This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

Basada en células Individual tipo no colonia monocapa (NCM) cultura de hPSCs. 1. Preparación <ol><li> Hacer 500 ml de medio de cultivo de fibroblastos embrionarios de ratón (MEFs): medio DMEM suplementado con 10% de FBS, 2 mM de L-glutamina, y 0,1 mM de aminoácidos no esenciales (NEAA). </li><li> Aislar fibroblastos embrionarios de ratón (MEFs) células derivadas de la cepa CF1 siguiendo un protocolo de rutina 16 y la cultura MEFs en 0,1% 6-así placa de cultivo celular recubierto con gelatina en medio DMEM. Alternativamente, comprar acciones del MEF en el paso 3 de los recursos comerciales. </li><li> Preparar placas Matrigel. <ol><li> Diluir 5 ml de células madre calificado Matrigel acción con 5 ml de medio DMEM/F12 (refrigerado a ~ 4 ° C) y la tienda de un 50% de alícuotas en un -20 ° C congelador. Descongelar la Matrigel de stock congelado en un refrigerador (~ 4 ° C) O / N y más diluir el Matrigel en medio DMEM/F12 frío (para dar lugar a una concentración de trabajo 2,5%). </li><li&gt; Coat 6-y placas de cultivo celular con 1,5 ml de 2,5% de Matrigel por pocillo, almacenar las placas recubiertas en el refrigerador O / N (Nota: utilizar la placa recubierta con Matrigel dentro de 2 semanas). </li><li> Retire la placa de Matrigel del refrigerador, deje que la placa se caliente a temperatura ambiente en la campana de cultivo de células de 10 a 30 min (Nota: no calentar la placa en una incubadora de cultivo celular), y aspirar DMEM medio antes de chapado hPSCs. </li></ol></li><li> Hacer 500 ml de medio de células madre: 80% medio DMEM/F12, 20% KSR, 2 mM de L-glutamina, 0,1 mM de aminoácidos no esenciales, 0,1 mM de β-mercaptoetanol, y 4 ng / ml de FGF-2. </li><li> Preparar 10 ml de 2X HPSC medio de congelación: 60% de SFB, 20% de DMSO y 20 M Y-27632 en medio mTeSR1, esterilizar por filtración, y utilizar el medio plazo de 1 semana. </li></ol> . 2 Protocolo 1 (Básico): Crecen HPSC Colonias en Alimentadores <ol><li> Use los números de paso del MEF a las 5 o 6 (designado como P5 y P6) para la cultura HPSC el fin de obtener resul consistentets. Mitóticamente inactivar MEFs mediante el tratamiento de las células con 10 g / ml de mitomicina C durante 3 horas a 37 ° C, se lavan las células 3 veces con fosfato solución salina tamponada con 1X de Dulbecco (D-PBS), y a continuación se disocian los MEFs con 0,05% de tripsina en 0,53 mM EDTA. Alternativamente, irradiar MEFs a una dosis de 8.000 rads con un irradiador de rayos X. </li><li> Contar el número de células usando el método de exclusión con tinción de azul de tripano bajo el microscopio. Como alternativa, utilice un contador de células automático. </li><li> Placa MEFs irradiadas en placas de poliestireno de 6 pocillos recubiertas con 0,1% de gelatina a una densidad de 1,88 x 10 5 células por pocillo (es decir, 1,96 x 10 4 células / cm 2). Se incuban las células a 37 ° C y 5% de CO 2 durante 24 horas. </li><li> Retire el medio de cultivo MEF mediante aspiración con una pipeta Pasteur (Nota: no hay pasos de lavado necesarios en este momento). </li><li> Colonias Triturar HPSC de cultivo previo en pequeños grupos, examinan los grumos con el microscopio para asegurar que sus tamaños que van from 50 a 100 micras de diámetro, y la placa grumos HPSC en la parte superior de las capas alimentadoras MEF en un pocillo que contiene 2 ml de medio HPSC. </li><li> Cambie hESC medio día durante 3 a 5 días, récord de crecimiento de colonias por fotografía, marcar cualquier colonias morfológicamente alterados o diferenciados (~ 5%), y quitar manualmente las colonias marcadas por aspiración suave con una pipeta Pasteur. </li><li> Enjuague las colonias restantes en MEFs dos veces (2 min cada uno con D-PBS), e incubar las colonias con 2 ml de 1 mg / ml de colagenasa IV en medio HPSC durante 10 a 30 min), y examinar el desprendimiento de colonias HPSC de la superficie recubierta-MEF (Nota: use un raspador para ayudar al proceso de desprendimiento sólo si las colonias están estrechamente unidos a la placa). </li><li> Añadir 5 ml de medio de HPSC a cada pocillo para minimizar las reacciones enzimáticas, las