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.

O protocolo descrito aqui utiliza camundongos recém-nascidos. Cuidados com os animais e as experiências sejam realizadas em conformidade com as diretrizes estabelecidas pela Divisão de Laboratório de Medicina Animal (DLAM) na Universidade da Carolina do Norte, Chapel Hill. 1. Preparação de amortecedores e Mídia <ol><li> Prepare de fibroblastos (FB) forma: Suplemento 500 ml de meio IMDM com 100 ml Soro Fetal Bovino (FBS) e 6 ml de penicilina / estreptomicina. </li><li> Prepare médio iCM: Suplemento 400 ml meio DMEM com 100 ml de mídia M199 e 50 ml FBS. </li><li> Prepare meio B27: Suplemento 490 ml de mídia RPMI1640 com 10 ml de meio B27. </li><li> Prepare Plat-E meio de cultura: 500 mL de Suplemento meio DMEM com 50 ml de FBS, 5 ml penicilina / estreptomicina e 5 ml de aminoácidos não-essenciais. </li><li> Prepare Plat-E meio de transfecção: Suplemento 500 ml meio DMEM com FBS a 50 ml e 5 ml de aminoácidos não-essenciais. </li><li> Prepare revestidos com gelatina placa de 24 poços: Adicionar 0,5 ml de solução de gelatina a 0,1% para um poço de placa de 24 poços, incubar a 37 ° C durante pelo menos 10 min e aspirado imediatamente antes da utilização. </li><li> Preparar tampão de marcação FACS: Suplemento 500 ml de DPBS com FBS a 10 ml e 2 ml de EDTA (0,5 M) até atingir uma concentração final de 2 mM. Filtrar através de um filtro de 0,22? M e armazenamento a 4 ° C. </li><li> Preparar tampão de triagem MACS: Suplemento 500 ml de DPBS com 2,5 g de BSA e 2 ml de EDTA (0,5 M) até atingir uma concentração final de 2 mM. Filtrar através de um filtro de 0,22? M e armazenamento a 4 ° C. </li><li> Preparar tampão HBS 2x para a transfecção: Suplemento 500 ml de tampão HEPES (50 mM) com NaCl 280 mM (8,1816 g), 10 mM de KCl (0,37275 g), 1,5 mM de Na 2 HPO 4 (0,10647 g), Glicose 12 mM (1,08096 g ). Adicionar cerca de 650 ul de NaOH 10 N para ajustar o pH a 7,05-7,12. Filtrar através de um filtro de 0,22? M de poro e armazenar a 4 ° C. </li><li> Prepare 2,5 M CaCl 2 solução para a transfecção: Adicione 18,376 g CaCl 2 a 50 ml de ddH 2 O. Filtrar através de um filtro de 0,22? M de poro e armazenar a 4 ° C. </li><li> Prepare TPI (imunocitoquímica) tampão de fixação: Adicionar 5 ml de paraformaldeído (PFA, 32%) a 35 ml de DPBS para se atingir uma concentração final de 4%. </li><li> Preparar tampão de permeabilização ICC: Adicionar 0,1 mL de Triton a 100 ml de DPBS para se atingir uma concentração final de 0,1%. </li><li> Preparar um tampão de bloqueio ICC: Adicionar 5 g de albumina de soro bovino (BSA) a 100 ml de DPBS para se atingir uma concentração final de 5%. </li><li> Preparar tampão de coloração ICC: Adicionar 1 g de BSA para 100 ml de DPBS para se atingir uma concentração final de 1%. </li></ol> 2. Geração de Neonatais mouse fibroblastos cardíacos <ol style="margin-left: 40px;"><li> Gerar fibroblastos cardíacos de método de cultura de explante <ol><li> Limpe neonatal αMHC-GFP camundongo transgênico (P1 a P2) com etanol 75%. Cortar a cabeça com uma tesoura esterilizada. Em seguida, faça uma incisão horizontal debaixo de uma axila para o outro. Use um estérilfórceps sem corte e dobrados para dissecar o coração e colocá-lo em um poço da placa de 24 poços contendo tampão PBS gelado. <ol><li> Alternativamente, espremer o coração após a incisão horizontal. </li></ol></li><li> Verifique expressão da GFP no coração por microscópio de fluorescência. Uma vez que a expressão da GFP é accionado por αMHC promotor que é um promotor específico cardíaco, o coração de ratinho transgénico deve ser em verde 15, enquanto o coração é negativa fraca em qualquer fluorescência. </li><li> Tome 3-4 GFP corações positivos em 60 mm centro bem prato de cultura e pique-os em pedaços pequenos menos de 1 mm 3 em tamanho por tesouras e pinças esterilizadas. </li><li> Coloque 3-4 corações picada em uma placa de 10 cm com 2 ml de mídia FB. Deixe os tecidos se estabelecer durante 3 horas. </li><li> Lentamente, adicionar 8 ml de mídia FB pré-aquecido para os pratos que contenham tecidos cardíacos. Não perturbe os tecidos por três dias. </li><li> Substitua o material a cada três dias. </li><li> No dia 7, ASPIRcomeu o meio de cultura e lavar as células com DPBS. Adicionar 3 ml de 0,05% de tripsina a cada placa e digerir a 37 ° C durante 5 min. </li><li> Adicionar 5 ml de mídia FB para saciar a tripsina. Suavemente separar as células com um raspador de células. Pipetar os meios de comunicação cima e para baixo para dissociar ainda mais mecanicamente