This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.

1. Preparación del Sótano matriz de la membrana y de revestimiento de Material de plástico <ol><li> Coloque la matriz de la membrana basal madre congelada de -20 ° C en hielo y descongelar la noche a 4 ° C. </li><li> Después de la descongelación, la pipeta 2 mg alícuotas de matriz de membrana basal en tubos Eppendorf pre-refrigerados. Almacenar estos en -20 ° C hasta que se necesite. </li><li> Para preparar las placas de matriz recubierto de la membrana basal, descongelar una alícuota sobre hielo hasta el último trozo de hielo en la que desaparezca tubo Eppendorf (normalmente dentro de ~ 2 h). </li><li> Después de la descongelación, añadir matriz de membrana basal a 12 ml de frío (4 ° C) DMEM / F12 para obtener una solución de revestimiento de matriz de la membrana basal. </li><li> Añadir solución de matriz de membrana basal al recipiente de cultivo apropiado. Para una placa de 6 pocillos, prescindir de 1 ml por pocillo. Agite la placa para asegurarse de que la solución de la matriz de la membrana basal cubre completamente cada pocillo. </li><li> Parafilm sellar la membrana basal de la matriz recubierta de placa / placa, y se incuba a Room temperatura durante 1 hora antes de su uso. Alternativamente, las placas de matriz recubiertos tienda membrana basal / platos a 4 ° C y utilizar dentro de 2 semanas de recubrimiento. NOTA: Añadir DMEM extra / F12 a la membrana basal de la matriz recubierta de placa / placa para evitar el secado. Antes de usar 4 ° C membrana basal almacenado matriz recubiertos plato / plato, colóquelo en una cabina de seguridad biológica y deje que llegue a temperatura ambiente durante al menos 30 minutos. </li><li> Aspirar matriz de membrana basal completamente antes de la adición de células y medio. </li></ol> 2. Preparación del medio E8 <ol><li> Preparar medio de cultivo E8 descongelando suplemento E8 la noche a 4 ° C. </li><li> Extraiga 10 ml de medio basal E8 de la población de 500 ml y desechar. </li><li> Pipetear todo el vial de 10 ml de suplemento E8 directamente en 490 ml de medio basal E8. No entibie medio completo E8 en un baño de agua a 37 ° C, como las fluctuaciones de temperatura repetidos pueden degradar el bFGF en el complemedio te E8. </li><li> Utilice un medio completo E8 dentro de 2 semanas de la adición del suplemento. </li></ol> 3. La descongelación de iPSCs <ol><li> Retirar un vial de iPSCs congelados de nitrógeno líquido y colocar en hielo seco. </li><li> Rápidamente descongelar el vial en un baño de agua a 37 ° C; agitar el vial en el baño de agua hasta que sólo un pequeño fragmento de los restos de hielo. </li><li> Rocíe el vial con etanol y la transferencia del 70% de una cabina de seguridad biológica. </li><li> Añadir 1 ml de la temperatura ambiente E8 medio gota a gota directamente al vial. </li><li> Usando una pipeta de 2 ml, transferir la suspensión celular en gota a gota 9 ml de medio de E8 en un tubo cónico de 15 ml. Agitar el tubo de frecuencia para asegurarse de que las células y el medio se mezclan bien rápidamente. </li><li> Centrifugar las células a 200 xg durante 5 min. </li><li> Aspirar el sobrenadante y resuspender el sedimento celular en un volumen apropiado de E8 suplementado con 10 mM Y-27632. </li><li> Añadir las células a un número adecuado de sótano membrane matriz recubiertos pozos, y colóquelo en un 37 ° C, 5% de CO2 durante la noche. Se recomienda a la placa al menos 0,2 x 10 6 IPSC por pocillo de una placa de 6 pocillos para permitir una rápida recuperación después de la descongelación. </li></ol> 4. Mantenimiento y pases de rutina de iPSCs <ol><li> Actualizar medio E8 diaria. </li><li> Monitorear la morfología y la confluencia de las células con un microscopio invertido. iPSCs de alta calidad crecen en colonias planas con bordes bien definidos; colonias individuales poseen una apariencia &quot;empedrado&quot;. </li><li> Passage las células iPSCs cuando llegan a ~ 70% de confluencia. </li><li> Preparar una solución pases EDTA mediante la adición de 0,9 g de NaCl y 500 l 0,5 M EDTA a 500 ml de DPBS. Mezclar bien para disolver NaCl, y el filtro de vacío para esterilizar. Calentar una parte alícuota de la solución de los pases en un 37 ° C baño de agua antes de pases. </li><li> Para pasaje, aspirado pasó medio de cultivo y lavar las células una vez con un volumen igual de pas calientessaging solución. Aspirar, y pipetear suficiente de solución EDTA pases para recubrir las células (1 ml por pocillo de una placa de 6 pocillos). </li><li> Coloque las células bajo un microscopio invertido y observar las colonias de IPSC. La aparición de agujeros dentro de