colonias de la transferencia a un tubo de 15 ml, permitir colonias HPSC sedimentar durante 3 a 5 min a TA, y garantizar la sedimentación de colonias por visualización directa de la Cells en la parte inferior del tubo (Nota: No centrifugue el tubo en este paso). </li><li> Retire los sobrenadantes que contienen MEFs residuales y disociada células individuales, volver a suspender las colonias con 5 ml de medio de células madre, y repetir la etapa de sedimentación en dos ocasiones. </li><li> Retire las medianas, las colonias se tritura HPSC en pequeños grupos, tienda en 1X HPSC medio de congelación (o medio de congelación CryoStore CS10) para la crioconservación (1 confluentes bien por vial congelado) o la placa en una placa de 6 pocillos de pases celulares. </li></ol> 3 Protocolo 2:. Convertir HPSC Colonias de Comederos para NCM <ol><li> Enjuague pellets de HPSC en D-PBS una vez, incubar los gránulos con 1 ml de 1X Accutase durante 10 min, y examinar la reacción enzimática bajo un microscopio para asegurarse de disociación de una sola célula. </li><li> Terminar las reacciones enzimáticas resuspendiendo suavemente las células en 5 ml de medio mTeSR1 que contiene 10 mM de Y-27632 seguido por centrifugación a 200 x g a temperatura ambiente durante 5 min(Nota, no utilice fuerzas de centrifugación excesivas que causan daño celular). </li><li> Añadir 5 ml de medio mTeSR1 al sedimento, resuspender el precipitado como células individuales, y el filtro se disoció las células a través de un filtro de células de 40 m (para eliminar cualquier agregados celulares residuales). </li><li> Semillas 1,3-2 x 10 6 hPSCs en un pocillo (1.4 a 2.1 x 10 5 células / cm 2) de una placa de 6 pocillos recubiertos con el 2,5%-hESC calificado Matrigel, añadir medio mTeSR1 hasta 2,5 ml, y se compone de 10 M Y-27632 en el medio (para facilitar la 24 hr placa inicial de una sola célula). Como alternativa, utilice los siguientes moléculas pequeñas para reemplazar 10 M Y-27632 para mejorar chapado de una sola célula: 1 M Y-39983 (ROCK I inhibidor), 1 phenylbenzodioxane mM (inhibidor ROCA II), 1 mM thiazovivin (un inhibidor de la ROCA novela) , y el inhibidor de JAK 2 M 1. </li><li> Sustituya el medio con medio mTeSR1 libre de drogas, al día siguiente (dentro de las 24 horas), disociar las células de una extraasí, y contar el número de células para determinar eficiencia de plaqueo de una sola célula (Nota: aproximadamente, 50 a 90% de eficiencia de deposición electrolítica de una sola célula que se puede lograr en esta etapa). </li><li> Permita que las células crezcan como una sola célula-formado monocapa durante unos días con 3 ml de medio mTeSR1. Cambio de medio al día. </li><li> Empíricamente determinar el calendario de pases adaptado NCM dependiendo de la densidad celular. Pasaje cuando el crecimiento celular alcanza la confluencia en el día 3 o 4 días, o en el punto de tiempo de 4 horas después de la desaparición de las fronteras de célula a célula. Disociar las células en 1 ml de Accutase, utilizar una relación de división de 1 a 3 (es decir, 1 confluentes bien de células cultivadas en placas en 3 pocillos de placa de 6 pocillos) para los pases de células, y estabilizar adaptado NCM por 5 pasajes. </li><li> Congelación de la célula <ol><li> Utilizar un pozo confluente (~ 5-6 x10 6 células) para un vial congelado: disociar las células de un confluente bien con 1 ml de Accutase durante 10 min. </li><li> Diluir wITH 5 ml de células medianas y centrifugar mTeSR1 como se describió anteriormente. </li><li> Resuspender el sedimento en 500 l de medio de mTeSR1 y luego añadir lentamente 500 l de 2X HPSC medio de congelación (que contienen 20 mM de Y-27632) en un vial de crioconservación. Alternativamente, resuspender las células en 1X HPSC medio de congelación que contienen inhibidor de JAK 2 M 1 o medio 1X CryoStor CS10 en presencia de ya sea 10 M Y-27632 o 2 inhibidor de JAK M 1. </li><li> Coloque los viales congelados en un recipiente criogénico enfriada con hielo (abajo llena con isopranol) y transferir el recipiente criogénico a -80 ° C congelador inmediatamente. </li><li> Células de transferencia del -80 ° C congelador a un tanque de nitrógeno líquido (al día siguiente) para la crioconservación a largo plazo. </li></ol></li><li> Descongelación celular <ol><li> Utilizar un vial de crioconservación para recubrir 1 pocillo de una placa de 6 pocillos. </li><li> Medio mTeSR1 Pre-caliente en un baño de agua a 37 ° C durante 10 min, las células de deshielo en el mismo baño de agua durante 2 min,gota a gota añadir células a 5 ml de medio mTeSR1 pre-calentado que contienen 10 mM de Y-27632, y se centrifuga como se describió anteriormente. </li><li> Resuspender suavemente los sedimentos de células con 2 ml de medio mTeSR1 que contienen 10 mM de Y-27632 y transferir lentamente las células a un pocillo de pre-recubierta con Matrigel (Nota: evitar la producción de burbujas de aire). </li><li> Cambiar medio diario y esperamos que el crecimiento HPSC alcanzar confluencia en el día 3 o 4. </li></ol></li></ol> 4 Protocolo 3:. Convertir HPSC colonias en Matrigel a NCM Cultura <ol><li> Quitar una placa recubierta con Matrigel del refrigerador, coloque la placa en la campana de cultivo de tejidos de 10 a 30 min, se elimina el medio de cada pocillo de la placa, y añadir 2 ml de medio mTeSR1 precalentado a cada pocillo. </li><li> Transferencia trituró grupos HPSC de la cultura alimentador (Paso 2,10 del protocolo básico) a la placa de Matrigel anteriormente. Añadir medio mTeSR1 hasta 2,5 ml de volumen final en cada pocillo. </li><li> Cambie medio diario del 3 al 4 díass hasta que las colonias alcanzan 80 a 90% de confluencia. </li><li> Para pases de células: aclarar las colonias con D-PBS dos veces, el tratamiento de las células con 1 ml de 2 mg / ml dispasa a 37 ° C durante 15 a 20 minutos, y de sedimentos y de paso de células en forma de grumos. </li><li> Para la cultura NCM: repita los pasos 3.1 a 3.9 describen en el Protocolo 2. </li></ol> 5 Protocolo 4:. NCM Cultura de hPSCs sobre LN-521 <ol><li> Descongelar la solución recombinante LN-521 a 4 ° C antes de que el día de uso y hacer que la solución de revestimiento laminina (LCS) diluyendo la laminina descongelado con 1X D-PBS (que contiene Ca 2 + / Mg 2 +) a una concentración final de 10 g / ml. </li><li> Añadir 1 ml de la LCS a un pocillo de placa de 6 pocillos (nota: evitar en seco durante el proceso de recubrimiento). </li><li> Sellar la placa recubierta con Parafilm para evitar la evaporación y almacenar el plato en el refrigerador (4 ° C) O / N (nota: utilizar la placa dentro de 1 semana). </li><li> Retire con cuidado los LCS con una pipeta Pasteur y sin queuching la superficie recubierta y añadir 2 ml de medio mTeSR1 a un pocillo. </li><li> Calentar todas las soluciones de cultivo (incluyendo D-PBS) en un baño de agua a 37 ° C durante 25 min. </li><li> Convertir colonias HPSC a NCM <ol><li> Disociar agregados celulares de células individuales con Accutase como se describe en el Protocolo 2. </li><li> Semillas 1,3-2 x 10 6 hPSCs en una sola LN-521 con revestimiento de pozo (1.4 a 2.1 x 10 5 células / cm 2), sin la presencia de inhibidores de ROCK. </li><li> Cambio de medio al día durante 3 a 4 días. </li><li> Disociar las células en 1 ml de Accutase en el día 3 o 4 para la próxima pases de células utilizando una relación de división de 1 a 3. </li></ol></li></ol> . 6 Protocolo 5: NCM cultura para el ADN plásmido transfección <ol><li> Adaptar colonias HPSC a la cultura NCM como se describe en los Protocolos 2 a 4. </li><li> Placa disociado hPSCs en placa de 12 pocillos con una densidad celular de 7,5 x 10 5 células / pocillo en medio mTeSR1 en presencia de 10 mM de Y-27632. </li><li> Reemplace medio mTeSR1 con medio mTeSR1 libre de drogas entre 4-8 horas después de la siembra de las células. </li><li> Para cada transfección, diluir 2,5 g de plásmidos de expresión (por ejemplo, pmaxGFP) y 5 l de Lipofectamine 2000 en tubos Eppendorf separados en 125 l de cada uno de Opti-MEM medio de suero reducido. </li><li> Después de 5 min, mezclar los reactivos diluidos y se incuba durante 20 min a TA (para formar complejos de transfección). </li><li> Agregue los complejos de transfección mu l 250 a cada pocillo que contiene hPSCs en 1 ml de medio mTeSR1 e incubar las células a 37 ° C en una incubadora de CO 2 durante 24 horas. </li><li> Examinar la eficacia de la transfección con un microscopio de fluorescencia y la fotografía al azar para el cálculo de la eficiencia de transfección real. </li></ol> . 