We are grateful for expert technical assistance from the UNC Flow Cytometry Core and UNC Microscopy Core. We thank members of the Qian lab and the Liu lab for helpful discussions and critical reviews of the manuscript. This study was supported by NIH/NHLBI R00 HL109079 grant to Dr. Liu and American Heart Association (AHA) Scientist Development Grant 13SDG17060010 and the Ellison Medical Foundation (EMF) New Scholar Grant AG-NS-1064-13 to Dr. Qian.

El protocolo descrito aquí utiliza ratones recién nacidos. Cuidado de los animales y los experimentos se realizan de acuerdo con los lineamientos establecidos por la División de Laboratorio de Medicina Animal (DLAM) en la Universidad de Carolina del Norte, Chapel Hill. 1. Preparación de tampones y Medios de Comunicación <ol><li> Preparar fibroblastos (FB) medio: Suplemento de 500 ml de medio IMDM con 100 ml de suero fetal bovino (FBS) y 6 ml de penicilina / estreptomicina. </li><li> Prepare medio iCM: Suplemento de 400 ml de medio DMEM con 100 ml de medio M199 y 50 ml de FBS. </li><li> Preparar medio B27: Suplemento 490 ml RPMI 1640 los medios de comunicación con 10 ml B27 medios. </li><li> Preparar medio de cultivo Plat-E: Suplemento 500 ml DMEM medios con 50 ml de FBS, 5 ml de penicilina / estreptomicina y 5 ml aminoácidos no esenciales. </li><li> Preparar medio de transfección Plat-E: Suplemento 500 ml de DMEM los medios de comunicación con 50 ml de FBS y 5 ml de aminoácidos no esenciales. </li><li> Prepare gelatina revestido placa de 24 pocillos: Añadir solución de gelatina 0,5 ml 0,1% a un pocillo de placa de 24 pocillos, se incuba a 37 ° C durante al menos 10 minutos y aspirado de la derecha antes de su uso. </li><li> Preparar tampón FACS etiquetado: Suplemento 500 ml de DPBS con 10 ml de FBS y 2 ml de EDTA (0,5 M) para alcanzar una concentración final de 2 mM. Pasar a través de un filtro y tienda de 0,22 micras a 4 ° C. </li><li> Preparar tampón MACS de clasificación: Suplemento 500 ml de DPBS con 2,5 g de BSA y 2 ml de EDTA (0,5 M) para alcanzar una concentración final de 2 mM. Pasar a través de un filtro y tienda de 0,22 micras a 4 ° C. </li><li> Preparar tampón HBS 2x para la transfección: Suplemento 500 ml de tampón HEPES (50 mM) con NaCl 280 mM (8,1816 g), mM KCl 10 (0,37275 g), 1,5 mM Na 2 HPO 4 (0,10647 g), 12 mM de glucosa (1,08096 g ). Añadir alrededor de 650 l 10 N NaOH para ajustar el pH a 7.5 a 7.12. Filtrar a través de un filtro de 0,22 micras de poro y se almacena a 4 ° C. </li><li> Preparar M CaCl solución 2,5 2 para la transfección: Agregar 18,376 g de CaCl 2 a 50 ml ddH2O Filtrar a través de un filtro de 0,22 micras de poro y se almacena a 4 ° C. </li><li> Preparar ICC (inmunocitoquímica) Tampón de fijación: Añadir 5 ml de paraformaldehído (PFA, 32%) a 35 ml de DPBS para llegar a una concentración final de 4%. </li><li> Preparar tampón de permeabilización ICC: Añadir 0,1 ml de Triton 100 ml de DPBS para llegar a una concentración final de 0,1%. </li><li> Preparar tampón de bloqueo ICC: Añadir 5 g de albúmina de suero bovino (BSA) a 100 ml de DPBS para llegar a una concentración final de 5%. </li><li> Preparar tampón de tinción ICC: Añadir 1 g de BSA a 100 ml de DPBS para llegar a una concentración final de 1%. </li></ol> 2. Generación de neonatales mouse cardíacos fibroblastos <ol style="margin-left: 40px;"><li> Generar fibroblastos cardíacos de método de cultivo de explante <ol><li> Clean neonatal αMHC-GFP ratón transgénico (P1 a P2) con etanol 75%. Corta la cabeza con unas tijeras estériles. A continuación, hacer una incisión horizontal desde debajo de una axila a la otra. Use un estérilfórceps romos y se inclinó para diseccionar el corazón y la colocan en un pocillo de la placa de 24 pocillos que contiene tampón PBS helado. <ol><li> Alternativamente, exprimir el corazón después de la incisión horizontal. </li></ol></li><li> Compruebe la expresión de GFP en el corazón por el microscopio de fluorescencia. Dado que la expresión de GFP es impulsado por el promotor αMHC que es un promotor específico cardíaco, el corazón de ratón transgénico debe estar en verde 15, mientras que el corazón negativo es tenue en cualquiera de fluorescencia. </li><li> Tome 3-4 GFP corazones positivos en un pozo central placa de cultivo de 60 mm y picar en trozos pequeños de menos de 1 mm 3 en el tamaño de tijeras y pinzas estériles. </li><li> Coloca 3-4 corazones picados en una sola placa de 10 cm con 2 ml de medio de FB. Deje que los tejidos se establecen durante 3 horas. </li><li> Agregue lentamente 8 ml precalentado medios FB a los platos que contienen los tejidos del corazón. No moleste a los tejidos durante tres días. </li><li> Vuelva a colocar los medios de comunicación cada tres días. </li><li> El día 7, aspircomió el medio de cultivo y lavar las células con DPBS. Añadir 