Los autores desean agradecer a las siguientes subvenciones del NIH para el apoyo a esta investigación: K08DK082783 (AR), P30DK56465 (BTS), U01HL099993 (BTS), T32HL00715036 (SKS), y K12HL0806406 (SKS); y la Fundación JP McCarthy (AR).

Consentimiento informado por escrito, según lo aprobado por las juntas de revisión institucional del Centro de Investigación del Cáncer Fred Hutchinson y los niños &quot;s Hospital of Philadelphia, se obtuvo de los pacientes antes de la recogida de muestras de sangre periférica. Se observaron Todas las directrices institucionales. Todos los experimentos con animales, incluyendo la generación de MEF y formación de teratomas fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional. 1. Ficoll separación de PBMC y Expansión de Eritroblastos - Día 0 <ol><li> Diluir la sangre periférica 1: 1 con tampón fosfato salino de Dulbecco (DPBS). En un tubo de poliestireno de fondo redondo de 15 ml, la capa cuidadosamente 7 ml de sangre diluida sobre 3 ml de temperatura ambiente Ficoll-Hypaque. </li><li> Centrifugar la muestra a 400 xg durante 30 min. </li><li> Las PBMCs se han asentado a la interfaz entre el plasma y el Ficoll-Hypaque, visto como una capa blanca turbia. Con una pipeta de transferencia estéril, recoger el PBMCs en uno 15 ml tubo cónico. Llevar el volumen a 10 ml usando DPBS. </li><li> El uso de un hemocitómetro, contar las CMSP. </li><li> Pipetear 2 × 10 6 PBMCs en un tubo cónico de 15 ml separado y se centrifuga a 300 xg durante 5 min. Centrifugar las células restantes y congelar a una concentración de 2 × 10 6 PBMC / ml en suero bovino fetal al 90% (FBS) sulfóxido / 10% de dimetilo (DMSO). </li><li> Preparar Media Expansión: medio libre de suero QBSF-60 (proteger de la luz) + penicilina / estreptomicina (1%) + ácido ascórbico (50 ng / ml) + factor de células madre (SCF, 50 ng / ml) + interleucina 3 (IL- 3, 10 ng / ml) + eritropoyetina (EPO, 2 U / ml) + insulina como factor de crecimiento 1 (IGF-1, 40 ng / ml) + dexametasona (1 M; protegerlo de la luz; descongelar una alícuota fresca cada medio cambiar). </li><li> Resuspender 2 × 10 6 PBMC en 2 ml de medio de expansión recién hecho y el lugar en un pocillo de una placa de cultivo de tejidos de 12 pocillos y se incuban en un incubador humidificado 37 ° con una atmósfera de 5% O 2, 5% de CO 2, y 90% de N 2. </li><li> En el día 3 y el día 6, sustituir los medios de comunicación. <ol><li> Recoger las células y lavar el pocillo con 2 ml de QBSF-60 para recoger las células restantes. </li><li> Centrifugar la suspensión de células a 300 xg durante 5 min y aspirar el sobrenadante. </li><li> Resuspender las células en 2 ml de nuevo medio de expansión y devolverlos a la misma placa de 12 pocillos. </li></ol></li></ol> 2. Las células reprogramación por Nucleofection - Día 9 <ol><li> Pipetear los plásmidos de reprogramación en un tubo Eppendorf estéril de 1,5 ml (Tabla 1). </li><li> Mezclar la solución con nucleofection Suplemento (suministrado por el fabricante) y pipeta en un tubo Eppendorf de 1,5 ml estéril. </li><li> Pipetear 2 ml de Media Expansión en un pocillo de una placa de 12 y colocarla en un humidificado 37 ° C incubadora (5% O 2, 5% de CO 2, y 90% N 2), para el equilibrado antes de añadir las células. </li><li> Recoger elcélulas cultivadas y lavar el pocillo con 2 ml de QBSF-60 para recoger las células restantes. </li><li> Centrifugar las células a 300 xg durante 5 minutos y aspirar el sobrenadante. </li><li> Lavar las células mediante resuspensión en 5 ml de DPBS y centrifugando a 300 xg durante 5 min. </li><li> Aspirar el sobrenadante. </li><li> Resuspender el sedimento celular en 100 l de solución de nucleofection + Suplemento, teniendo cuidado de evitar la generación de burbujas. </li><li> Pipetear la suspensión de células en el tubo que contiene los plásmidos reprogramación, pipeta arriba y hacia abajo una vez, y pipetear la muestra en la parte inferior de la cubeta nucleofection. </li><li> Colocar la cubeta en la máquina nucleofection y ejecutar el Programa T-019. </li><li> Transferencia de 100 l de la expansión precalentado mediano (de la placa equilibrante en el Paso 2.3) en la cubeta nucleofection. </li><li> Inmediatamente recoger toda la muestra y el depósito en el pozo