Este trabajo fue apoyado por becas de los Institutos Nacionales de Salud (CA127574, CA107040 y CA09071) para WM

1. Xenoinjertos de la cosecha de los ratones <ol><li> Prepare la disociación celular de amortiguación: Dulbecco modificado de Eagle (DMEM) suplementado con 10% de suero fetal bovino (FCS), penicilina-estreptomicina, 200 U / ml de colagenasa tipo IV, y el 0,6 dispasa U / mL. </li><li> Un atímicos (nu + / nu +) ratón albergar una inyección subcutánea humanos xenoinjertos de cáncer de páncreas que es menos de 1 cm de diámetro mayor es la eutanasia por asfixia de dióxido de carbono y la dislocación cervical. </li><li> El tumor se extrae por vía subcutánea en el flanco del ratón mediante disección roma con unas pinzas y tijeras y se coloca inmediatamente en el buffer de disociación celular. </li><li> Los tumores se pesan y se coloca de nuevo en 10 ml de células buffer de disociación por 1 gramo de tejido del tumor. </li></ol> 2. Generar una única suspensión de células a partir de los xenoinjertos <ol><li> Trabajo en un recipiente estéril de cabinas de seguridad, el tumor se transfiere a un 100 x 20 mm placa de cultivo con 1 ml de células buffer de disociación. Los tumores son mecánicamente picada con cuchillas de afeitar y pinzas estériles. La suspensión de las células tumorales se tritura a través de una pipeta serológica de 5 ml de 10 veces. Las piezas del tumor debe ser lo suficientemente pequeño como para no obstruir la pipeta serológica de 5 ml. </li><li> La suspensión de las células tumorales se transfiere a un tubo cónico de 50 ml que contiene el volumen restante de disociación celular de amortiguación (llena de menos del 50%) y vortex a máxima velocidad durante 1 minuto. </li><li> La suspensión de las células tumorales se incuba a 37 ° C durante 2 horas con agitación intermitente durante 1 minuto cada 20 minutos. </li></ol> 3. Agotan la suspensión de células de restos de tejido y las células muertas <ol><li> Después de la incubación de 2 horas la suspensión celular se agita a la máxima velocidad durante 1 minuto. </li><li> La suspensión celular se hace pasar por un filtro de 70 micras, y se recoge en un nuevo máximo de 50 ml tubo cónico y ajustado a un volumen final de 15 ml por tubo. </li><li> A fin de separar las células viables de las células muertas y restos de tejido, la suspensión de las células tumorales se separa por centrifugación de densidad con Ficoll-Paque Plus. Una capa de base de Ficoll-Paque Plus es creado por pipeta 15 ml de Ficoll-Paque Plus lentamente por debajo de la suspensión celular con cuidado de no mezclar las dos capas. </li><li> La suspensión de células y capas de Ficoll se se centrifuga a 500x g durante 30 minutos con el freno desactivado. </li><li> Las células en la interfase del Plus Ficoll-Paque y el tampón disociación celular se recogen y se transfieren a un nuevo máximo de 50 ml tubo cónico. </li><li> DMEM suplementado con 10% FCS, se añade a la suspensión celular para diluirla 1:3 (por ejemplo, 20 ml de medio fresco a 10 ml de suspensión celular). </li><li> Las células se recogieron por centrifugación a 450x g durante 10 minutos, los medios de decantación y el pellet de células se resuspende en 1 ml de solución tampón ALDEFLUOR. </li><li> Diez microlitros de la suspensión celular se diluye con 10 l azul tripano y las células viables se contaron usando un hemocitómetro. Si un gran número de células muertas o restos de tejido se observó durante este paso, entonces la separación de células por centrifugación de densidad se puede repetir (pasos 3,3 hasta 3,7). </li></ol> 4. Tinción de las células de clasificación celular activado por fluorescencia <ol><li> Etiqueta de siete de 12 x 75 mm tubos de ensayo de poliestireno de la siguiente manera: <ol><li> Sin mancha </li><li> CD44-APC </li><li> CD24-PE </li><li> ratón CD31-biotina + ratón linaje-biotina + ratón H-biotina-2Kd </li><li> ALDEFLUOR </li><li> ALDEFLUOR + + IgG DEAB 2bκ-APC + IgG 2aκ-PE </li><li> ALDEFLUOR + CD44 + CD24-APC-PE + ratón + CD31-biotina ratón linaje-biotina + ratón H-2Kd-biotina. </li></ol></li><li> Diluir la suspensión de células en 2 ml de solución tampón ALDEFLUOR a una concentración final de 5-10 millones de células / ml y mantener en hielo. Añadir 100 ml de la suspensión celular a los tubos 1, 2, 3 y 4 y mantener en hielo. Añadir un DEAB l de tubo n º 6 y mantener en hielo. Añadir el reactivo ALDEFLUOR a la suspensión celular diluida restante 1000 - veces (por ejemplo, añadir 1,6 l de reactivo ALDEFLUOR a 1,6 ml de suspensión celular), mezclar bien y colocar en hielo. Transferencia de 100 l de esta mezcla en los tubos # 5 y 6, y el resto de la mezcla (1,4 ml) al tubo # 7. Incubar todos los tubos en un baño de agua 37 ° C durante 40 minutos. Mezcle la suspensión de células cada 10 minutos con el fin de evitar que las células se depositen en el fondo de los tubos. </li><li> Centrifugar las células a 450x g y 4 ° C durante 10 minutos y luego