las colonias y bordes elevados debe ser evidentes dentro de 2 a 5 min. </li><li> Aspirar con cuidado la solución passaging EDTA. </li><li> Usando una pipeta de 10 ml, dispensar 4 ml de medio E8 (si se utiliza una placa de 6 pocillos) a alta presión directamente en cada pocillo para ser pasado. </li><li> Recoge los grupos IPSC, y dividido en un número apropiado de pocillos en función de la relación de división de 1: 8 a 1:12. No sobre-pipeta, como desagregación de los grupos de células que puede reducirse el viabilidad. </li><li> Colocar la placa en una incubadora, y el rock de la placa posterior y hacia adelante y de lado a lado varias veces para dispersar las células. </li></ol> 5. Preparación de MEFs y iPSCs para Tansfection <ol><li> 48 horas antes de la transfection, iPSCs paso a ~ 1: 6 en cuatro o más pocillos de una placa de 6 pocillos de la membrana basal de la matriz recubiertos, por lo que serán 70% confluentes aquí a dos días. </li><li> El día siguiente, se descongele DR4 MEFs en el medio de MEF que consiste en DMEM (alta glucosa) suplementado con 10% de FBS y 1x MEM-NEAA. </li><li> Placa DR4 MEFs en dos placas de 10 cm a ~ 2 x 10 4 células / cm 2 y se incuba durante la noche a 37 ° C. </li><li> En el día de la transfección, cambio de medio MEF a E8 suplementado con 10 mM Y-27632 30 min antes de realizar la transfección en células iPS. </li><li> Opcional: Si se desea análisis de citometría de flujo de iPSCs después de la transfección, quitar una placa de matriz recubiertos con membrana basal de 4 ° C y el lugar a temperatura ambiente. </li><li> Opcional: 4 h antes de la transfección, suplemento pre-transfección cultura IPSC con Y-27632 en la concentración final de 10 mM. </li></ol> 6.-La disociación celular Gentle reactivo de tratamiento unand La transfección de células iPS usando un sistema de electroporación <ol><li> Retire la solución de transfección de células primarias P3 de 4 ° C y deje que llegue a temperatura ambiente durante ~ 30 min. Añadir todo el suplemento de 100 l a la solución de transfección antes de su uso. </li><li> Calentamiento celular suave reactivo de disociación en un baño a 37 ° C. </li><li> Obtener AAVS1 Talens (pZT-AAVS1-L1 y pZT-AAVS1-R1) y AAVS1-CAG-EGFP donante de -20 ° C. </li><li> Retire iPSCs del incubador y lavar una vez con DPBS. </li><li> Añadir 1 ml de reactivo de disociación de células suave por pocillo, y se incuba iPSCs a 37 ° C durante 5 min, o hasta mayor que 50% de las células se han disociado del recipiente de cultivo. </li><li> Pipetear las células arriba y abajo un par de veces con una pipeta p1000 disociar las células restantes del recipiente de cultivo, y para romper los grumos IPSC. </li><li> Añadir 2 ml de medio E8 a cada pocillo, y pipeta hacia arriba y abajo varias veces con una pipeta de 10 ml a Fürther deshacer los agregados celulares en células individuales NOTA: La eficacia de transfección disminuye significativamente si los agregados celulares no son suficientemente desagregada. </li><li> Recoger iPSCs en un tubo cónico de 15 ml y centrifugar a 100 xg durante 3 min. </li><li> Aspirar el sobrenadante y volver a suspender las células en una cantidad mínima de medio de E8. </li><li> Contar las células utilizando un hemocitómetro después de la aplicación de un colorante vital, tales como 0,4% de azul de tripano. Asegúrese de que las células están suficientemente disociados mientras cuenta (1-3 células por &quot;grupo&quot;). </li><li> Prescindir de 3 x 10 6 células en cada uno de dos tubos de 15 ml cónicos, y girar de nuevo a 100 g durante 3 min. NOTA: centrifugación a baja velocidad reduce el estrés celular y permite un fácil resuspensión de iPSCs antes de la electroporación. </li><li> Ajuste el sistema de electroporación para el programa específico de tipo celular de la línea de células madre embrionarias humanas H9 (Programa CB-150). </li><li> Después de la centrifugación, aspirar el sobrenadante de la cell pellets. Para una pastilla, añadir 10 g de la donante de recursos humanos como muestra de control. Para el otro pellet añadir 10 g de la donante HR, junto con 5 g de cada uno TALEN (PZT-AAVS1-L1 y PZT-AAVS1-R1) como muestra experimental. </li><li> Resuspender cada sedimento celular en 100 l de solución de transfección de células primarias P3, y transferir a una cubeta. </li><li> Realice la transfección y añadir inmediatamente 500 l de medio E8 temperatura ambiente a cada cubeta. </li><li> Transferir el iPSCs gota a gota transfectadas a uno de 10 cm de plato que contiene MEFs DR4 preparados en la etapa 5.4. </li><li> Opcional: Añadir varias gotas de cada experimento en un pocillo de una placa de 6 pocillos