3 ml 0,05% de tripsina a cada placa y digerir a 37 ° C durante 5 min. </li><li> Añadir 5 ml de medio de FB para saciar la tripsina. Separar suavemente las células con un raspador de células. Pipetear los medios de comunicación de arriba a abajo para disociar más mecánicamente el tejido. </li><li> Recoger las células y filtrar a través de 40 micras coladores celulares para evitar la contaminación de fragmentos de tejido del corazón, y luego sedimentar las células mediante centrifugación a 200 xg durante 5 min. </li><li> Lavar las células una vez con tampón MACS y las células están listas para la clasificación. </li></ol></li><li> Generar fibroblastos cardíacos de método de digestión enzimática <ol><li> Cosecha GFP corazones positivos como 2.1.1. Transferir todos los corazones en un plato de 10 cm con 10 ml de DPBS enfriado con hielo. </li><li> Apriete ventrículos con pinzas estériles para quitar la sangre y enjuagar una vez con DPBS enfriado con hielo. </li><li> Recorte los corazones de estar libre de otros tejidos y grasa. </li><li> Corte cada corazón en aproximadamente 4 piezas que todavía están conectados vagamente. </li><li> Si menos de 20 corazones, transfiera los corazones en un tubo cónico de 15 ml con 8 ml cálida 0,05% de tripsina-EDTA, y se incuba a 37 ° C durante 15 min. <ol><li> Si hay 20-30 corazones, transferir los corazones en un tubo cónico de 50 ml con 10 ml cálida 0,05% de tripsina-EDTA, y se incuba a 37 ° C durante 15 min. </li></ol></li><li> Deseche la tripsina y añadir 5 ml (por menos de 20 corazones) o 10 ml (para 20-30 corazones) cálida colagenasa tipo II (0,5 mg / ml) en HBSS. </li><li> Vortex el tubo de vórtice durante 1 minuto a un nivel de velocidad alrededor de 4 a 6 (si el líquido no se levante a la tapa, entonces la velocidad está muy bien). </li><li> Incubar el tubo en 37 ° C baño de agua durante 3-5 minutos. </li><li> Vortex el tubo durante 1 min. </li><li> Sedimentos durante 1 minuto por gravedad hasta que los trozos de tejido se calmen, recoger el líquido en un nuevo tubo que contiene 5 ml de medio FB frío. </li><li> Repita los pasos 2.2.6-2.2.10 3-5 veces. </li><li> Combinar todas las colecciones mediante la filtración a través de filtro de células de 40 micrashacer suspensión de células individuales. </li><li> Centrifugar a 200 xg durante 5 min a 4 ° C. </li><li> Resuspender las células en 10 ml de tampón MACS, y centrifugar a 200 xg durante 5 min a 4 ° C. </li><li> Opcional: Si hay una gran cantidad de células sanguíneas, Resuspender las células con 1 ml de RBC tampón de lisis (150 mM NH 4 Cl, 10 mM KHCO3, y EDTA 0,1 mM), mantener en hielo durante 1 min, luego se diluye con 10 ml MACS tampón y se centrifuga a 200 xg durante 5 min. Lavar una vez más con tampón MACS. </li></ol></li><li> Aislamiento de Thy1.2 + fibroblastos por MACS (magnético clasificación de células activadas) <ol><li> Determinar el número de células viables mediante tinción con azul de tripano. <ol><li> Tomar 10 células mu l a partir de la suspensión de células de 10 ml en el paso 2.2.14. Mezclar con solución de azul de 10 l 0,4% de tripano. </li><li> Añadir la mezcla a un hemocitómetro. Deje reposar durante 3-5 minutos y luego examinar de inmediato con un microscopio. Las células muertas se tiñen en las células azules y viables son sin mancha. </li><li&gt; contar todas las células viables en cuatro cuadrados de 1 mm de esquina del hemocitómetro. Determinar el número de células viables: el recuento medio por metro cuadrado x 2 (factor de dilución) x 10 4 x 10 (volumen total de la suspensión celular). </li></ol></li><li> Por menos de 1 x 10 7 células, las células volver a suspender las microesferas con 10 l Thy1.2 en 90 l enfriaron tampón MACS. Añadir más cuentas proporcionalmente si hay más de 1 x 10 7 células. Mezclar bien e incubar en nevera (2-8 ° C) durante 30 minutos. </li><li> Añadir 10 ml de tampón MACS y muestras giran a 200 xg durante 5 min; lavar una vez con tampón MACS 10 ml y centrifugar de nuevo. </li><li> Traiga volumen de 2 ml en tampón MACS. </li><li> Establecer un separador de MACS en el capó. Inserte una columna LS al separador. Aplicar 3 ml MACS buffer para la columna que se equilibren. </li><li> Pasar la suspensión celular a través de la columna LS equilibrada. </li><li> Lavar 3 veces con 2 ml de tampón MACS cada vez. </li><li> Tome la columna de la LS fuera tque Separador y eluir 3 veces con 2 ml MACS amortiguan en un nuevo tubo de 50 ml. </li><li> Girar a 200 xg durante 5 min. </li><li> Resuspender las células en 10 ml de medio FB, cuentan las células y se siembran en placas a una densidad adecuada. Para fibroblastos generados por el método de cultivo de explante, la densidad de células de siembra podría ser alrededor de 2.3 x 10 4 células / pocillo de placa de 24 pocillos. Para fibroblastos