decantar el buffer. Volver a suspender las pastillas en tubos de 1 y 5 de cada 100 l de amortiguación ALDEFLUOR. A los tubos # 2, 3, 4 y 6, añadir 100 ml de solución tampón ALDEFLUOR que contiene los anticuerpos adecuados diluido de la siguiente manera: 1:20 de la IgG 2bΚ-APC, IgG 2aΚ-PE, CD44-APC, y CD24-PEanticuerpos; 1:100 para el ratón CD31-biotina, el ratón linaje-biotina, y el ratón H-biotina-2Kd anticuerpos. Al tubo # 7 añadir 1,4 ml de solución tampón que contiene ALDEFLUOR una dilución 1:20 de CD44-APC, y los anticuerpos CD24-PE y una dilución de 1:100 de ratón CD31-biotina, el ratón linaje-biotina, y el ratón H-biotina-2Kd anticuerpos. Se incuban las células en suspensión en hielo durante 10 minutos. </li><li> Centrifugar las células a 450x g y 4 ° C durante 10 minutos y luego decantar el buffer. Volver a suspender las pastillas en tubos de 1, 2, 3, 5 y 6 de cada 100 l de amortiguación ALDEFLUOR. Preparar 1.5 mL de tampón ALDEFLUOR que contiene una dilución 1:100 de estreptavidina-PerCP proteínas. Añadir 100 ml de tubo n º 4 y añadir 1,4 ml al tubo # 7. Se incuban las células en suspensión en hielo durante 10 minutos. </li><li> Centrifugar las células a 450x g y 4 ° C durante 10 minutos y luego decantar el buffer. Volver a suspender las pastillas en tubos de 1, 2, 3, 4, 5 y 6 de cada 200 l de amortiguación ALDEFLUOR. Resuspender el precipitado en el tubo # 7 en 2,8 ml de solución tampón que contiene ALDEFLUOR 2 mg / ml de yoduro de propidio. Mantenga los tubos en hielo en todo momento. </li><li> Pasar las células a través de 35 micras filtro con 12 x 75 mm tubos de ensayo de poliestireno con tapas de filtro. </li></ol> 5. Aislar células madre del cáncer de clasificación celular activado por fluorescencia <ol><li> Un citómetro de flujo FACSAria se prepara para la clasificación de la célula. </li><li> Controles de compensación se establecen con las células de los tubos 1, 2, 3, 4 y 5. </li><li> Gates se crean sobre la base de proyecciones y los parámetros de dispersión lateral para recoger células de tirantes. </li><li> No-ratón (PerCP-negativo) y células viables (no teñidas de propidio yoduro) son cerradas mediante el canal FL-3. </li><li> ALDH + células se obtienen sobre la base de las puertas creadas con DEAB las células tratadas (tubo n º 6) y el canal FL-1. </li><li> CD44 + CD24 + células se obtienen sobre la base de las puertas creadas con células teñidas con ALDEFLUOR, IgG 2bΚ-APC, y la IgG 2aκ-PE (tubo n º 6) con el FL-4 y los canales de FL-2. </li><li> Las células se recolectan en 12 x 75 mm tubos de ensayo de poliestireno que contiene 1 ml de DMEM suplementado con 10% FCS. </li><li> Después las células han sido ordenados, centrifugar la suspensión celular a 450x g durante 10 minutos, decantar los medios de comunicación, y volver a suspender las células en 500 l DMEM suplementado con 10% FCS. </li><li> Cuente el número de células seleccionadas utilizando un hemocitómetro como se describe en el paso 3.8. </li></ol> 6. Resultados representante Este protocolo conducirá al aislamiento de ALDH + y CD44 + CD24 + de páncreas células madre cancerosas. El porcentaje de células derivadas de ratón es variable, pero hemos encontrado que la mayoría de los xenoinjertos de cáncer humano de páncreas contienen un 20-60% de las células derivadas de ratón con el ratón CD31, cóctel de ratón linaje, y el ratón H-biotina 2Kd anticuerpos conjugados (Figura 1 ). Asimismo, la frecuencia de cada población CSC de xenoinjertos diferentes es variable, pero en general hemos encontrado que 4.1% de la población total de células ALDH es + (fig. 1B) y 0,2-5% es CD44 + CD24 + (fig. 1C). En forma rutinaria manual recuento de células ordenados utilizando un hemocitómetro desde la máquina cuenta FACSAria son a menudo inexactas debido a la no-celular partículas detectadas por el FACSAria. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="Figura 1" /> Figura 1. Trama de citometría de flujo que representa la población específica de un xenoinjerto de cáncer de páncreas. (A) Las células de tinción positiva en el canal FL-3 (yoduro de propidio +, CD31 + ratón + linaje de ratón o mouse H-2Kd +) están excluidos a fin de aislar sólo viables y no las células derivadas de ratones. (B) la actividad de la ALDH en un xenoinjerto de cáncer de páncreas humanos se midió por citometría de flujo utilizando el reactivo ALDEFLUOR en presencia y ausencia del inhibidor ALDH1 dietilamino-benzaldehído (DEAB). Los cuadros representan las puertas que representan las células ALDH + que se han creado sobre la base de las células tratadas con DEAB y luego se aplican a las células no tratadas. Los porcentajes de células + ALDH se muestran en la puerta. (C) El CD44 + CD24 + puerta fue creado a partir de células teñidas con ALDEFLUOR, y los anticuerpos IgG 2bκ-APC (control isotípico de CD44-APC) y la IgG 2aκ-PE (control isotípico de CD24-PE). Los porcentajes de células CD44 + CD24 + se muestran en la puerta.