7 Protocolo 6: NCM Cultura para la transfección de microARNs <ol><li> Disociar hPSCs semi-confluentes en el día 2, en condiciones NCM añadiendo 1 ml de Accutase por pocillo en 6<html> Y placa. </li><li> Añadir 10 ml de medio mTeSR1 para diluir las reacciones enzimáticas y centrifugar a 200 xg durante 5 min. </li><li> Resuspender el sedimento celular con medio mTeSR1, sembrar las células a una densidad de 7,5 x 10 5 células por pocillo en una placa de 12 pocillos en un medio que contiene 10 mM mTeSR1 Y-27632, y dejar que las células se adhieren durante 4 horas. </li><li> Sustituya el medio con medio Y27632 libre mTeSR1. </li><li> Utilice disponible comercialmente microRNAs (por ejemplo, un miRIDIAN miRNA control de transfección no dirigido etiquetado con Dy547). Valorar concentraciones que van desde 0 hasta 160 nM usando los reactivos proporcionados y siga las instrucciones del fabricante. </li><li> Optimizar la eficacia de transfección para cada concentración en líneas HPSC por imágenes de la fluorescencia Dy547 de las células vivas con un microscopio a las 24 horas después de la transfección. </li><li> Cálculo de las eficiencias de transfección basados ​​en el porcentaje de células positivas Dy547 sobre todas las células de imágenes. </li></ol>. tle &quot;&gt; 8 Protocolo 7: NCM Cultura para la transducción de vector lentiviral <ol><li> Repita los pasos 7.1 a 7.4 del Protocolo 6. </li><li> Calcule las cantidades de partículas virales que se utilizarán para la transducción: Utilice la siguiente fórmula: TU = (MOI x CN) / VT, mientras que TU denota el número total de unidades de transformación, MOI es igual a la multiplicidad deseada de la infección en el pozo (MOI o TU / célula), CN especifica el número de células en cada pocillo, y VT indica los títulos virales de archivo (TU / ml). Por ejemplo, dado que los títulos virales de acciones para SMART-shRNA vector es igual a 1 x 10 9 TU / ml (unidades de transformación por ml), 7,5 x 10 5 células por pocillo en el momento de la transducción, y una MOI esperado de 20: uso de 15 l de las partículas virales de valores para cada bien (nota: se recomienda esta condición para la inducción lentiviral transitorio). </li><li> Pre-caliente 300 l de medio de mTeSR1 con 10 mg / ml de polibreno durante 30 min. </li><li> Añadir 15 l de partículas virales de acciones enel medio mTeSR1 precalentado que contiene polibreno y mezclar suavemente la solución. </li><li> Reemplazar el medio de cultivo celular con 300 l de medio precalentado que contienen las partículas virales. </li><li> Después de 4 horas de incubación, examinar turboGFP fluorescencia (una máquina para la expresión shRNA) para determinar la eficacia de la transducción. </li><li> Añadir 300 l de medio de mTeSR1 adicional a la transducción bien cuando las células comienzan a expresar turboGFP. </li><li> Después de 12-16 horas de incubación, examinar de nuevo la expresión turbo-GFP. </li><li> Lleve a cabo los experimentos de seguimiento deseados con estas células transducidas dentro de 72 horas. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenicity+as+a+clinical+hurdle+for+pluripotent+stem+cell+therapies.">Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies.</a> Nat Med. 19, 998-1004 (2013).</li><li>Domogatskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin-511+but+not+-332,+-111,+or+-411+enables+mouse+embryonic+stem+cell+self-renewal+in+vitro.">Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro.</a> Stem Cells. 26, 2800-2809 (2008).</li><li>Domogatskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+laminins.">Functional diversity of laminins.</a> Annu Rev Cell Dev Biol. 28, 523-553 (2012).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nat Biotechnol. 28, 611-615 (2010).</li><li>Liew, C. 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The authors declare that they have no competing financial interests.