generados por método de digestión enzimática, la densidad de las células podría ser alrededor de 5.4 x 10 4 células / pocillo de placa de 24 pocillos. </li></ol></li></ol> 3. Generación de retrovirus para iCM Reprogramación Nota: Realice los siguientes pasos en un Gabinete de Seguridad Biológica BSL2 en condiciones estériles. Se recomienda la eliminación adecuada de las células transfectadas, puntas de pipeta y tubos para evitar el riesgo de los riesgos ambientales y de salud. <ol style="margin-left: 40px;"><li> Mantener células Plat-E en Plat-E medio suplementado con 1 mg / ml de puromicina y 10 mg / ml blasticidin a 37 ° C con 5% de CO 2. </li><li> El día 2, sustituir el medio por el medio de cultivo Plat-E carente puromicina y blasticidina. </li><li> En el día 0, dividir celdas Plat-E en placas de 10 cm el día antes de la transfección en aproximadamente 4-5 x 10 6 células por placa. Las células deben ser ~ 80-90% de confluencia en el día de la transfección. </li><li> El día 1, los medios de comunicación el cambio de cultura a los medios de transfección de las células dentro de 1 hora antes de la transfección. Procedimiento de transfección es la siguiente: <ol><li> La transfección utilizando kits comerciales <ol><li> Mezclar 20 l de reactivo de transfección (Lipofectamine por ejemplo) y 500 l de suero reducido medios de comunicación (por ejemplo, Opti-MEM). Añadir 10 g del plásmido retroviral a otros 500 l reducidos medio de suero. Incubar cada mezcla a temperatura ambiente (RT) durante 5 min. </li><li> Poco a poco agregue la mezcla de plásmido a la mezcla de transfección. Este paso puede tardar 15-30 seg. Incubar la mezcla durante 20 min a RT (solución puede aparecer turbia). </li&gt; <li> Añadir gota sabia mezcla de las células. </li><li> Incubar toda la noche en una incubadora a 37 °. </li></ol></li><li> La transfección utilizando fosfato de calcio. <ol><li> Mezclar 40 l 2,5 M CaCl2 y 440 l ddH2O, a continuación, añadir 20 g de plásmido retroviral (ajustar la cantidad de ddH2O de manera que el volumen total es de 500 l). </li><li> Añadir 500 l 2x HBS a un tubo cónico de 15 ml. </li><li> Vortex la HBS y añada su parte la mezcla de ADN de calcio a la HBS gota a gota. Este paso lleva alrededor de 30 segundos para cada muestra. Así máximamente hacer 3-4 muestras simultáneamente. </li><li> Incubar a temperatura ambiente durante 3 min de incubación durante más tiempo lleva al precipitado más grande y menos células asumirá el precipitado. </li><li> Añadir la mezcla gota a gota a las células y observar bajo el microscopio 20X. Precipitados finos (un montón de pequeñas son buenas pero algunas grandes no son buenos) deben ser vistos. </li><li> Incubar durante la noche a 37 ° C incubadora. </li&gt; </ol></li></ol></li><li> El día 2, cambiar el medio sobre las células con 8 ml precalentada medios de cultivo. Incubar durante 24 horas. </li><li> El día 3 y el día 4, recoger el sobrenadante del cultivo de las placas mediante el uso de una jeringa desechable de 10 ml estéril, filtrándola a través de un filtro de acetato de celulosa de tamaño de poro 0,45 micras, y transferir a un tubo de 50 ml. Añadir 2 ml de solución de precipitación retrovirus a cada sobrenadante que contiene virus-8 ml. Suavemente mezclar e incubar durante la noche a 4 ° C. </li><li> El día 5, girar la mezcla viral a 1500 × g a 4 ° C durante 30 min. Las partículas virales deben aparecer en pellet blanco en la parte inferior del tubo. </li><li> Eliminar el sobrenadante. Centrifugar la solución residual por centrifugación a 1500 xg durante 5 min. Quite todos los rastros de líquido por aspiración, teniendo mucho cuidado de no molestar a las partículas retrovirales precipitados en tabletas. </li><li> Resuspender gránulos retrovirales con tampón DPBS 100 l por cada 8 ml de sobrenadante viral. Alícuotael virus en hielo. Utilice inmediatamente o almacenar a -80 ° C. </li></ol> 4. La reprogramación de fibroblastos cardíacos <ol><li> Añadir 4 l de solución de polibreno 10 mg / ml en el medio de iCM 10 ml, y mezclar suavemente pipeteando arriba y abajo. La concentración final de polibreno es 4 mg / ml. </li><li> Añadir 500 l medios iCM que contiene polibreno a cada pocillo de placa de 24 pocillos sembrada con fibroblastos. </li><li> Añadir 10 l retrovirus a cada pocillo que contienen ya sea 2-3 x 10 4 células de cultivo de explantes o 4-5 x 10 4 células de método de digestión enzimática. La cantidad de virus se debe aumentar proporcionalmente con el aumento de número de células en diferentes placas o placas de cultivo. Poner la placa en una incubadora a 37ºC durante 24 a 48 horas para permitir que la infección viral. La línea de tiempo para la reprogramación cardiaca se muestra en la Figura 1. </li><li> Después de la transducción viral, reemplace virus medios que contienen