o tecido. </li><li> Recolher as células e filtrar através de filtros de 40 uM de células para evitar a contaminação de fragmentos de tecido do coração, e, em seguida, peletizar as células por centrifugação a 200 xg durante 5 min. </li><li> Lave as células uma vez com MACS tampão e células estão prontas para a classificação. </li></ol></li><li> Gerar fibroblastos cardíacos de método de digestão enzimática <ol><li> Colheita GFP corações positivos como 2.1.1. Transferir todos os corações em uma placa de 10 cm com 10 ml DPBS geladas. </li><li> Esprema ventrículos com uma pinça estéril para remover o sangue e lavar uma vez com DPBS geladas. </li><li> Apare os corações para ser livre de outros tecidos e gordura. </li><li> Corte cada coração em cerca de 4 peças que ainda são frouxamente ligados. </li><li> Se menos de 20 corações, transferir os corações em um tubo de 15 ml com 8 ml quente 0,05% de tripsina-EDTA, e incuba-se a 37 ° C durante 15 min. <ol><li> Se houver 20-30 corações, transferir os corações em um tubo de 50 ml com 10 ml quente 0,05% de tripsina-EDTA, e incuba-se a 37 ° C durante 15 min. </li></ol></li><li> Descartar a tripsina e adicionam-se 5 ml (para menos de 20 corações) ou (10 ml) durante 20-30 corações quente colagenase tipo II (0,5 mg / ml) em HBSS. </li><li> Vortex o tubo em vortex durante 1 min a um nível de velocidade de cerca de 4 a 6 (se o líquido não se levantar para a tampa, em seguida, a velocidade é fina). </li><li> Incubar o tubo 37 ° C em banho de água durante 3-5 min. </li><li> Vortex o tubo durante 1 min. </li><li> Sedimento durante 1 min por gravidade até que os pedaços de tecido sossegar, recolher o líquido em um novo tubo contendo 5 ml de meio FB frio. </li><li> Repita os passos 3-5 2.2.6-2.2.10 vezes. </li><li> Combine todas as coleções, filtrando através de 40 um filtro de célulaspara fazer a suspensão de célula única. </li><li> Centrifuga-se a 200 xg durante 5 min a 4 ° C. </li><li> Ressuspender as células em 10 ml de tampão MACS, e centrifugar a 200 xg durante 5 min a 4 ° C. </li><li> Opcional: Se há um grande número de células do sangue, Ressuspender as células com 1 ml de tampão de lise de RBC (150 mM de NH4Cl, 10 KHCO 3 mM, e EDTA 0,1 mM), manter em gelo durante 1 min, em seguida, dilui-se com 10 ml MACS tampão e centrifugar a 200 xg durante 5 min. Lave mais uma vez com tampão de MACS. </li></ol></li><li> Isolamento de fibroblastos THY1.2 + por MACS (triagem magnética-activada de células) <ol><li> Determinar o número de células viáveis ​​através de coloração azul de tripano. <ol><li> Tome 10 células ul a partir de suspensão de células 10 ml na etapa 2.2.14. Misturá-lo com solução de azul de 10 mL de 0,4% tripano. </li><li> Adicione a mistura para um hemocitômetro. Deixe-a repousar durante 3-5 minutos e depois examinar imediatamente sob um microscópio. As células mortas são manchadas em células azuis e viáveis ​​são imaculado. </li><li&gt; Conte todas as células viáveis ​​em quatro quadrados 1 milímetro de canto do hemocitômetro. Determinar o número de células viáveis: a contagem média por metro quadrado x 2 (factor de diluição) x 10 4 x 10 (volume total de suspensão de células). </li></ol></li><li> Por menos de 1 x 10 7 células, as células ressuspender com 10 ul microbeads THY1.2 em 90 ul refrigerados tampão MACS. Adicionar mais grânulos proporcionalmente se houver mais do que 1 x 10 7 células. Misturar bem e incubar num frigorífico (2-8 ° C) durante 30 min. </li><li> Adicionar 10 ml de tampão e MACS amostras de rotação a 200 g durante 5 min; lavar uma vez com tampão de MACS 10 ml e centrifugar novamente. </li><li> Levar o volume a 2 ml em tampão MACS. </li><li> Configurar uma Separator MACS na capa. Inserir uma coluna LS para o separador. Aplicar 3 ml MACS tampão para a coluna para equilibrar isso. </li><li> Passar a suspensão de células através da coluna LS equilibrada. </li><li> Lavar 3 vezes com 2 ml de tampão de MACS cada vez. </li><li> Tome a coluna LS fora tSeparador ele e elui-se 3 vezes com 2 ml de tampão MACS para um novo tubo de 50 ml. </li><li> Gire a 200 xg durante 5 min. </li><li> Ressuspender as células em 10 ml de mídia FB, contar as células e sementes em placas a densidade adequada. Para fibroblastos gerados a partir de método de cultura de explante, a densidade de sementeira de células poderia ser de cerca de 2-3 x 10 4 células / poço de placa de 24 poços. Para fibroblastos gerados a partir de método de digestão enzimática, a densidade de células poderá ser cerca de 4-5 x 10 4 células / poço de placa