Hay dos formas principales para hPSCs de cultivo in vitro: cultura convencional de tipo colonia (de las células en los comederos o matrices extracelulares) y de cultivo en suspensión de hPSCs como agregados sin alimentadores 6. Las limitaciones de los métodos de cultivo, tanto de tipo colonia y suspensión incluyen la heterogeneidad acumulada y los cambios epigenéticos heredables. Cultura NCM, basada tanto en los pases de una sola célula y de las planchas de células de alta densidad, r...

La capacidad de hPSCs para diferenciar hacia los tejidos adultos multilinaje ha abierto nuevas vías para el tratamiento de pacientes que sufren de enfermedades graves que involucran cardiovasculares, hepáticas, pancreáticas, y los sistemas neurológicos 1-4. Varios tipos de células derivadas de hPSCs también proporcionarían plataformas móviles robustos para el modelado de la enfermedad, la ingeniería genética, la detección de drogas y 1,4 pruebas toxicológicas. La cuestión clave que garantiza sus futuras aplicaciones clínicas y farmacológicas es la generación de un gran número de hPSCs de grado clínico a través de cultivo celular in vitro. Sin embargo, los sistemas de cultivo actuales son insuficientes o intrínsecamente variables, diversos cultivos de alimentación y libres de alimentador de hPSCs como colonias 5,6. Crecimiento de tipo colonia de acciones hPSCs muchas características estructurales de la masa celular interna (ICM) de embriones de mamíferos tempranos. El ICM es propenso a diferenciarse en las tres capas germinalesen un entorno multicelular debido a la existencia de gradientes de señalización heterogéneos. Por lo tanto, la adquisición de la heterogeneidad en el desarrollo embrionario temprano se considera como un proceso necesario para la diferenciación, pero una característica no deseada de la cultura HPSC. La heterogeneidad en la cultura HPSC es a menudo inducida por señales apoptóticas excesivas y diferenciación espontánea debido a las condiciones de crecimiento subóptimas. Por lo tanto, en el tipo de colonia de cultivo, las células heterogéneas se observan a menudo en la periferia de las colonias 7,8. También se ha demostrado que las células en células madre embrionarias (células madre) colonias de respuestas diferenciales de exposición a moléculas de señalización tales como BMP-9 4. Por otra parte, los métodos de cultivo de colonias producen rendimientos bajos de células, así como las tasas de recuperación muy bajo de células de la criopreservación, debido a las tasas de crecimiento incontrolable y señalización apoptótica pathways 6,9. En los últimos años, diversos cultivos en suspensión han sido desarrollados para hPSCs de cultivo, particulArly para la expansión de grandes cantidades de hPSCs en subordinado, y las condiciones de matriz libre 6,10-13. Obviamente, diferentes sistemas de cultivo tienen sus propias ventajas y desventajas. En general, la naturaleza heterogénea de hPSCs representa uno de los principales inconvenientes en los métodos de tipo colonia y cultura agregada, que son subóptimas para la entrega de materiales de ADN y de ARN en hPSCs para la ingeniería genética 6. Claramente, existe una necesidad imperiosa de desarrollar nuevos sistemas que evitan algunas deficiencias de los métodos de cultivo actuales. Los descubrimientos de inhibidores de moléculas pequeñas (como el inhibidor de la ROCA Y-27632 y JAK inhibidor 1) que mejoran la supervivencia de una sola célula preparan el terreno para el cultivo disociado-HPSC 14,15. Con el uso de estas moléculas pequeñas, hemos desarrollado recientemente un método de cultivo basado en el tipo no-colonia (NCM) de crecimiento de-hPSCs disociadas 9. Este método de cultivo de la novela combina pases de una sola célula y de alta densidadchapado métodos, que nos permite producir grandes cantidades de hPSCs homogéneas bajo ciclos de crecimiento consistentes sin mayores anomalías cromosómicas 9. Alternativamente, la cultura NCM podría ser implementado con diferentes moléculas pequeñas y matrices definidas (tales como lamininas) con el fin de optimizar el método de cultivo para amplias aplicaciones. A continuación, presentamos varios protocolos de detalle sobre la base de la cultura NCM y delinear los procedimientos detallados para la ingeniería genética. Para demostrar la versatilidad de los protocolos de NCM, también a prueba la cultura NCM con inhibidores de ROCK diversos y con la única isoforma laminina 521 (es decir, LN-521).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Countess automated cell counter&#160;&#160;</td>
			<td style="width:220px;">Invitrogen Inc.&#160;</td>
			<td style="width:187px;">C10227</td>
			<td style="width:187px;">Automatic cell counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Faxitron Cabinet X-ray System</td>
			<td style="width:220px;">Faxitron X-ray Corporation, Wheeling, IL&#160;</td>
			<td style="width:187px;">Model RX-650</td>
			<td style="width:187px;">X-ray irradiation of MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">MULTIWELL six-well plates&#160;</td>
			<td style="width:220px;">Becton Dickinson Labware</td>
			<td style="width:187px;">353046</td>
			<td style="width:187px;">Polystyrene plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11965&#8211;092</td>
			<td style="width:187px;">For MEF medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">mitomycin C</td>
			<td style="width:220px;">Roche&#160;</td>
			<td style="width:187px;">107 409</td>
