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 platos de la cultura CELLstart recubierto <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> 990 l </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) medio 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. no. 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-FF medio 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 para hasta una semana. </li></ol> La adaptación a CMPi KSR-FF Medio <ol><li> Antes de comenzar, pre-equilibrar sus platos Geltrex revestido a temperatura ambiente en el capó. </li><li> La cultura de la CMPi en células alimentadoras MEF hasta que el 70-80% confluente. Por favor, consulte el manual GIBCO de fibroblastos de ratón embrionario para los protocolos de la cultura basada en el MEF. </li&gt; <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 1 a continuación para obtener información sobre los volúmenes requeridos. </li><li> Aspirar el medio de las placas de cultivo, y añadir una cantidad apropiada de solución Dispasa. Incubar las placas a 37 ° C durante 3-5 minutos. </li><li> Aspirar la solución Dispasa de cada recipiente de cultivo y lavar las células alimentadoras MEF suavemente con D-PBS (2 a 3 veces). </li><li> Añadir una cantidad apropiada de medio completo SR golpe de gracia a cada recipiente de cultivo. Use un raspador celular o una pipeta de 5 ml para raspar suavemente las células de la superficie del recipiente de cultivo. Nota: véase el apéndice para los protocolos de adaptación alternativa para las líneas celulares duro para adaptarse iPS, </li><li> Recoger la suspensión de células de cada placa de cultivo en distintos tubos de 15 ml cónicos. Enjuague cada recipiente de cultivo con una cantidad apropiada de medio completo SR golpe de gracia, y añadir el D-PBS enjuague medio en los tubos de 15 ml cónicos que contengan la suspensión celular. Tenga cuidado de no romper los cúmulos de células en células individuales </li><li> Centrífuga de 15 ml en tubos cónicos de 200 xg durante 5 minutos para que sedimenten las CMPi. </li><li> Aspirar el sobrenadante del precipitado de IPSC. Resuspender el precipitado en una cantidad adecuada de KSR-FF medio de acuerdo con la relación de división (tabla 1). No romper los grumos de células de un tamaño más pequeño, porque las matas más pequeñas no se adhieren bien a la superficie. Nota: Se recomienda una relación de división de 1:2 para los 3 primeros pasos después de la CMPi han pasajes directamente desde el medio de cultivo iPSC MEF para KSR-FF medio. Normalmente, una relación de separación 01:03-01:05 es apropiado, pero los pases en 01:02 garantiza la mayor densidad de células necesarias para la adaptación de una cultura de enlace de forma gratuita. </li><li> Antes de la chapa en los platos CMPi Geltrex recubierto, aspirar residual solución Geltrex del plato pre-revestidos, y se añade lentamente una cantidad apropiada de la suspensión celular a cada plato de cultivo. Nota: No enjuague los platos antes de la galvanoplastia. </li><li> Mover el plato de la cultura de ida y vuelta y de lado a lado varias veces para dispersar las células en la superficie del plato. Con cuidado, coloque la placa de cultivo en una incubadora a 37 ° C con una atmósfera húmeda de CO 4 al 6% 2 en el aire. Sustituya el medio pasó con KSR-FF todos los días. </li></ol> Pases células iPS humanas con KSR-FF <ol start="13"><li> Tenga en cuenta las CMPi humanos crecen en completa KSR-FF bajo el microscopio para confirmar que las células son del 70-80% confluentes y listo para ser subcultivaron. Refiérase a la Figura 1. Nota: Si las colonias es muy densa o demasiado grande, mayor diferenciación se produce. </li><li> Cortar y eliminar cualquier colonias iPSC diferenciadas antes de pases de la cultura. </li><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 1 a continuación 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> Agitar suavemente el tubo para desalojar completamente el pellet de células de la parte inferior del tubo. </li><li> Resuspenda suavemente las células pre-equilibrio KSR-FF con una pipeta serológica de 5 ml. No triturar. Nota: es fundamental en el paso para volver a suspender suavemente las células sin usar la fuerza para evitar daños. </li><li> Transferencia de las células a un nuevo máximo de 60 mm Geltrex recubiertos plato en la relación de división que desee y mover la placa de cultivo de ida y vuelta y de lado a lado varias veces para dispersar las células a través de su superficie. </li><li> Coloque el plato de cultivo en una incubadora a 37 ° C con una atmósfera húmeda de4 a 6% de CO 2 en el aire. </li><li> Al día siguiente, reemplazar el cuidado medio gastado con KSR-FF para eliminar los restos celulares. Vuelva a colocar el medio gastado todos los días a partir de entonces. Observar las células iPS diario y el paso cuando sea necesario (aproximadamente cada 4-5 días). Pases se recomienda cuando las células alcanzan el 70-80% de confluencia. Para la criopreservación de células iPS y deshielo, se refieren a nuestro protocolo titulado &quot;criopreservación y recuperación de células iPS humanas con alimentador de Libre Completa golpe de gracia suero sustitución Medio&quot;. </li></ol> Resultados esperados <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Figura 1" /> Figura 1. La imagen de contraste de fase muestra CMPi cultivadas en placas de cultivo Geltrex recubiertos con alimentador de Libre completa golpe de gracia en suero de sustitución medio. La exposición CMPi morfología similar a hESCs, que se caracteriza por núcleos grandes y escaso citoplasma. (40 aumentos) <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 valign="top"><td> Completa golpe de gracia SR medio </td><td> 2 mL </td><td> 4 ml </td><td> 10 ml </td></tr><tr valign="top"><td> Geltrex Solución </td><td> 1 mL </td><td> 1,5 ml </td><td> 4.5 mL </td></tr><tr valign="top"><td> Dispasa </td><td> 0,5 ml </td><td> 1 mL </td><td> 3.4 mL </td></tr><tr valign="top"><td> D-PBS para enjuagar </td><td> 2 mL </td><td> 4 ml </td><td> 10 ml </td></tr></tbody></table> Tabla 1. Los volúmenes recomendados <a href="http://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">Por favor, 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>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products</td>
</tr>
<tr>
<td>GlutaMAX-I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. No. 35050-061</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR, Cat. no. 10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR-GFC, Cat. no. A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>bFGF, Cat. no. 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>Cat. no. 21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 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>Cat. no. 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>Cat. no. 14190-144</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>60mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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feeder-free adaptation, culture and passaging 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 fácilmente la transición de su cadera para cultivos de células de alimentación libre, manteniendo el uso de SR golpe de gracia.

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

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

El siguiente protocolo proporciona instrucciones para la adaptación humana células madre pluripotentes inducidas (iPS) celdas para alimentación libre de la cultura a través del alimentador de Libre completa golpe de gracia suero sustitución medio (KSR-FF). Una vez adaptado, instrucciones para el mantenimiento continuo también se proporcionan.

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

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 ...

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

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES pases madre de embriones humanos líneas celulares con tripsina

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Investigación

Biología

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células madre embrionarias humanas: Cultura a partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Pases células madre neurales humanas

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Cryopreserving and Recovering 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 and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

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

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 ...