<ol>
	<li>Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells--perspectives+on+current+status+and+future+directions:+AACR+Workshop+on+cancer+stem+cells.">Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells.</a> Cancer Res. 66, 9339-9344 (2006).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 3983-3988 (2003).</li><li>Ginestier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALDH1+Is+a+Marker+of+Normal+and+Malignant+Human+Mammary+Stem+Cells+and+a+Predictor+of+Poor+Clinical+Outcome.">ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome.</a> Cell stem cell. 1, 555-567 (2007).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+clonotypic+B+cells+in+classic+Hodgkin+lymphoma.">Circulating clonotypic B cells in classic Hodgkin lymphoma.</a> Blood. 113, 5920-5926 (2009).</li><li>Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+multiple+myeloma+progenitors,+stem+cell+properties,+and+drug+resistance.">Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance.</a> Cancer Res. 68, 190-197 (2008).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pancreatic+cancer+stem+cells.">Identification of pancreatic cancer stem cells.</a> Cancer Res. 67, 1030-1037 (2007).</li><li>Hermann, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+cancer+stem+cells+determine+tumor+growth+and+metastatic+activity+in+human+pancreatic+cancer.">Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.</a> Cell stem cell. 1, 313-323 (2007).</li><li>Rasheed, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+tumorigenic+cells+with+mesenchymal+features+in+pancreatic+adenocarcinoma.">Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.</a> J Natl Cancer Inst. 102, 340-351 (2010).</li><li>Rubio-Viqueira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+platform+for+translational+drug+development+in+pancreatic+cancer.">An in vivo platform for translational drug development in pancreatic cancer.</a> Clin Cancer Res. 12, 4652-4661 (2006).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+aldehyde+dehydrogenase+in+viable+cells.">Assessment of aldehyde dehydrogenase in viable cells.</a> Blood. 85, 2742-2746 (1995).</li></ol>