			<td style="width:187px;">Mitotic inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Trypsin</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">25300-054</td>
			<td style="width:187px;">For MEF dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM/F12&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11330&#8211;032</td>
			<td style="width:187px;">For hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Opti-MEM I Reduced Serum Medium&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">31985-062</td>
			<td style="width:187px;">For hPSC transfection</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Heat-inactivated FBS</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">16000&#8211;044</td>
			<td style="width:187px;">Component of MEF medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Knockout Serum Replacer&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">10828&#8211;028</td>
			<td style="width:187px;">KSR, Component of hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">14190-144</td>
			<td style="width:187px;">D-PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Non-essential amino acids&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">11140&#8211;050</td>
			<td style="width:187px;">NEAA, component of hPSC medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">L-Glutamine&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">25030&#8211;081</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:203px;">mTeSR1 &#38; Supplements</td>
			<td rowspan="2" style="width:220px;">StemCell Technologies</td>
			<td rowspan="2" style="width:187px;">5850</td>
			<td style="width:187px;">Animal protein-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TeSR2 &#38; Supplements</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">5860</td>
			<td style="width:187px;">Xeno-free medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:220px;">Sigma&#160;</td>
			<td style="width:187px;">7522</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;"> 
			MEF (CF-1) ATCC</td>
			<td style="width:220px;">American Type Culture Collection (ATCC)&#160;</td>
			<td style="width:187px;">SCRC-1040</td>
			<td style="width:187px;">For feeder culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">hESC-qualified Matrigel</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">354277</td>
			<td style="width:187px;">For feeder-free culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Laminin-521</td>
			<td style="width:220px;">BioLamina</td>
			<td style="width:187px;">LN521-02</td>
			<td style="width:187px;">Human recombinant protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">FGF-2 (recombinant FGF, basic)</td>
			<td style="width:220px;">R&#38;D Systems, MN</td>
			<td style="width:187px;">223-FB</td>
			<td style="width:187px;">Growth factor in hPSC medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CryoStor CA10&#160;</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">7930</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Accutase</td>
			<td style="width:220px;">Innovative Cell Technologies</td>
			<td style="width:187px;">AT-104</td>
			<td style="width:187px;">1X mixed enzymatic solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">JAK inhibitor I</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">420099</td>
			<td style="width:187px;">An inhibitor of Janus kinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">688000</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0012</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-39983</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0029</td>
			<td style="width:187px;">ROCK I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Phenylbenzodioxane&#160;</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0030</td>
			<td style="width:187px;">ROCK II inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Thiazovivin</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0017</td>
			<td style="width:187px;">A novel ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">BD Falcon Cell Strainer&#160;</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">352340</td>
			<td style="width:187px;">40-&#181;m cell strainer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nalgene 5100-0001 Cryo 1&#176;C</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">C6516F-1</td>
			<td style="width:187px;">&#8220;Mr. Frosty&#8221; Freezing Container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Lipofectamine 2000&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11668-027</td>
			<td style="width:187px;">Transfection reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DharmaFECT Duo&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">T-2010-02</td>
			<td style="width:187px;">Transfection reagent</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Non-targeting miRIDIAN miRNA Transfection Control</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">IP-004500-01-05</td>
			<td style="width:187px;">Labeled with Dy547, to monitor the delivery of microRNAs&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">SMART-shRNA</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">To be determined</td>
			<td style="width:187px;">Lentiviral vector</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">pmaxGFP</td>
			<td>amaxa Inc (Lonza)</td>
			<td>Included in every transfection kit</td>