de pre-calentado medio de expansión. Evite recoger el blanco, flotando los restos celulares muertos. </li&gt; <li> Colocar la placa en un humidificado 37 ° C incubadora (5% O 2, 5% de CO 2, y 90% N 2) para el crecimiento. </li></ol> 3. Revestimiento células alimentadoras - Día 10 o 11 <ol><li> El día 10, las células alimentadoras placa. <ol><li> Escudo uno de 6 pocillos placa de cultivo de tejidos con gelatina. <ol><li> Pipetear 1 m de 0,1% de gelatina en solución salina tamponada de Hank (HBSS) en cada pocillo de la placa. </li><li> Colocar la placa en un humidificado incubadora a 37ºC durante al menos 5 min. </li><li> Inmediatamente antes de su uso, aspirar la solución de gelatina de la placa. </li></ol></li><li> Descongelar un vial de 2 × 10 6 fibroblastos embrionarios de ratón irradiados a 3000 cGy en 10 ml de precalentado MEF Medios (Medio Eagle Modificado de Dulbecco (DMEM) + FBS (10%) + penicilina / estreptomicina (1%)). </li><li> Centrifugar las células a 300 xg durante 5 minutos y aspirar el sobrenadante. Resuspender las células en 12 ml de MEF Medios y pipeta de 2 mlnto cada pocillo de la placa de 6 así revestida con gelatina. Colocar la placa en un humidificado 37 ° C incubadora (5% O 2, 5% de CO 2, y 90% N 2) hasta el día 12. </li></ol></li></ol> 4. Revestimiento células en células alimentadoras y Cambio Medio - Día 12 <ol><li> Recoger las células nucleofected en uno 15 ml tubo cónico y recoger cualquier resto de las células por lavado de las bien con 2 ml de QBSF-60. Centrifugar la muestra a 300 xg durante 5 min. </li><li> Preparar reprogramación Medio: Iscove medio de Dulbecco modificado (IMDM) + FBS (10%) + no animal L-glutamina (1 mM) + no esenciales aminoácidos (NEAA, 1%) + penicilina / estreptomicina (1%) + β mercaptoetanol (0,1 mM) + factor de crecimiento de fibroblastos básico (bFGF, añaden fresco en la alimentación, 10 ng / ml) + ácido ascórbico (añadido en fresco de alimentación, 50 g / ml). </li><li> Aspirar el sobrenadante y resuspender las células en 12 ml de Medio de reprogramación. De pipeta 2 ml por pocillo de la suspensión celular enla placa de 6 pocillos de células de alimentación (como se ha preparado en la Sección 3). Centrifugar la placa a 50 xg durante 30 min a temperatura ambiente. </li><li> Colocar la placa en un humidificado 37 ° C incubadora (5% O 2, 5% de CO 2, y 90% N 2) para el crecimiento. Cambie la Reprogramación Medio cada dos días hasta que las colonias IPSC comienzan a aparecer (1-2 semanas). </li><li> Preparar hESC Medio: DMEM / F12 1: 1 + Knockout suero sustitución (20%) + penicilina / estreptomicina (1%) + NEAA (1%) + no animal L-glutamina (1 mM) + piruvato de sodio (1%) + bicarbonato de sodio (0,12%) + β-mercaptoetanol (0,1 mM) + bFGF (añadidos fresco en la alimentación, 10 ng / ml) </li><li> Prepare los inhibidores de HDAC: butirato sódico (añadido a la alimentación fresca, 100 M), ácido suberanilohydroxamic (SAHA, añadió fresca en la alimentación, 200 nM). Una vez que aparecen colonias IPSC, comienzan a alimentarse de las células con inhibidores de células madre Mediana + HDAC, el cambio de medio cada día. NOTA: butirato de sodio es una pequeña molécula que se adsorbe a tubos de plástico, tor lo tanto guárdelo en un tubo de vidrio de borosilicato a 4 ° C. </li></ol> 5. Aislamiento de clones IPSC <ol><li> Una vez que las colonias IPSC son lo suficientemente grandes como para ser aislado (20-25 células), ellos recoger con una pipeta P20. </li><li> Preparar los inhibidores de HDAC: butirato de sodio (añadido en fresco de alimentación, 100 mM), SAHA (añadido en fresco de alimentación, 200 nM) </li><li> Coloque las colonias aisladas en iMEFs en platos individuales de 35 mm que contienen inhibidores de la HDAC hESC Medio + + La clonación y recuperación del Suplemento. </li><li> El día después de recoger las colonias, cambie el medio a los inhibidores de células madre Mediana + HDAC. </li><li> Cambie el medio todos los días hasta que las colonias IPSC están listos para los pases. </li><li> Eliminar los inhibidores de HDAC del medio de colonias una vez estabilizan en el paso 2-3. </li></ol>

<ol>
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Los autores declaran que no tienen intereses financieros en competencia.