de la membrana basal de la matriz recubierto si se desea citometría de flujo anaylsis de la eficiencia de la transfección. </li><li> Repita los pasos 06.15 a 06.17 para terminar ambas muestras. El día siguiente, lavar las células con DPBS dos veces y cambiar el medio de cultivo a NutriStem, que parece apoyar la cultura IPSC en alimentadores mejor que E8. </li><li> Trpicos de expresión EGFP ansient en de 48 a 72 horas después de la transfección; evaluar la eficiencia de transfección como se desee. </li></ol> 7. Puromicina Selección de iPSCs Targeted <ol><li> Comience selección puromicina 72 horas después de la transfección cuando las iPSCs alcanzan el 70% de confluencia. </li><li> Para NCRM5 iPSCs, comenzará la selección puromicina a 0,5 mg / ml de puromicina (1/2 de la dosis completa) diluido en medio de cultivo NutriStem. La concentración óptima puromicina puede variar desde 0,25 hasta 1 mg / mIL para algunas líneas de IPSC. </li><li> IPSCs Cultura en NutriStem + 0,5 mg / ml de puromicina durante 3 días, refrescantes del medio día. </li><li> Después de 3 días, aumentar la concentración de puromicina a 1 mg / ml. </li><li> IPSCs Cultura menores de 1 mg / ml puromycin selección durante otros 7 a 8 días, o hasta que las colonias distintas parecen lo suficientemente grande para picking colonia. </li></ol> 8. Colonia Picking y ampliación de iPS específicas <ol><li> Uso de la membrana basalmatriz para recubrir una placa de 96 pocillos mediante la supresión de 50 l de solución de revestimiento de matriz de la membrana basal (2 mg matriz de membrana basal diluido en 12 ml de DMEM / F12) en cada pocillo. Guarde la placa a 4 ° C durante la noche antes de su uso. </li><li> Después de permitir que la placa de matriz recubiertos con membrana basal para llegar a la temperatura ambiente, aspirar la matriz de la membrana basal y dispensar 100 l E8 suplementado con 10 mM Y-27632 en cada pocillo. </li><li> Publicar un microscopio invertido en una cabina de seguridad biológica, y rociar ligeramente con EtOH al 70% para esterilizar. </li><li> Saque una pipeta Pasteur de vidrio sobre un mechero Bunsen para obtener una herramienta de colonias de picking ideal que tiene un &quot;gancho&quot; minúscula en la punta. Esterilizar con EtOH al 70%, y el lugar en la campana para secar. </li><li> Retirar la cápsula que contiene colonias IPSC dirigidos de la incubadora, y colocar en el capó bajo el microscopio. </li><li> Escoja iPSCs trazando un círculo alrededor de la frontera colonia con la herramienta de colonias de picking.</li><li> Use el &quot;gancho&quot; de la herramienta de colonias de picking para raspar suavemente y quitar la colonia IPSC de la superficie de la placa. Para las colonias más grandes, dibujando una X con la herramienta de colonias de recoger a trimestre la colonia facilitará el crecimiento celular después de la re-enchapado. </li><li> Una vez que grupos de células se separan y libremente flotante, utilice una pipeta p200 ajustado a ~ 30 l para recoger las células iPS. Células de la placa directamente en la placa de 96 pocillos. </li><li> Opcional: utilizar una pipeta p20 ajustado a ~ 5 l para recoger las células iPS en un tubo Eppendorf, a continuación, añadir 50 l de reactivo de disociación de células suave de digerir durante 5 minutos a 37 ° C. Después de la digestión, añadir 500 l medio E8 en el tubo eppendorf para diluir el reactivo de disociación de células suave y centrifugar las células a 200 xg en una microcentrífuga de sobremesa estándar para 5 min. Entonces aspirar la mayor parte del medio y dejar ~ 30 l de volver a suspender y transferir las células en la placa de 96 pocillos. NOTA: Esteenfoque facilitará la disociación del grupo celular y la rápida expansión de la colonia recogido. </li><li> Continuar picking colonia hasta que se hayan recogido un número suficiente de colonias IPSC (más o menos se recomienda 20 a 30 colonias por experimento). </li><li> Después de la recolección de colonias, coloque la placa de 96 pocillos en una incubadora durante la noche. Actualizar con medio completo E8 el día siguiente, y la cultura hasta ~ 70% de confluencia. </li><li> Células pasaje de los 96 pozos en un 24 y, a continuación, en un 6 pocillos, como se describe en los pasos 04.05 a 04.10. NOTA: los volúmenes de solución de EDTA y medio E8 deben reducirse proporcionalmente cuando se utiliza pozos más pequeños que una placa de 6 pocillos. </li><li> Evaluar la orientación éxito por la unión de PCR y análisis de transferencia de Southern para confirmar la integración casete específica y ausencia de vectores insertados al azar 16. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