con 500 l de medios regulares iCM. </li><li> Cambie los medios de comunicación cada 2-3 días. Para la selección positiva de células transducidas virales, añadir medios ICM suplementados con puromicina a 2 mg / ml a las células diana. Manténgalo durante tres días. Después de eso, mantener la puromicina en el medio iCM a la concentración de 1 mg / ml. </li><li> Tres días después de la infección viral, tomar la placa hacia fuera de la incubadora y colocarlo bajo un microscopio de fluorescencia invertida (20X) para observar la expresión de GFP. Nota: Diez días después de la infección viral, las células pueden ser cosechadas para el análisis FACS y análisis de la CPI para determinar la eficiencia de reprogramación (en el paso 5 y 6). </li><li> Catorce días después de la infección viral, reemplace medios iCM con medios B27. Cambie los medios de comunicación cada tres días. Espontánea loci celular latido puede aparecer a partir de tres (células de método de digestión enzimática) o cuatro (células de método de cultivo de explante) semanas después de la transducción viral. </li></ol> 5. Inmunocitoquímico Análisis de Eficiencia Reprogramación <ol><li> Enjuague las célulascon el hielo frío PBS tres veces; eliminar el exceso de solución. </li><li> Añadir tampón de fijación 0,5 ml de la CPI a cada pocillo de placas de 24 pocillos. Fijar las células a temperatura ambiente durante 15-20 min. </li><li> Enjuagar las células con PBS tres veces. </li><li> Añadir 0,5 ml de tampón de permeabilización ICC a cada pocillo de las placas de 24 pocillos. Permeabilizar las células a temperatura ambiente durante 15 min. </li><li> Enjuagar las células con PBS tres veces. </li><li> Añadir 0,5 ml de la CPI tampón de bloqueo a cada pocillo de placa de 24 pocillos, se incuba a temperatura ambiente durante 1 hora. </li><li> Anticuerpos primarios diluidos con tampón de tinción ICC. Añadir 200 l soluciones de anticuerpo a cada pocillo e incubar durante la noche a 4 ° C. Los factores de dilución de anticuerpo se enumeran en Materiales. </li><li> En el día siguiente, lavar las células con PBS tres veces. </li><li> Añadir 200 soluciones de anticuerpos secundarios mu l a las células y se incuba durante 1 hora a TA en la oscuridad. </li><li> Lavar las células con PBS tres veces. </li><li> Añadir 150 l solución que contiene DAPI a cada pocillo de montaje. Permitir a manchar núcleos durante 1 min. entoncescubrir cada pocillo con hoja de la cubierta de cristal redondo y presione suavemente el deslizamiento contra la superficie inferior con unas pinzas para quitar las burbujas de aire. </li><li> Aspirar el exceso de solución y las muestras están listas para la imagen. Las imágenes representativas que muestran las células reprogramadas que expresan marcadores cardíacos cTnT y αActinin en d14 se muestran en la Figura 2B. </li></ol> 6. FACS análisis de la eficiencia de reprogramación <ol><li> Lave las células una vez con DPBS. Añadir 0,3 ml de tripsina (0,05%) a cada pocillo y se incuba a 37 ° C durante 5 min. </li><li> Golpee suavemente la placa para facilitar el levantamiento de las células. Compruebe el desprendimiento de células bajo el microscopio. Añadir 1 ml de tampón FACS a cada pocillo cuando se disocian mayoría de las células. Pipetear hacia arriba y abajo para disociar más mecánicamente las células. </li><li> Transferir las células de la placa de 24 pocillos de una placa de 96 pozos profundos bien a bien. Centrifugado a 200 xg durante 5 min a 4 ° C para recoger las células. </li><li> Lavar las células una vez conTampón FACS. </li><li> Añadir 100 soluciones de fijación / permeabilización mu l para resuspender las células durante 20 min a 4 ° C. </li><li> Lavar las células dos veces con solución de lavado 1x, pellets, y retirar el sobrenadante. </li><li> Prepare la solución de anticuerpo primario contra la GFP y cTnT en tampón de lavado 1x. Resuspender a fondo cada muestra con una solución de anticuerpo 50 l y se incuba a 4 ° C durante 30 min. </li><li> Lave las células una vez en solución de lavado 1x, pellets, y retirar el sobrenadante. </li><li> Añadir 50 solución de anticuerpo secundario que contiene l Alexa Fluor 488 burro conjugado IgG anti-conejo y Alexa Fluor 647 burro conjugado anti-IgG de ratón a cada muestra. Se incuba a 4 ° C durante 30 min en la oscuridad. </li><li> Lave las células una vez en solución de lavado 1x, pellets, y retirar el sobrenadante. </li><li> Resuspender las células en 400 l de tampón de fijación (DPBS con 1% PFA). La transferencia de células en el tubo de FACS con el casquillo de filtro de células. Las muestras están listas para la detección FACS. El análisis FACS representante de una eficiencia de reprogramacióny usando pMxs-puromicina-MGT construir con o sin selección puromicina se muestra en la Figura 2A. </li></ol>