Para exitosa generación de IPSC cuando se utiliza este protocolo, hay varias advertencias importantes que deben ser considerados. Durante la fase de expansión eritroblasto, el programa de cambio de los medios de comunicación debe ser estrictamente seguida, como desviaciones pueden conducir a la estimulación ineficiente de la población de células progenitoras diana y una menor eficiencia de la generación de IPSC. Es importante hacer nuevo medio de expansión con dexametasona fresca con cada cambio de los medios de...

iPSCs se derivan de tejidos somáticos a través de la expresión ectópica de un conjunto mínimo de genes pluripotencia. Esta técnica se demostró inicialmente por la transducción retroviral de los fibroblastos humanos con Oct4, Sox2, Klf4, y cMYC, que son altamente expresado en el estado pluripotente 1. Estos transitoriamente expresadas &quot;factores de reprogramación&quot; alteran el perfil de expresión génica y epigenética paisaje de la célula diana análoga a las células madre de embriones humanos 2. Una vez creado, iPSCs potencialmente puede diferenciarse en cualquier tipo de tejido para análisis adicionales. Por lo tanto, mantienen la promesa para el uso en la medicina regenerativa, el modelado de la enfermedad, y aplicaciones de terapia génica. Sin embargo, lo que altera el genoma con virus que se integran tiene el potencial de alterar la expresión del gen endógeno, la influencia fenotipo celular, y en última instancia sesgan los resultados científicos. Además, integraciones virales aleatorias pueden conducir a celular e deletéreofectos, incluyendo la posibilidad de la transformación maligna 3 o re-expresión de los transgenes oncogénicos 4. Aplicaciones clínicas futuras requerirán-integración no generación de IPSC. Episomas, que son moléculas de ADN circular cromosómicas adicionales, ofrecen una estrategia para generar, CMPI-integración libre de costo efectivo 5. La combinación de vectores episomales que se muestran en la Tabla 1 expresa la reprogramación factores de Oct4, Sox2, Klf4, MYCL, y LIN28A. El plásmido pCXLE_hOCT3 / 4-F-shp53 también contiene una p53 shRNA para la supresión temporal de TP53 para mejorar la reprogramación celular 6. El vector deficiente pCXWB-EBNA1 replicación promueve la amplificación de factores de reprogramación y el aumento de la eficiencia de reprogramación proporcionando un aumento transitorio en la expresión de EBNA1 7. El plásmido pCXLE_EGFP se puede añadir a la mezcla de nucleofection con el propósito de determining la eficacia de transfección o para aplicaciones de células de clasificación. Con la excepción de p CXWB-EBNA1, los plásmidos episomales utilizados en este protocolo contienen el origen del virus de Epstein-Barr de la replicación viral y el gen EBNA1, que median la replicación y la partición del episoma durante la división de la célula huésped 8. Los episomas se pierden espontáneamente con la expansión sucesiva IPSC 7. Subclonación y caracterización de iPSCs con pérdida vector episomal, que se puede inferir a partir de la pérdida de la expresión de eGFP, pueden conducir a la integración completamente iPSCs gratuitas para futuras aplicaciones clínicas. Inherente al proceso de generación de IPSC es la supresión de genes específicos de linaje y la reactivación de los genes de pluripotencia asociada. Regulación de la expresión del gen se produce en múltiples niveles dentro del núcleo, incluyendo modificaciones en el ADN y la cromatina para permitir que factores de transcripción, elementos de ADN reguladores, y el acceso de la ARN polimerasa para orientargenes. Remodelación del paisaje epigenético de la cromatina a través de modificaciones globales es un componente clave para la re-expresión del programa genético pluripotencia. Una modificación de la cromatina específica que es importante en la regulación de la expresión génica es la acetilación de las histonas en particular los residuos de lisina, que permite el acceso a genes diana a través de disminución de la tensión de la bobina de ADN-histona. Inhibidores de HDAC son pequeñas moléculas que se han demostrado para mejorar IPSC reprogramación y células madre auto-renovación 9,10, probablemente debido al apoyo del estado acetilado 11. El protocolo se describe a continuación, adaptada de una publicación anterior utilizando la integración de los lentivirus 12, proporciona un método paso a paso para la generación de IPSC optimizado a partir de sangre periférica utilizando episomas y los inhibidores de HDAC. Las concentraciones de inhibidor de HDAC utilizados aquí son la mitad de los descritos por Ware, y otros han llevado habitualmente a un aumento de 2 veces en colonias IPSC totalmente reprogramadas más estándar. 9, yprotocolos de reprogramación episomales sin inhibidores de HDAC. Este nivel de reprogramación es a la par con la eficiencia que observamos con métodos de lentivirus. El uso de este protocolo, se han generado de manera eficiente IPSC de individuos como viejos como 87 años de edad.