Este protocolo describe el aislamiento de células madre cancerosas del páncreas de xenoinjertos humanos. Varios pasos clave en este procedimiento mejorará el rendimiento de los viables CSC. La recuperación de una población pura de células madre cancerosas del páncreas depende de la pureza de las células viables presentes en la suspensión celular antes de la clasificación en el FACSAria. Asegurándose de que utilizan los xenoinjertos de menos de 1 cm de diámetro mayor ayuda a reducir la cantidad de tejido necrótico en el centro del tumor. Además, el agotamiento de la suspensión de células de restos de tejido y las células muertas durante la centrifugación en gradiente de Ficoll-Paque es crítica y puede repetirse si un gran número de restos de tejido o células no viables se detectan cuando las células se contó con el hemocitómetro. El protocolo de tinción descrito aquí utiliza el sistema de reactivos para detectar la actividad ALDEFLUOR ALDH, así como los anticuerpos conjugados con fluorocromo para detectar antígenos específicos de la superficie celular. Tinción ALDEFLUOR se realiza a 37 ° C y, por tanto, debe ser completado antes de la tinción de anticuerpos (sobre hielo) para evitar la posibilidad de limitación de anticuerpos, lo que puede ocurrir a 37 º C. Dado que las células pueden eflujo reactivo ALDEFLUOR, es importante para mantener las células en ALDEFLUOR buffer a 4 ° C después de la tinción ALDEFLUOR se ha completado. El buffer ALDEFLUOR contiene un inhibidor de flujo de salida de la droga que ayuda a mantener los niveles intracelulares de ALDEFLUOR. Dado que este método aísla viable páncreas CSC se pueden aplicar en una serie de experimentos que miden la función fisiológica de estas células. Hemos utilizado estas células para los ensayos de la iniciación del tumor, la utilización de ratones inmunodeprimidos y en ensayos de células in vitro de migración / invasión. Además, el ARN, el ADN y las proteínas se pueden obtener de estas células para la comparación de los estudios analíticos CSC y las poblaciones no-CSC.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM </td>
<td>&nbsp;</td>
<td>Invitrogen (Carlsbad, CA) </td>
<td>11965-118 </td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum </td>
<td>&nbsp;</td>
<td>Harlan (Indianapolis, IN)</td>
<td>BT-9501</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15140-122 </td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type IV </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>17104-019 </td>
<td>&nbsp;</td>
<tr>
<td>Dispase </td>
<td>&nbsp;</td>
<td>Sigma (St. Louis, MO) </td>
<td>D4818 </td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm culture dish </td>
<td>&nbsp;</td>
<td>BD Biosciences (San Jose, CA) </td>
<td>353003 </td>
<td>&nbsp;</td>
<tr>
<td>70-&mu;m filter </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352350 </td>
<td>&nbsp;</td>
<tr>
<td>50-mL conical tube </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352070 </td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque Plus </td>
<td>&nbsp;</td>
<td>GE Healthcare (Upsala, Sweden) </td>
<td>17-1440 </td>
<td>&nbsp;</td>
<tr>
<td>ALDEFLUOR reagent system </td>
<td>&nbsp;</td>
<td>Stem Cell Technologies (Vancouver, BC, Canada) </td>
<td>01700 </td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15250-061 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352054 </td>
<td>&nbsp;</td>
<tr>
<td>CD44-APC (clone G44-26) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>559942 </td>
<td>&nbsp;</td>
<tr>
<td>CD24-PE (clone ML5) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555428 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse CD31-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553371 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse lineage-biotin </td>
<td>&nbsp;</td>
<td>Miltenyi Biotec (Auburn, CA) </td>
<td>130-092-613 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse H-2Kd-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553564 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2b&#x03BA;-APC</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555745 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2a&#x03BA;-PE</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555574 </td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin-PerCP protein </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>554064 </td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4170 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes with strainer caps </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352235 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of stem cells from human pancreatic cancer xenografts