de 24 poços. </li></ol></li></ol> 3. Geração de Retrovirus para iCM Reprogramação Nota: Execute as seguintes etapas em um BSL2 Gabinete de Segurança Biológica em condições estéreis. O descarte adequado de células transfectadas, pontas para pipetas e tubos é recomendado para evitar o risco de riscos ambientais e de saúde. <ol style="margin-left: 40px;"><li> Manter células Plat-E em meio Plat-E suplementado com 1 ug / ml de puromicina e 10 ug / mL blasticidin a 37 ° C com 5% de CO 2. </li><li> No dia 2, substituir o meio por Plat-E meio de cultura sem puromicina e blasticidina. </li><li> No dia 0, dividir células Plat-E em placas de 10 cm no dia antes da transfecção, aproximadamente 4-5 x 10 6 culas por placa. As células devem ser ~ 80-90% confluentes no dia da transfecção. </li><li> No dia 1, os meios para mudança de cultura meio de transfecção em células dentro de 1 h antes da transfecção. Procedimento de transfecção é o seguinte: <ol><li> A transfecção usando kits comerciais <ol><li> Misturar 20 ul de reagente de transfecção (por exemplo, de Lipofectamine) e 500 ul de meio de soro reduzido (por exemplo, Opti-MEM). Adiciona-se 10 ug do plasmídeo retroviral para mais 500 ul de meio de soro reduzido. Incubar cada mistura à temperatura ambiente (TA) durante 5 min. </li><li> Adiciona-se lentamente a mistura de plasmídeo a mistura de transfecção. Esta etapa pode demorar 15-30 segundos. Incubar a mistura durante 20 min à temperatura ambiente (solução pode aspecto turvo). </li&gt; <li> Adicionar gota mistura sábia para as células. </li><li> Incubar durante a noite numa incubadora a 37 ° C. </li></ol></li><li> A transfecção com fosfato de cálcio. <ol><li> Misturar 40 ul de 2,5 M de CaCl2 e 440 uL ddH2O, em seguida, adiciona-se 20 ug do plasmídeo retroviral (ajustar a quantidade de ddH2O modo que o volume total seja de 500 uL). </li><li> Adicionar 500 mL 2x HBS a um tubo de 15 ml. </li><li> Vortex da HBS e, entretanto, adicione a mistura de cálcio-ADN para a HBS gota a gota. Esta etapa demora cerca de 30 segundos para cada amostra. Assim maximamente fazer 3-4 amostras simultaneamente. </li><li> Incubar à temperatura ambiente durante 3 minutos de incubação por mais tempo leva a maior precipitado e menos células vai ocupar o precipitado. </li><li> Adicione a mistura gota a gota às células e observar ao microscópio 20X. Precipitados finos (um monte de pequenos são bons, mas alguns grandes não são bons) deve ser visto. </li><li> Incubar durante a noite a 37 ° C incubadora. </li&gt; </ol></li></ol></li><li> No dia 2, mudar meios sobre as células, com 8 ml de meio de cultura previamente aquecido. Incubar durante 24 h. </li><li> No dia 3 e no dia 4, recolher o sobrenadante da cultura a partir das placas, utilizando uma seringa descartável de 10 ml estéril, filtrando-a através de um filtro de acetato de celulose de 0,45 um de tamanho de poro, e transferir para um tubo de 50 ml. Adicionar 2 ml de solução de precipitação de retrovírus para cada sobrenadante contendo vírus de 8 ml. Suavemente misturá-lo e incubar durante a noite a 4 ° C. </li><li> No dia 5, a mistura viral girar a 1.500 x g a 4 ° C durante 30 min. As partículas virais devem aparecer no sedimento branco no fundo do tubo. </li><li> Desprezar o sobrenadante. Girar solução residual por centrifugação a 1500 × g durante 5 min. Remover todos os vestígios de líquido por aspiração, tomando muito cuidado para não perturbar as partículas retrovirais precipitados em pelota. </li><li> Ressuspender retrovirais peletes com tampão de DPBS 100 uL para cada 8 ml de sobrenadante viral. Alíquotao vírus em gelo. Utilizar imediatamente ou armazenar a -80 ° C. </li></ol> 4. A reprogramação de fibroblastos cardíacos <ol><li> Adicione 4 ml de solução polibrene 10 mg / ml para o meio iCM 10 ml, e misture delicadamente por pipetagem cima e para baixo. A concentração final de polibreno é de 4 ug / ml. </li><li> Adicionar 500 ul de meios iCM contendo polibreno a cada poço da placa de 24 poços semeada com fibroblastos. </li><li> Adicionar 10 ul a cada poço retrovírus contendo os 2-3 x 10 4 células de cultura de explante ou 4-5 x 10 células de 4 método de digestão enzimática. A quantidade de vírus deve ser aumentada proporcionalmente com o aumento do número de células em placas de cultura ou diferentes pratos. Colocar a placa numa incubadora a 37 ° C durante 24-48 h, para permitir que a infecção virai. A linha do tempo para a reprogramação cardíaca é mostrado na Figura 1. </li><li> Depois de transdução viral, substitua vírus contendo mídia