Agradecimentos especiais ao Dr. Boris Greber para configurar o Activin Um protocolo de ELISA, publicada no Greber et al. De 2007. Estamos particularmente gratos à Sra. Monica Shevack para preparar a visão geral gráfica. Somos muito gratos ao Dr. Heiko Fuchs por toda a ajuda e sugestões valiosas, antes e durante as filmagens. Gostaríamos de agradecer a todos os membros do laboratório Adjaye, especialmente Elisabeth Socha para manter um fornecimento constante de MEFs e CM. Reconhecemos também os nossos colegas no biotério do MPIMG por seu apoio permanente. Este trabalho foi parcialmente financiado pela Sociedade Max Planck ea [BMBF; concessão 0315717A número], parceiros da iniciativa ERASysBio + apoiado no âmbito do EU ERA-NET Plus esquema no 7 º PQ.

1. Isolamento de fibroblastos embrionários (MEFs) Os dois passos seguintes são realizadas sob condições não assépticas. <ol><li> Sacrifique um rato grávida (CF1, Harlan, EUA) em 13 ou 14 dpc (dia pós-coitum) por deslocamento cervical. </li><li> Dissecar os cornos uterinos, brevemente enxaguar em 70% (v / v) de etanol e local dentro de um tubo Falcon contendo PBS sem Ca 2 + Mg 2 + (Gibco, Invitrogen). </li></ol> Os passos seguintes são realizadas em um capuz de cultura de tecido sob condições assépticas e utilizando instrumentos estéreis. <ol start="3"><li> Coloque cornos uterinos em uma placa de Petri e separar cada embrião a partir da sua placenta e do saco embrionário. </li><li> Dissecar cabeça e os órgãos vermelhas, lave em PBS e colocar todos os embriões em placas de Petri limpa. Finamente moída o tecido usando uma lâmina de barbear estéril até que se torne possível a pipeta. </li><li> Adicionar 1 ml de 0,05% de tripsina / EDTA (Gibco, Invitrogen), including 100 unidades Kunitz de DNase I (USB), por embrião. </li><li> Transferir o tecido em um tubo Falcon 50 ml e incubar durante 15 min a 37 ° C. Após cada 5 min de incubação, dissociar as células por pipetagem de cima e para baixo completamente. </li><li> Inactivar a tripsina, adicionando cerca de 1 volume do meio de MEF recentemente preparada. MEF meio de cultura (componentes para fazer 500 ml de meio, misturar todos os componentes e filtro): 450 ml de DMEM, 50 ml de FBS (10% (v / v)), 5 ml de 200 mM de L-glutamina (1/100 (v / v)), 5 ml de penicilina-estreptomicina (1/100 (v / v)). </li><li> Centrifugar as células com baixa velocidade (300 xg), 5 min, cuidadosamente remover o sobrenadante e ressuspender o pellet em meio de células MEF quente. </li><li> Placa de aproximadamente um número de células que é equivalente a 3-4 embriões em cada balão T150 (TPP) revestidas com gelatina a 0,2% (A gelatina de pele de bovino, do Tipo B, Sigma) durante 2 horas. Os fibroblastos (P0, passagem 0) são as únicas células que têm a capacidade para ligar aos frascos revestidos a gelatina. </li&gt; <li> Idealmente, as células são de 80-90% confluentes após 24 hr e, nesta fase, uma parte importante do P0 células é congelado para uso futuro. </li><li> Expandir o balão T150 restante (s) de P0 células até P3 ou P4, em seguida, inactivar e utilização como alimentadores para replate hESCs ou para produzir o meio condicionado (CM). </li></ol> 2. Inativação e Plating MEFs (Preparação células alimentadoras) Todos os passos são realizados em uma capa de cultura de tecido sob condições assépticas. <ol><li> Casaco T150 frascos com gelatina a 0,2% e incubar à temperatura ambiente durante pelo menos 2 horas. </li><li> Diluir a mitomicina C em PBS (1 mg / ml) e filtrar. </li><li> Aspirar a mídia MEFs e lavar com PBS sem Ca 2 + Mg 2 +. </li><li> Colocar 20 ml de meio contendo 10 ug / ml de mitomicina C em MEFs. </li><li> Incubar durante 2 horas a 37 ° C com mitomicina C, em seguida, contendo o meio de lavar duas vezes com PBS, trypsinize, de centrifugação (durante 5 min a 300 xg) e Ressuspender as células em meio quente. </li><li>Contar as células e em placas a uma densidade de 56.000 células / cm 2 no T150 frascos e utilizar para a produção de CM para os seguintes 6 dias. </li></ol> 3. Meio condicionado Preparação (CM) Todos os passos são realizados em uma capa de cultura de tecido sob condições assépticas. <ol><li> No dia após plaqueamento MEFs inactivada a uma densidade de 56.000 células / cm 2 substituir o meio com meio MEF hESC (UM, meio não condicionado) (0,5 ml / cm 2) suplementado com recém-4 ng / ml de FGF2. </li><li> Collect CM partir de frascos de alimentação após 24 h de incubação e adicionar meio hESC fresco contendo 4 ng / ml de FGF2 para os