Las células madre del cáncer (CCC) se han identificado en un número cada vez mayor de enfermedades malignas y son funcionalmente definido por su capacidad para someterse a la auto-renovación y producir una progenie diferenciada. Estas propiedades permiten CSC para recapitular el tumor original cuando se inyectan en ratones inmunodeficientes. CSC dentro de un tumor maligno del epitelio fueron descritos por primera vez en el cáncer de mama y se encontró para mostrar la superficie específica de expresión de células antígeno (CD44 + CD24 baja / -) 2. Desde entonces, el CSC se han identificado en un número creciente de otras neoplasias humanas con CD44 y CD24, así como una serie de antígenos de superficie. Propiedades fisiológicas, incluyendo la aldehído deshidrogenasa (ALDH) la actividad, también se han utilizado para aislar células madre cancerosas en los tejidos malignos 3-5. Recientemente, y otros han identificado células madre cancerosas del adenocarcinoma de páncreas sobre la base de la actividad de la ALDH y la expresión de los antígenos de superficie celular CD44 y CD24, CD133 y 6-8. Estas poblaciones altamente tumorigénico pueden o no pueden superponerse y mostrar otras funciones. Hemos encontrado que ALDH + y CD44 + CD24 + páncreas CSC son igualmente tumorigénico, pero las células ALDH + son relativamente más invasiva 8. En este protocolo se describe un método para aislar células madre cancerosas del páncreas viable de xenoinjertos de bajo tránsito humano 9. Tumores transplantados son cosechados de los ratones y se convierte en una suspensión de una sola célula. Restos de tejido y las células muertas se separan de las células vivas y luego se tiñen con anticuerpos contra CD44 y CD24 y usando el reactivo ALDEFLUOR, un sustrato fluorescente de ALDH 10. CSC se aíslan por clasificación celular activado por fluorescencia. Aislado células madre cancerosas puede ser usado para los ensayos analíticos o funcionales que requieren las células viables.

Watch this Scientific Journal Video about Isolation of Stem Cells from Human Pancreatic Cancer Xenografts at JoVE.com

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Las células madre del cáncer (CCC) se han identificado en un número de tumores malignos. En este protocolo se describe un método de citometría de flujo utilizando la actividad de aldehído deshidrogenasa y la expresión de CD44 y CD24 para aislar células madre cancerosas de xenoinjertos de adenocarcinoma pancreático. Estas células viables se pueden utilizar en los estudios funcionales y análisis.

El aislamiento de células madre de cáncer de páncreas xenoinjertos

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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22.2K vistas.  Johns Hopkins University School of Medicine. Las células madre del cáncer (CCC) se han identificado en un número de tumores malignos. En este protocolo se describe un método de citometría de flujo utilizando la actividad de aldehído deshidrogenasa y la expresión de CD44 y CD24 para aislar células madre cancerosas de xenoinjertos de adenocarcinoma pancreático. Estas células viables se pueden utilizar en los estudios funcionales y análisis.

Video: Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Aislamiento y análisis de las células madre hematopoyéticas de la placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Investigación

Biología

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Aislamiento humanos islote pancreático: Parte I: La digestión y la acumulación de tejido pancreático

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selección y aislamiento de las colonias de humanos células madre pluripotentes inducidas a partir de fibroblastos adultos Reprogramado

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

El aislamiento de las células inmunes de tumores primarios

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Aislamiento Manual de células madre derivadas de tejido adiposo de Lipoaspirates Humanos

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...