The most critical steps for the successful generation of AAVS1 safe-harbor targeted human iPSCs are: (1) efficiently delivering TALEN and donor plasmids into iPSCs by transfection; (2) optimizing dissociation of iPSCs into single cells before transfection and plating density after transfection; (3) optimizing dose and time of drug-selection based on the growth of iPSC line; (4) carefully dissecting and picking targeted colonies and transferring to new plate/well. Compared to similar methods used in Hockemeyer&#8217;s pap...

La capacidad de reprogramar células somáticas humanas en células madre pluripotentes inducidas similares a células madre embrionarias (células iPS) fue descubierto por primera vez por Takahashi et al. En 2007 1. Fibroblastos dérmicos humanos transducidas con retrovirus que expresa cuatro factores de transcripción (El Yamanaka así apodado-Factores Oct3 / 4, Sox2, se muestra c-Myc, y Klf4) para ser muy similares a las células madre embrionarias humanas (hESCs) basándose en la morfología, proliferación, expresión génica, y el estado epigenética; crucialmente, iPSCs también son capaces de diferenciarse en células de las tres capas germinales 1. La capacidad potencial y la diferenciación de proliferación de células iPS los hace herramientas muy atractivos; mediante la reprogramación de células de pacientes que sufren de enfermedades específicas, iPSCs se pueden utilizar tanto como sistemas modelo in vitro de la enfermedad y como agentes terapéuticos potenciales. Para este último propósito, varios asuntos deben ser abordados antes de que el pleno potencial de iPSCsen un entorno clínico puede realizarse; el potencial tumorigénico de hESCs cultivadas in vitro y CMPI, el uso de derivados xenogénicos durante la reprogramación celular y el mantenimiento, y la necesidad de realizar un seguimiento de las células trasplantadas in vivo son todos los obstáculos cruciales para la aplicación clínica de células madre pluripotentes (Revisado por Hentze et al. 2). Una solución ideal a la necesidad para el seguimiento de las células diferenciadas post-trasplante implicaría un marcador visualmente detectable que se resiste a silenciar y variegación independientemente de la aplicación. Expresión robusta y sostenida de transgenes integrados es más fácilmente alcanzable cuando se introduce ADN exógeno en loci puerto seguro; es decir, sitios genómicos que permiten la transcripción suficiente de un vector integrado, mientras que al mismo tiempo mitigar perturbaciones de expresión en genes vecinos 3. Uno de esos sitios que ha sido muy bien caracterizado desde su descubrimiento es la integr virus adeno-asociadoEl sitio ación 1 (AAVS1), en el primer intrón de la 1 12C subunidad reguladora de genes (PPP1R12C) la proteína fosfatasa. Este locus ha sido demostrado no sólo para permitir la expresión robusta de transgenes integrados a través del tiempo extendido en la cultura y en la diferenciación in vitro sostenido y 3, sino también para proteger rodea genes de perturbación transcripcional 4; ambas características se cree que es debido a la presencia de elementos aislantes de la cromatina endógenos que flanquean el sitio AAVS1 5. Los avances en las herramientas de ingeniería del genoma más sólo la última década han facilitado en gran medida la facilidad y eficiencia con que las manipulaciones genéticas en cualquier tipo de célula se puede lograr. Mientras que los experimentos exitosos primeros basaron en extremadamente bajos niveles de recombinación homóloga endógena (HR) con un donante introducido para lograr la orientación de genes en los CES 6,7, el uso de nucleasas específicas de sitio, tales como nucleasas de dedos de zinc (ZFNs), que signinificativamente inducir la recombinación homóloga a través de la generación de una rotura del DNA de doble cadena ha aumentado en gran medida la eficiencia de tales experimentos 8,9. La reutilización de ambos tipo activador de transcripción efectores (cuentos) de planta patógeno Xanthomonas géneros y los agrupados interspaced regularmente repeticiones palindrómicas cortas procariotas (CRISPR) / sistema Cas9 en nucleasas de diseño específicos del sitio eficientes ha hecho la orientación de genes en células madre pluripotentes un accesible y metodología practicable 10-13. Un reciente documento describe un método eficaz para la integración estable de un reportero de cassette verde fluorescente en el AAVS1 locus puerto seguro en iPS humanas utilizando nucleasas TALE (Talens) 14. Estos iPSCs dirigidos mantuvieron su fluorescencia incluso después de la diferenciación dirigida a cardiomiocitos y trasplante en un modelo de ratón de infarto de miocardio (MI), proporcionando una fuerte evidencia de la utilidad de talesestablemente células madre pluripotentes fluorescente 14. Para obtener colonias específicas, se utilizó un método para atrapar genes en el que un corte y empalme-aceptor (SA), secuencia de péptido autoescindible 2A coloca el puromicina N-acetil-transferasa gen (PAC) bajo el control del promotor endógeno PPP1R12C; Por lo tanto, sólo iPSCs que han incorporado el donante de ADN en el locus AAVS1 expresan PAC, haciéndolos seleccionable basado en puromicina-resistencia; (Figura 1, 15). Este protocolo detalla los procedimientos de generación de AAVS1-GFP iPSCs informó en el documento reciente 14, incluido el proceso de transfección de células iPS con Talens y un donante de 9,8 kb para integrar un fragmento de ADN de 4,2 kb en el locus AAVS1 puerto seguro, la selección de iPSCs basados ​​en puromicina-resistencia, y las colonias de la cosecha por la expansión clonal. Las técnicas descritas en el presente documento pueden aplicarse a muchos experimentos de ingeniería del genoma.