com 500 mL de mídia regulares iCM. </li><li> Mudança de mídia a cada 2-3 dias. Para a selecção positiva de células transduzidas virais, adicionar ICM meios suplementados com puromicina a 2 ug / ml para as células alvo. Mantê-lo por três dias. Depois disso, manter a puromicina na forma iCM na concentração de 1 ug / ml. </li><li> Três dias após a infecção virai, tomar a placa para fora da incubadora e colocá-lo sob um microscópio de fluorescência invertido (20X) para observar a expressão da GFP. Nota: Dez dias após a infecção virai, as células podem ser colhidas para análise FACS e análise ICC para determinar a eficiência de reprogramação (no passo 5 e 6). </li><li> Quatorze dias após a infecção viral, substituir a mídia iCM com a mídia B27. Mudança de mídia a cada três dias. Batimento espontâneo loci célula pode aparecer a partir de três células (método de digestão enzimática) ou quatro (células de cultura método explante) semanas após a transdução virai. </li></ol> 5. Immunocytochemical Análise de Eficiência Reprogramação <ol><li> Células de enxágüecom PBS gelado três vezes; remover o excesso de solução. </li><li> Adicionar 0,5 ml de tampão de fixação ICC para cada poço de placas de 24 poços. Fixar as células à temperatura ambiente durante 15-20 min. </li><li> Enxaguar as células três vezes com PBS. </li><li> Adicionar 0,5 ml de tampão de permeabilização ICC para cada poço das placas de poços 24. Permeabilizar as células à TA durante 15 min. </li><li> Enxaguar as células três vezes com PBS. </li><li> Adicionar 0,5 ml de ICC tampão de bloqueio a cada poço da placa de 24 poços, incubar à TA durante 1 h. </li><li> Anticorpos primários diluídos com tampão de coloração TPI. Adicionar 200 ul de soluções de anticorpo a cada poço e incubar durante a noite a 4 ° C. Os factores de diluição de anticorpo estão listados em materiais. </li><li> No dia seguinte, lavar as células com PBS três vezes. </li><li> Adicionar 200 ul de soluções de anticorpo secundário para as células e incuba-se durante 1 h à TA em escuro. </li><li> Lavar as células com PBS três vezes. </li><li> Adicionar 150 ul de solução contendo DAPI para cada cavidade de montagem. Permitir que a mancha núcleos durante 1 min. Entãocobrir cada poço com rodada tampa de vidro deslizante e pressione suavemente o deslizamento contra a superfície inferior com uma pinça para remover bolhas de ar. </li><li> Aspirar o excesso de solução e as amostras estão prontas para imagiologia. As imagens representativas que mostram as células que expressam o marcador reprogramadas cardíaca e TnT αActinin em D14 são mostrados na Figura 2B. </li></ol> 6. FACS análise da eficiência Reprogramação <ol><li> Lave as células uma vez com DPBS. Adicionar 0,3 ml de tripsina (0,05%) a cada poço e incuba-se a 37 ° C durante 5 min. </li><li> Bata suavemente na placa para facilitar o levantamento das células. Verifique o desprendimento de células ao microscópio. Adicionar 1 mL de tampão de FACS a cada poço quando a maioria das células são dissociadas. Pipeta-se para baixo e para dissociar mais mecanicamente as células. </li><li> Transferir as células na placa de 24 poços para uma placa de 96 poços poço profundo poço a poço. Rotação a 200 xg durante 5 min a 4 ° C para recolher as células. </li><li> Lavar as células uma vez comTampão FACS. </li><li> Adicionar soluções de fixação 100 / ul permeabilização para ressuspender as células durante 20 min a 4 ° C. </li><li> Lave as células duas vezes com solução de lavagem 1x, pelota, e remover o sobrenadante. </li><li> Prepare solução de anticorpo primário contra GFP e cTnT em tampão de lavagem 1x. Ressuspender cuidadosamente cada uma das amostras com uma solução de anticorpo de 50 ul e incubar a 4 ° C durante 30 min. </li><li> Lave as células uma vez em solução de lavagem 1x, pelota, e remover o sobrenadante. </li><li> Adicionar 50 ul de solução de anticorpo secundário contendo Alexa Fluor 488 conjugado de IgG de burro anti-coelho Alexa Fluor 647 e burro conjugado anti-IgG de ratinho a cada amostra. Incubar a 4 ° C durante 30 min no escuro. </li><li> Lave as células uma vez em solução de lavagem 1x, pelota, e remover o sobrenadante. </li><li> Ressuspender as células em tampão 400 ul de fixação (DPBS com 1% de PFA). Transferência de células para dentro do tubo FACS com tampa de filtro de células. As amostras estão prontos para a detecção de FACS. A análise FACS representante de reprogramação eficy usando pMxs-puromicina-construção MGT com ou sem puromicina selecção é mostrado na Figura 2A. </li></ol>