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D., 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=Direct+reprogramming+of+human+fibroblasts+toward+a+cardiomyocyte-like+state.">Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.</a> Stem Cell Reports. 1, 235-247 (2013).</li><li>Hirai, H., Katoku-Kikyo, N., Keirstead, S. A., Kikyo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+direct+reprogramming+of+fibroblasts+into+cardiomyocyte-like+cells+with+the+MyoD+transactivation+domain.">Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain.</a> Cardiovasc Res. 100, 105-113 (2013).</li><li>Ieda, M., 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=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142, 375-386 (2010).</li><li>Inagawa, K., 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=Induction+of+cardiomyocyte-like+cells+in+infarct+hearts+by+gene+transfer+of+Gata4,+Mef2c,+and+Tbx5.">Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.</a> Circ Res. 111, 1147-1156 (2012).</li><li>Islas, J. F., 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=Transcription+factors+ETS2+and+MESP1+transdifferentiate+human+dermal+fibroblasts+into+cardiac+progenitors.">Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.</a> Proc Natl Acad Sci U.S.A. 109, 13016-13021 (2012).</li><li>Jayawardena, T. M., 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=MicroRNA-mediated+in+vitro+and+in+vivo+direct+reprogramming+of+cardiac+fibroblasts+to+cardiomyocytes.">MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes.</a> Circ Res. 110, 1465-1473 (2012).</li><li>Muraoka, N., 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=MiR-133+promotes+cardiac+reprogramming+by+directly+repressing+Snai1+and+silencing+fibroblast+signatures.">MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.</a> Embo J. 33, 1565-1581 (2014).</li><li>Nam, Y. J., 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=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141, 4267-4278 (2014).</li><li>Nam, Y. J., 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=Reprogramming+of+human+fibroblasts+toward+a+cardiac+fate.">Reprogramming of human fibroblasts toward a cardiac fate.</a> Proc Natl Acad Sci USA. 110, 5588-5593 (2013).</li><li>Protze, 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=A+new+approach+to+transcription+factor+screening+for+reprogramming+of+fibroblasts+to+cardiomyocyte-like+cells.">A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells.</a> J Mol Cell Cardiol. 53, 323-332 (2012).</li><li>Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+mouse+fibroblasts+into+cardiomyocyte-like+cells+in+vitro.">Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.</a> Nat Protoc. 8, 1204-1215 (2013).</li><li>Wang, H., 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=Small+Molecules+Enable+Cardiac+Reprogramming+of+Mouse+Fibroblasts+with+a+Single+Factor,+Oct4.">Small Molecules Enable Cardiac Reprogramming of Mouse Fibroblasts with a Single Factor, Oct4.</a> Cell Rep. , (2014).</li><li>Wang, L., 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=Stoichiometry+of+Gata4,+Mef2c,+and+Tbx5+Influences+the+Efficiency+and+Quality+of+Induced+Cardiac+Myocyte+Reprogramming.">Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming.</a> Circ Res. , (2014).</li><li>Song, K., 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=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485, 599-604 (2012).</li><li>Qian, L., 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=In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes.">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485, 593-598 (2012).</li><li>Mathison, M., 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=In+vivo+cardiac+cellular+reprogramming+efficacy+is+enhanced+by+angiogenic+preconditioning+of+the+infarcted+myocardium+with+vascular+endothelial+growth+factor.">In vivo cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the infarcted myocardium with vascular endothelial growth factor.</a> J Am Heart Assoc. 1, e005652 (2012).</li><li>Srivastava, D., Ieda, M., Fu, J., Qian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+repair+with+thymosin+beta4+and+cardiac+reprogramming+factors.">Cardiac repair with thymosin beta4 and cardiac reprogramming factors.</a> Ann NY Acad Sci. 1270, 66-72 (2012).</li><li>Muraoka, N., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+transcription+factors+is+critical+for+cardiac+reprogramming.">Stoichiometry of transcription factors is critical for cardiac reprogramming.</a> Circ Res. , 116-216 (2015).</li><li>Srivastava, D., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+factors+for+cardiac+reprogramming.">Critical factors for cardiac reprogramming.</a> Circ Res. 111, 5-8 (2012).</li></ol>