alimentadores. </li><li> Repita este procedimento para os próximos 6 dias. Cada loja dia coletados CM a -20 ° C. </li><li> Após 6 dias misturar todos os alíquotas de médio e de filtro (Corning, 0,22, PAS). Fazer alíquotas de 50 ml e armazenar a -80 ° C. </li><li> Suplemento CM com 4 ng adicional / ml de FGF2 antes de adicionar a hESCs cultivadas em Matrigel. &lt;/ Li&gt; </ol> 4. Medição da Activina A em meios condicionados (ELISA) 15 <ol><li> Traga todas as amostras e reagentes à temperatura ambiente. </li><li> Diluir o anticorpo capturar (Humanos \ Mouse \ Rat Activina A MAB, R e D Systems) em PBS com BSA a 1%, adicionar a microplaca (100 uL / ​​poço) e incubar durante a noite à RT. </li><li> Após 24 hr lavar os poços três vezes com PBST (PBS com 0,05% de Tween 20) (300 ul / poço) então o bloco (1% BSA / PBS, 300 ul / poço) durante 1 h à RT. </li><li> Neste tempo preparar uma Um Activina (R e D Systems) curva padrão, incluindo 7 diluições (concentração inferior a 30 ng / ml) e amostra em branco. A gama linear de trabalho do Activina A é entre 0,25 e 32 ng / ml. </li><li> Adicionar duplicados de padrões e amostras aos poços (100 ul / poço) e incubar durante 2 horas à RT. </li><li> Lavar os poços três vezes (300 uL de PBST). </li><li> Adicione o anticorpo (biotinilado) secundário (Mouse / Humano / Rato biotinilado Activina A MAB, R &amp; D Systems) (0.25 ug / ml em 1% de BSA / PBS) e incubar durante 2 horas à RT. </li><li> Lavar os poços três vezes (300 uL de PBST), adicionar Estreptavidina-HRP (diluído em BSA a 1% / PBS, R e D Systems) e incubar durante 20 min à TA. </li><li> Lavar os poços três vezes (300 uL de PBST), adicionar 100 uL de solução de substrato (Quantikine, R e D Systems) e incubar durante 30 min à temperatura ambiente no escuro. </li><li> Adicione 100 ml de solução de paragem (Quantikine, R &amp; D Systems) para cada poço e misture delicadamente. </li><li> Conjunto Microplate Reader (Molecular Devices Spectra Max 250, Instrumentação Médica Global, Inc., Minnesota) a 450 nm, com correcção de comprimento de onda a 540 ou 570 nm, para determinar a densidade óptica de cada poço. </li></ol> 5. Os resultados representativos O esquema geral do procedimento de isolamento é apresentada na Figura 1. A morfologia típica de hESCs e hiPSCs cultivados em condições diferentes é apresentado na Figura 2. A morfologia do<html> MEFs e células alimentadoras inactivadas utilizados para preparar CM é apresentada na Figura 3. Em geral, as células devem ser confluente de 24 horas pós-isolamento e pronto para ser congelada ou expandido. No entanto, às vezes isso pode levar 2-3 dias antes da obtenção de culturas confluentes. O CM deve ser preparados a partir de células em passagem 4 e não depois. Isto é crucial porque as células primárias só pode ser expandido para 4-5 passagens antes do início da senescência. A citoquina, Activina A, é considerado como o factor mais crítico secretada por células de alimentação para o suporte do crescimento de células indiferenciadas pluripotentes 14. A medição do nível de Activina A em CM (Figura 4) é um ensaio muito conveniente quantitativo para controlar a qualidade dos MEFs. <img alt="A Figura 1" src="/files/ftp_upload/3854/3854fig1.jpg" /> Figura 1. Uma representação esquemática do procedimento de isolamento MEFs. ONTEÚDO &quot;&gt; <img alt="A Figura 2" src="/files/ftp_upload/3854/3854fig2.jpg" /> Figura 2. A morfologia típica de hESCs indiferenciadas cultivadas na presença de células (A) de alimentação, (B) meio condicionado e médios (C) definido. A morfologia típica de hiPSCs cultivadas na presença de células (D, E) de alimentação. <img alt="A Figura 3" src="/files/ftp_upload/3854/3854fig3.jpg" /> Figura 3. A morfologia típica de fibroblastos de rato embrionários (MEFs). (A) Passagem 0 (P0), dois dias após o plaqueamento / isolamento, (B) camada alimentadora inactivada a uma densidade de 56.000 células / cm. 2 <img alt="A Figura 4" src="/files/ftp_upload/3854/3854fig4.jpg" /> Figura 4. Enzyme-linked immunosorbent assay (ELISA)-baseados medições da concentração de Activina A em meio condicionado (CM) prepared com fibroblastos de rato embrionários provenientes da estirpe de ratinhos CF1. CM foi recolhido durante 6 dias e depois reunidas. CM &quot;1&quot; CM e &quot;2&quot; se referem a diferentes lotes de meios de comunicação e da UM para a mídia incondicionados. Como a função do processo de condicionamento é Activina A secreção para o meio de pelo MEFs, Activina A é quase indetectável em UM.