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

efficient ips cell generation from blood using episomes and hdac inhibitors

Este manuscrito ilustra un protocolo para la creación de células de manera eficiente a la integración libre inducidos por el hombre madre pluripotentes (iPSCs) a partir de sangre periférica utilizando plásmidos episomales e inhibidores de la histona deacetilasa (HDAC). Las ventajas de este enfoque incluyen: (1) el uso de una cantidad mínima de sangre periférica como un material de origen; (2) nonintegrating vectores de reprogramación; (3) un método rentable para la generación de vectores iPSCs libres; (4) una sola transfección; y (5) el uso de pequeñas moléculas para facilitar la reprogramación epigenética. Brevemente, las células mononucleares de sangre periférica (PBMCs) se aislaron a partir de muestras de flebotomía de rutina y después se cultivaron en factores de crecimiento definidas para producir una población de células progenitoras de eritrocitos altamente proliferativa que es notablemente susceptibles de reprogramación. Nonintegrating, plásmidos episomales no transmisibles que expresan Oct4, Sox2, KLF4, MYCL, LIN28A, y un corto horquilla (sh) ARN p53 son introduced en los eritroblastos derivados través de un único nucleofection. La cotransfección de un episoma que expresa la proteína fluorescente verde mejorada (EGFP) permite una fácil identificación de las células transfectadas. Un plásmido de replicación deficiente separada expresar Epstein-Barr antígeno nuclear 1 (EBNA1) también se añade a la mezcla de reacción para aumentar la expresión de proteínas episomales. Las células transfectadas se sembraron sobre una capa de fibroblastos de embriones de ratón irradiados (iMEFs) para continuar la reprogramación. Tan pronto como colonias IPSC-como aparecen en aproximadamente doce días después de nucleofection, se añaden inhibidores de HDAC al medio para facilitar la remodelación epigenética. Hemos encontrado que la inclusión de los inhibidores de HDAC aumenta habitualmente la generación de colonias IPSC totalmente reprogramadas por 2 veces. Una vez que las colonias IPSC presentan la morfología típica de células madre embrionarias humanas (CMEH), se transfieren suavemente para placas de cultivo individuales recubiertos de tejido IMEF para el continuo crecimiento y expansión.

Tres días después de nucleofection y de las placas, las células nucleofected en iMEFs, la eficiencia de nucleofection exitoso debe ser estimado por microscopía de fluorescencia para eGFP. La Figura 1 muestra un experimento de nucleofection típico con aproximadamente 5-10% de la población total de células que expresan eGFP. Colonias reprogramado IPSC comenzarán a aparecer aproximadamente dos semanas después de nucleofection. Las colonias son generalmente circular con...

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

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Aquí se describe un protocolo para la generación de células madre pluripotentes inducidas humanas a partir de sangre periférica mediante una estrategia basada en la reprogramación episoma y los inhibidores de la histona deacetilasa.

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 ...

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

Research

JoVE Journal

Biology

12.7K vistas.  Fred Hutchinson Cancer Research Center.  Aquí se describe un protocolo para la generación de células madre pluripotentes inducidas humanas a partir de sangre periférica mediante una estrategia basada en la reprogramación episoma y los inhibidores de la histona deacetilasa. 

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

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

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

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

Extracción de sangre del cangrejo herradura de América, Limulus polyphemus

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

Investigación

Biología

Generation of Single-Cell Suspensions from Mouse Neural Tissue

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

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

Generación de un solo suspensiones de células de tejido de ratón Neural

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

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

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

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

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

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

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Generación de las células madre pluripotentes inducidas a partir de sangre periférica mediante el STEMCCA vector lentiviral

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Generación rápida y eficiente de las neuronas a partir de células madre pluripotentes en un formato de placa Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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

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

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

Evaluación de Generación de la Fuerza isométrica y excéntrica de los músculos esqueléticos Aislados de modelos murinos de Distrofias Musculares

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

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

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

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

Rápido y Eficiente de pez cebra Genotipado Utilizando PCR con análisis del derretimiento de alta resolución

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

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

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

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

Aislamiento de Vaso sanguíneo derivado Multipotentes Precursores de músculo esquelético humano

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

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente generación, por actividad humana células madre pluripotentes de células somáticas humanas con virus Sendai-

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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

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

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

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, ...