The authors have nothing to disclose.

Para exitosa generación iCM al utilizar este protocolo, hay varios factores importantes que tienen un impacto en la eficiencia global. Particularmente las condiciones de fibroblastos de partida y la calidad de codificación retrovirus MGT pueden afectar en gran medida la eficiencia de la reprogramación. Es importante generar fibroblastos tan fresco y saludable posible. Para el método de cultivo de explantes, los fibroblastos se pueden utilizar antes de siete días después de los explante...

Cardiovascular disease remains the leading cause of death worldwide, accounting for 17.3 million deaths per year1. Loss of cardiomyocytes resulting from myocardial infarction (MI) or progressive heart failure is a major cause of morbidity and mortality2. Due to limited regenerative capacity, adult mammalian hearts usually suffer from impaired pump function and heart failure following injury3-6. As such, efficient (re)generation of cardiomyocytes in vivo and in vitro for treatment of heart disease and for disease modeling is a critical issue needing to be addressed.
Recent development of direct reprogramming, which directly reprograms cells from one differentiated phenotype to another without transitioning through the pluripotent state, offers a promising alternative approach for regenerative medicine. The mammalian heart contains abundant cardiac fibroblasts (CFs), which account for approximately half of the cells in heart and massively proliferate upon injury7-9. Thus, the vast pool of CFs could serve as an endogenous source of new CMs for regenerative therapy if they could be directly reprogrammed into functional CMs. It has been shown that a combination of transcription factors, such as Gata4 (G), Mef2c (M) and Tbx5 (T), with or without microRNAs or small molecules can reprogram fibroblasts into iCMs10-26. Importantly, this conversion can also be induced in vivo, and results in an improvement in cardiac function and a reduction in scar size in an infarcted heart16,27-29. These studies indicate that direct cardiac reprogramming may be a potential avenue to heal an injured heart. However, the low efficiency of iCM reprogramming has become a major hurdle for further mechanistic studies. In addition, the reproducibility of cardiac reprogramming is another controversial issue of this technology11,30,31. 
Very recently, we generated a complete set of polycistronic constructs encoding G,M,T in all possible splicing orders with identical 2A sequences in a single mRNA. These polycistronic constructs yielded varied G, M and T protein expression levels, which led to significantly different reprogramming efficiency25. The most efficient construct, named MGT, which showed a relatively high Mef2c and low Gata4 and Tbx5 expression, significantly improved reprogramming efficiency and produced large amounts of iCMs with CM markers expression, robust calcium oscillation and spontaneous beating25. Moreover, by using MGT polycistronic construct, our study avoided the use of multiple vectors and generated cells with homogenous expression ratio of G,M,T, thus providing an improved platform for cardiac reprogramming research. To increase experimental reproducibility, here we describe in detail how to isolate fibroblasts, produce retrovirus carrying MGT cassette, generate iCMs and evaluate the reprogramming efficiency.