<table border="0" cellpadding="0" cellspacing="0" width="1437">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:588px;">NAME OF MATERIAL/EQUIPMENT</td>
			<td style="width:123px;">COMPANY</td>
			<td style="width:123px;">CATALOG #</td>
			<td style="width:603px;">COMMENTS/DESCRIPTION</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Essential 8 Medium</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td>Store basal medium at 4&#176;C. Store supplement at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Store at room temp. Once dissolved in H2O, store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DR4 MEF 2M IRR - Academic</td>
			<td>GlobalStem</td>
			<td>GSC-6204G</td>
			<td>Store in liquid Nitrogen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-040</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined Fetal Bovine Serum, US Origin</td>
			<td>HyClone</td>
			<td>SH30070.03</td>
			<td>Store at -20&#176;C. Thaw at 4&#176;C overnight and aliquot. Store aliquots at -20&#176;C until needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector Core unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001B</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector X unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001X</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3024</td>
			<td>Upon arrival, remove Primary Cell Solution and supplement and store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C and warm an aliquot in a 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NutriStem XF/FF Culture Medium</td>
			<td>Stemgent</td>
			<td>01-0005</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td>
			<td>Addgene</td>
			<td>52637 and 52638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1-CAG-EGFP Homologous Recombination donor</td>
			<td>Addgene</td>
			<td>22212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Store at -20&#176;C. Prepare working aliquots of 1 mg/mL in ddH2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend XTR (Refrigerated), 120V 60Hz</td>
			<td>Thermo Scientific</td>
			<td>75-004-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TX-750 4 &#215; 750mL Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