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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 a geração iCM bem sucedido ao usar este protocolo, existem vários fatores importantes que têm um impacto sobre a eficiência global. Particularmente as condições de fibroblastos de partida e da qualidade de codificação retrovírus MGT pode afetar consideravelmente a eficiência de reprogramação. É importante para gerar fibroblastos tão fresco e saudável possível. Para o método de cultura de explante, os fibroblastos podem ser usadas antes de sete dias depois de os explant...

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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EVOS&#174; FL Auto Cell Imaging System</td>
			<td>Life Technologies</td>
			<td style="width:200px;">AMAFD1000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MidiMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
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improved generation of induced cardiomyocytes using a polycistronic construct expressing optimal ratio of gata4, mef2c and tbx5

Conversão directa de fibroblastos cardíacos (CFS) em cardiomiócitos induzida (ICMS) tem um grande potencial para a medicina regenerativa, oferecendo estratégias alternativas para o tratamento de doenças cardíacas. Esta conversão foi conseguida através da expressão forçada de factores tais como definidos GATA4 (L), MEF2C (M) e Tbx5 (T). Tradicionalmente, iCMS são gerados por um cocktail de vírus que expressam esses factores individuais. No entanto, a eficiência da reprogramação é relativamente baixa e a maior parte do in vitro, G, M, T transduzidas fibroblastos não se tornem totalmente reprogramado, o que torna difícil estudar os mecanismos de reprogramação. Recentemente, têm mostrado que a estequiometria de L, M, T, é crucial para a eficiente reprogramação MIC. Um estequiometria óptima de G, M, T com alto nível relativo de M e baixos níveis de G e T obtidos usando nosso vetor MGT policistrónico (doravante referida como MGT) aumentou significativamente a eficiência de reprogramação e melhoria da qualidade iCM in vitro. Aqui nós fornecemos uma descrição detalhada da metodologia utilizada para gerar ICMS com MGT construção de fibroblastos cardíacos. Isolamento de fibroblastos cardíacos, geração de vírus de reprogramação e avaliação do processo de reprogramação também estão incluídos para fornecer uma plataforma para a geração eficiente e reprodutível de ICMS.