<table border="0" cellpadding="0" cellspacing="0" width="1088">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">anti-cardiac troponin T</td>
			<td style="width:236px;">Thermo Scientific</td>
			<td style="width:200px;">MS-295-PO</td>
			<td style="width:191px;">1:200 for FACS and 1:400 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-GFP</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11122</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti- aActinin</td>
			<td>Sigma-Aldrich</td>
			<td>A7811</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Connexin43</td>
			<td>Sigma-Aldrich</td>
			<td>C6219</td>
			<td>1:500 for&#160; ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anit-Mef2c</td>
			<td>Abcam</td>
			<td style="width:200px;">ab64644</td>
			<td>1:1000 for ICC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Gata4</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:200px;">sc-1237</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Tbx5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17866</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 488&#8211;conjugated donkey anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>711-545-152</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 647&#8211;conjugated donkey anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>715-605-150</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Cytofix/Cytoperm kit for intracellular staining</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">554722</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rhod-3 Calcium Imaging Kit</td>
			<td>Life Technologies</td>
			<td style="width:200px;">R10145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thy1.2 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-049-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectashield solution with DAPI</td>
			<td>Vector labs</td>
			<td style="width:200px;">H-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td style="width:200px;">F-2442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">25-052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PRMI1640 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Life Technologies</td>
			<td style="width:200px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td style="width:200px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">30-002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11130-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11668500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">blasticidin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11139-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Millipore</td>
			<td style="width:200px;">TR-1003-G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td style="width:236px;">Fisher</td>
			<td style="width:200px;">BP151-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>PX1405</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:200px;">15714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Retrovirus Precipitation Solution</td>
			<td>ALSTEM</td>
			<td style="width:200px;">VC-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4%Trypan blue solution</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">G1393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS, 1x)</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS (Hanks Balanced Salt Solution)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">21022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LS column</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;m cellulose acetate filter&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">190-2545</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well plates</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm Tissue culture dishes&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">172958</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mm center well culture dish&#160;&#160;</td>
			<td>Corning</td>
			<td style="width:200px;">3260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Clear V-Bottom 2mL Polypropylene Deep Well Plate</td>
			<td>Denville Scientific</td>
			<td style="width:200px;">P9639</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Polystyrene round-bottom tubes with cell-strainer cap&#160;</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:200px;">5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortexer MINI</td>
			<td style="width:236px;">VWR</td>
			<td style="width:200px;">58816-121</td>
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			<td height="20" style="height:20px;">EVOS&#174; FL Auto Cell Imaging System</td>
			<td>Life Technologies</td>
			<td style="width:200px;">AMAFD1000</td>
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			<td height="20" style="height:20px;width:461px;">Round glass cover slip</td>
			<td style="width:236px;">Electron Microscopy Sciences</td>
			<td style="width:200px;">72195-15</td>
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improved generation of induced cardiomyocytes using a polycistronic construct expressing optimal ratio of gata4, mef2c and tbx5