			<td style="width:187px;">Expression plasmid for transfection control</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">4-Oct</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-5279</td>
			<td style="width:187px;">Mouse IgG2b, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-1</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21702</td>
			<td style="width:187px;">Mouse IgM, differentiation marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-4</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21704</td>
			<td style="width:187px;">Mouse IgG3, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-60</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21705&#160;</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-81</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21706</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">CK8 (C51)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8020</td>
			<td style="width:187px;">Mouse IgG1, against cytokeratin 8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">&#945;-fetoprotein</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8399</td>
			<td style="width:187px;">AFP, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF-3&#946; (P-19)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-9187</td>
			<td style="width:187px;">FOXA2, goat polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Troponin T (Av-1)</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">MS-295-P0</td>
			<td style="width:187px;">Mouse IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Desmin&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">RB-9014-P1</td>
			<td style="width:187px;">Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Anti-NANOG</td>
			<td style="width:220px;">ReproCELL Inc, Japan</td>
			<td style="width:187px;">RCAB0004P-F</td>
			<td style="width:187px;">Polyclonal antibody&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Rat anti-GFAP</td>
			<td style="width:220px;">Zymed</td>
			<td style="width:187px;">13-0300</td>
			<td style="width:187px;">Glial fibrillary acidic protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Albumin (clone HSA1/25.1.3)</td>
			<td style="width:220px;">Cedarlane Laboratories Ltd. (</td>
			<td style="width:187px;">CL2513A</td>
			<td style="width:187px;">Mouse IgG1,</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Smooth muscle actin (clone 1A4)</td>
			<td style="width:220px;">DakoCytomation Inc</td>
			<td style="width:187px;">IR611/IS611</td>
			<td style="width:187px;">Mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nestin</td>
			<td style="width:220px;">Chemicon International</td>
			<td style="width:187px;">MAB5326</td>
			<td style="width:187px;">Rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TUBB3</td>
			<td style="width:220px;">Convance Inc</td>
			<td style="width:187px;">MMS-435P</td>
			<td style="width:187px;">Tuj1, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF4&#945; (C11F12)</td>
			<td style="width:220px;">Cell Signaling Technologies</td>
			<td style="width:187px;">3113</td>
			<td style="width:187px;">Rabbit monoclonal antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Paraformaldehyde (solution)</td>
			<td style="width:220px;">Electron Microscopy Sciences</td>
			<td style="width:187px;">15710</td>
			<td style="width:187px;">PFA, fixative, diluted in D-PBS</td>
		</tr>
	</tbody>
</table>

alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Un esquema general de la cultura NCM La figura 1 representa un esquema típico de la cultura NCM que muestra los cambios dinámicos de hPSCs después de alta densidad de placas de una sola célula en presencia del inhibidor de Rock y-27632. Estos cambios morfológicos incluyen conexiones intercelulares después de la siembra, la formación de grupos celulares, y el crecimiento celular exponencial seguida de la condensación de células (Figura 1A).</...

Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis | Biology | Video

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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SCIENTISTS IN ACTION

12.2K vistas.  National Institutes of Health. Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Video: Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Ratón epidérmico de la cresta neural de células madre (EPI-NCSC) Culturas

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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Biología

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

La cultura y el mantenimiento de células estaminales embrionarias humanas

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

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparación de combinados lisado plaquetario humano (pHPL) como un suplemento eficaz de los animales libres de suero humano culturas de células madre

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfección y Nucleofecting Humanos células madre pluripotentes inducidas

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

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

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Producción automatizada de neuronas corticales y dopaminérgicas derivadas de células madre no inducidas por humanos con monitoreo integrado de células vivas

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Una estrategia de dos pasos que combina la modificación epigenética y las señales biomecánicas para generar células pluripotentes de mamíferos

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.