Os passos de reprogramação são resumidos por esquemática na Figura 1. Após a transdução MGT, a expressão da GFP em células reprogramação pode ser detectada tão cedo como o dia 3. A puromicina selecção de células transduzidas é iniciado a partir do dia 3 e é mantida durante as duas primeiras semanas, se pMX-puro constructo -MGT é usado. Por volta do dia 10 até ao dia 14, a expressão dos marcadores cardíacos como TnT e αActinin poderia ser detectado por ambos TPI <st...

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.

Melhoria Geração de Induced Cardiomyocytes utilizando uma construção policistrónico Expressando proporção ideal de GATA4, MEF2C e 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 visualizações.  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. 

Vídeo: 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.

Derivação de Cardiomyocytes altamente purificada de induzidas pelo homem pluripotentes células estaminais usando pequenos Diferenciação modulada-Molecule e Subsequente Glucose Fome

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

Biologia do Desenvolvimento

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.

O isolamento e criopreservação de Neonatais Rato Cardiomyocytes

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.

Geração de pluripotentes induzidas a partir de células-tronco Congelado Buffy Coats usando não-integração epissomais Plasmids

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.

Geração de Integração Induced-livre Human células estaminais pluripotentes usando o cabelo derivado queratinócitos

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.

Geração de pluripotentes induzidas a partir de células-tronco periférico humano T células usando Sendai Virus em condições livres de Alimentador

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.

Geração de células-tronco pluripotentes induzida por Usando sinoviócitos semelhantes a fibroblastos Isolado de articulações de pacientes com artrite reumatóide

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.

Induzida Geração Stem Cell pluripotentes a partir de células de sangue com o vírus Sendai e centrifugação

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.

Geração de Integração-livres induzidas células-tronco pluripotentes a partir de células mononucleares de sangue periférico humano Usando epissï¿½ica Vectors

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.

Maturação de cardiomiócitos estaminais humanas derivadas de células em Biowires Usando Electrical Stimulation

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.

Geração de insulina tridimensional, livre de andaime expressando Pancreatoids do Mouse progenitoras pancreáticas em 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 ...