La conversión directa de los fibroblastos cardíacos (CFS) en cardiomiocitos inducidos (ICMS) tiene un gran potencial para la medicina regenerativa al ofrecer estrategias alternativas para el tratamiento de enfermedades del corazón. Esta conversión se ha logrado mediante la expresión forzada de los factores definidos, tales como Gata4 (G), MEF2C (M) y Tbx5 (T). Tradicionalmente, los ICMs son generados por un cóctel de virus que expresan estos factores individuales. Sin embargo, la reprogramación de la eficiencia es relativamente baja y la mayor parte de la in vitro G, M, T-transduced fibroblastos no se vuelven totalmente reprogramados, lo que dificulta el estudio de los mecanismos de reprogramación. Recientemente hemos demostrado que la estequiometría de G, M, T es crucial para una eficiente reprogramación iCM. Una estequiometría óptima de G, M, T con un alto nivel relativo de M y bajos niveles de G y T obtenidos mediante el uso de nuestro vector policistrónico MGT (en lo sucesivo referido como MGT) aumentó significativamente la eficiencia de reprogramación y mejora de la calidad iCM in vitro. Aquí le ofrecemos una descripción detallada de la metodología utilizada para generar ICMS con constructo MGT de fibroblastos cardíacos. El aislamiento de los fibroblastos cardíacos, generación de virus para la reprogramación y la evaluación del proceso de reprogramación también se incluyen para proporcionar una plataforma para la generación eficiente y reproducible de ICMS.

Los pasos de reprogramación se resumen por esquema de la Figura 1. Después de la transducción de MGT, la expresión de GFP en la reprogramación de células podrían ser detectados tan pronto como día 3. puromicina selección de células transducidas comienza a partir de 3 días y se mantiene durante las dos primeras semanas si PMX-puro se utiliza constructo -MGT. Por día 10 a día 14, la expresión de los marcadores cardíacos como cTnT y αActinin podría ser detectada tanto por ...

Watch this Scientific Journal Video about Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5 at JoVE.com

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Mejora de la generación de cardiomiocitos inducida uso de un constructo Polycistronic Expresando relación óptima de Gata4, MEF2C y Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

improved-generation-induced-cardiomyocytes-using-polycistronic

Research

JoVE Journal

Developmental Biology

8.6K vistas.  University of North Carolina, Chapel Hill. We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming. 

Video: Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Derivación de cardiomiocitos altamente purificada de células madre humanas pluripotentes inducidas Uso Pequeño Diferenciación molécula modulada y posterior glucosa inanición

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Investigación

Biología del Desarrollo

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

El aislamiento y criopreservación de rata neonatal cardiomiocitos

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generación de células madre pluripotentes inducidas a partir de Frozen Buffy Coats utilizando para no integrar episomal plásmidos

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generación de células madre Integración de libre Humanos pluripotentes inducidas Usando derivada de pelo queratinocitos

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Generación de pluripotentes inducidas células madre a partir de células T periféricas Humanos Uso de Sendai virus en Condiciones-Feeder gratuita

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Generación de células madre pluripotentes inducidas por el Uso de sinoviocitos similares a fibroblastos aislados de las articulaciones de pacientes con artritis reumatoide

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Inducida por la generación de células madre pluripotentes a partir de células de la sangre usando el virus Sendai y centrifugación

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generación de Integración libres de células madre pluripotentes inducidas a partir de células mononucleares de sangre periférica humana Utilizando vectores episomales

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

La maduración de madre humanas cardiomiocitos derivado de células en Biowires uso de la estimulación eléctrica

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Generación de insulina libre de andamios, tridimensional expresar Pancreatoids de progenitores pancreáticos de ratón In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...