Una visualización del protocolo se proporciona en la Figura 2, con períodos durante los cuales iPSCs se cultivan en medio diferente puesto de manifiesto por cualquiera de verde para E8 o azul para NutriStem. Es importante para transfectar sólo iPSCs de alta calidad; examinar placas de cultivo de todo el mantenimiento de rutina y verificar que las culturas IPSC contienen colonias principalmente distintas que llevan una morfología de adoquines como (Figura 3A); células diferenciadas ...

Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

La transfección, selección y Colonia-cosecha de células madre humanas pluripotentes inducidas TALEN dirigidos con un gen GFP en el puerto seguro AAVS1

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

transfection-selection-colony-picking-human-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

19.3K vistas.  National Institutes of Health. TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Video: Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

La cuantificación tridimensional de espinas dendríticas de piramidal neuronas derivadas de células madre humanas pluripotentes inducidas

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Investigación

Biología del Desarrollo

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Cultivar las células primarias epiteliales nasales de niños y reprogramar en células madre pluripotentes inducidas

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

Diferenciación in vitro de células madre pluripotentes humana en células trofoblásticas

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Protocolo para la diferenciación de células madre pluripotentes inducidas por humanos en cultivos mixtos de neuronas y glia para pruebas de neurotoxicidad

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Diferenciación rápida y dirigida de las células epiteliales del pigmento retiniano de humanos embrionarios o inducidas pluripotenciales células

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

Usando humanos inducida por células de hepatocito-como derivado de células madre pluripotentes de descubrimiento de fármacos

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Un modelo de ratón quimérico hígado humano de hipercolesterolemia familiar con hepatocitos derivados de células madre pluripotentes inducidas

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Basado en el ARN la reprogramación de fibroblastos primarios de humanos en células madre pluripotentes inducidas

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

Diferenciación eficiente de células madre pluripotentes humanas en células hepáticas

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generación de 3D piel organoide de cable derivadas de sangre inducida por las células de vástago Pluripotent

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...