF-que contiene un medio condicionado, o medio KSR para su uso con los alimentadores. Véase el apéndice para obtener instrucciones para la preparación de los medios de comunicación. Pre-caliente 10 ml de KSR-FF medio de un tubo de 50 ml. <ol><li> Equilibre la cantidad apropiada de Geltrex recubierto platos a la temperatura ambiente y se aspira el líquido de los platos antes de recubrimiento quer las células. </li><li> Retire con cuidado la células madre (o IPSC) frasco de líquido del tanque de almacenamiento de nitrógeno. </li><li> Rápido deshielo vial congelado de células en un baño de agua 37 ° C, hasta que sólo una parte pequeña se mantiene congelado en el vial. </li><li> Rocíe vial con 70% de isopropanol para descontaminar y su transferencia a la capilla de cultivo de tejido estéril. </li><li> Transferir asépticamente todo el contenido del vial en un tubo cónico de 15 ml con una pipeta de 5 ml. </li><li> Enjuague el vial con 1 mL de KSR-FF y agregue esto a la del tubo de centrífuga de 15 ml. </li><li> Poco a poco, añadir 4 ml de KSR-FF, gota a gota a las células en el tubo cónico de 15 ml. Al tiempo que añade el medio, mueva suavemente el tubo de ida y vuelta a mezclar las células. (Esto reduce el choque osmótico de las células). </li><li> Centrifugar el tubo de 15 ml con la suspensión celular a 1000 rpm (200 x g) durante 2 minutos a temperatura ambiente. </li><li> Con cuidado aspirar y descartar el sobrenadante sin perturbar el sedimento celular. </li><li> Vuelva a suspender el pellet de células en el pre-calentado KSR-FF (por ejemplo, 4mL/60 plato mm) con una pipeta de 5 ml y pipeta suavemente las células de arriba a abajo hasta que el sedimento celular está totalmente dispersa. A la viabilidad de más del 70% que se espera, sin embargo, si la viabilidad es inferior al 70%, puede tomar más tiempo para que las células para llegar a la confluencia. </li><li> Utilizando la misma pipeta, transferir la suspensión celular, gota a gota a la placa de cultivo Geltrex recubierto antes de alcanzar la temperatura ambiente. </li><li> Coloque el plato de cultivo en una incubadora a 37 ° C, con una atmósfera húmeda de CO 4 al 6% 2 en el aire. Cuidadosamente remolino en un vaso de norte a sur, de este a oeste patrón para distribuir uniformemente las células. </li><li> Suavemente el líquido de cambio de la placa de cultivo de 24 horas después de la descongelación y posteriormente a diario para eliminar los desechos celulares y para proporcionar los alimentos frescos hasta que el plato es de aproximadamente 70-80% confluente. Para el cultivo continuado y pases de las células iPS, se refieren a nuestro protocolo titulado &quot;alimentador-cultura libre y pases de células iPS humanas con suero golpe de gracia completa sustitución del alimentador de medio libre&quot; </li></ol> Resultados esperados Las células deben ser aproximadamente del 70-80% confluentes antes de la crioconservación. Dispasa resultados digestión en el curling y plegamiento de las colonias a lo largo de los márgenes de la colonia. Después de cosechar las células se congelan como pequeños grupos en los medios de criopreservación. Una vez que el vial se descongela, las células se recuperan y se unen al plato pre-recubiertos de pequeños grupos. Crecen rápidamente lleva a un plato de confluencia en 5-6 días. Tabla 1 - Volúmenes recomendados <table border="1"><tbody><tr><td> Componente </td><td> 35 mm del plato </td><td> Plato de 60mm </td><td> Plato de 100 mm </td></tr><tr><td> Completa golpe de gracia SR medio </td><td> 2 mL </td><td> 4 ml </td><td> 10 ml </td></tr><tr><td> Geltrex Solución </td><td> 1 mL </td><td> 1,5 ml </td><td> 4.5 mL </td></tr><tr><td> Dispasa </td><td> 0,5 ml </td><td> 1 mL </td><td> 3.4 mL </td></tr><tr><td> D-PBS para enjuagar </td><td> 2 mL </td><td> 4 ml </td><td> 10 ml </td></tr></tbody></table> Por favor, <a href="http://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">haga clic aquí para ver el apéndice</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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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<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>
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<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>
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cryopreserving and recovering of human ips cells using complete knockout serum replacement feeder-free medium

El descubrimiento en 2006 de que los fibroblastos humanos y de ratón podría ser reprogramado para generar células iPS de 1-3 con cualidades muy similares a las células madre embrionarias ha creado una nueva y valiosa fuente de células pluripotentes para el descubrimiento de fármacos, la terapia celular y la investigación básica. Los medios de comunicación GIBCO y reactivos han estado a la vanguardia de la investigación de células madre pluripotentes de años. Nocaut DMEM suplementado con suero de reemplazo golpe de gracia es el medio de elección para el crecimiento de células madre embrionarias, y ahora el cultivo de células iPS 3-9. Este estándar de oro del sistema de medios ahora se puede utilizar para alimentación libre de la cultura con la adición de cóctel golpe de gracia SR factor de crecimiento. ES humanas tradicionales y los métodos de cultivo de células iPS requieren el uso de ratón o humano capas de fibroblastos de alimentación o el alimentador de medio condicionado. Estos métodos de cultivo son mano de obra, difícil de escalar y que es difícil mantener las células indiferenciadas caderas debido a las condiciones indefinido. Invitrogen ha desarrollado SR golpe de gracia crecimiento Cocktail factor que le permite una fácil transición de las caderas y las culturas hES celular para alimentación libre, manteniendo el uso de SR golpe de gracia.

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

Este protocolo describe el procedimiento detallado para criopreservar las células iPS humanas en el medio de criopreservación golpe de gracia SR y la recuperación de estas células en el golpe de gracia completa alimentador gratis SR (KSR-FF) a medio o en el alimentador medio basado golpe de gracia SR.

Criopreservación y recuperación de células iPS humanas con anulación completa del alimentador de sustitución sin suero medio

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

Research

JoVE Journal

Biology

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Video: 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.

La predicción de la eficacia de la estrategia de reemplazo de la población Utilizando modelos matemáticos

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

Investigación

Biología

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.

La generación de células iPS a partir de MEFs través de la expresión forzada de Sox-2, Oct-4, c-Myc y 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.

Aislamiento y suero animal expansión libre de cordón umbilical humano derivado de células mesenquimales del estroma (MSC) y endoteliales formadoras de colonias Células Progenitoras (ECFCs)

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.

Alimentador de Libre adaptación, Cultura y pases de células iPS humanas con alimentador de Libre Completa golpe de gracia suero sustitución Media

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.

La generación de cardiomiocitos humanos: Protocolo de diferenciación de células madre pluripotentes inducidas humanas Feeder libres

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.

El aislamiento de los pequeños RNAs no codificantes de suero humano

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.

Perfiles de expresión de microARN de las células iPS humanas, de la retina del epitelio pigmentario Derivado de Ips y Fetal epitelio pigmentario de la retina

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.

Eficiente iPS Generación Celular de sangre usando episomas y HDAC inhibidores

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.

Análisis comparativo de la hormona de crecimiento humano en suero utilizando SPRI, Nano-SPRI y ELISA Ensayos

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.

Monitoreo in situ de conjuntos de chaperona molecular formadas transitoriamente en bacterias, levaduras y células humanas

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 ...