<ol>
	<li>Takahashi, K., Yamanaka, S. <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+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonally+derived+human+embryonic+stem+cell+lines+maintain+pluripotency+and+proliferative+potential+for+prolonged+periods+of+culture.">Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.</a> Dev. Biol. 227, 271-278 (2000).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Ludwig, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+in+defined+conditions.">Derivation of human embryonic stem cells in defined conditions.</a> Nat. Biotechnol. 24, 185-187 (2006).</li><li>Yao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+and+directed+differentiation+of+human+embryonic+stem+cells+in+chemically+defined+conditions.">Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 6907-6912 (2006).</li><li>Lu, J., Hou, R., Booth, C. J., Yang, S. H., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+culture+conditions+of+human+embryonic+stem+cells.">Defined culture conditions of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 5688-5693 (2006).</li><li>Skottman, H., Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+human+embryonic+stem+cells.">Culture conditions for human embryonic stem cells.</a> Reproduction. 132, 691-698 (2006).</li><li>Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeders+support+prolonged+undifferentiated+growth+of+human+inner+cell+masses+and+embryonic+stem+cells.">Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.</a> Nat. Biotechnol. 20, 933-936 (2002).</li><li>Richards, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+various+human+feeders+for+prolonged+undifferentiated+growth+of+human+embryonic+stem+cells.">Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.</a> Stem Cells. 21, 546-556 (2003).</li><li>Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+layers+for+human+embryonic+stem+cells.">Human feeder layers for human embryonic stem cells.</a> Biol. Reprod. 68, 2150-2156 (2003).</li><li>Inzunza, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cell+lines+in+serum+replacement+medium+using+postnatal+human+fibroblasts+as+feeder+cells.">Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells.</a> Stem Cells. 23, 544-549 (2005).</li><li>Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Human+Amniotic+Mesenchymal+Cells+as+Feeder+Layers+to+Sustain+Propagation+of+Human+Embryonic+Stem+Cells+in+the+Undifferentiated+State.">Utilization of Human Amniotic Mesenchymal Cells as Feeder Layers to Sustain Propagation of Human Embryonic Stem Cells in the Undifferentiated State.</a> Cell Reprogram. , (2011).</li><li>Eiselleova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+mouse+and+human+feeder+cells+for+human+embryonic+stem+cells.">Comparative study of mouse and human feeder cells for human embryonic stem cells.</a> Int. J. Dev. Biol. 52, 353-363 (2008).</li><li>Greber, B., Lehrach, H., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+2+modulates+transforming+growth+factor+beta+signaling+in+mouse+embryonic+fibroblasts+and+human+ESCs+(hESCs)+to+support+hESC+self-renewal.">Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal.</a> Stem Cells. 25, 455-464 (2007).</li><li>Vallier, L., Alexander, M., Pedersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin/Nodal+and+FGF+pathways+cooperate+to+maintain+pluripotency+of+human+embryonic+stem+cells.">Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells.</a> J. Cell Sci. 118, 4495-4509 (2005).</li></ol>

Não há conflitos de interesse declarados.

O processo de isolamento MEFs aqui apresentado permite o estabelecimento de uma condição de cultura padronizada para hESCs e hiPSCs. Além disso, o sistema de ELISA baseado utilizado para avaliar a produção de citocinas pelas células de alimentação é um indicador útil da qualidade dos meios de comunicação MEF derivados condicionados. A manutenção de rotina da estirpe de rato (CF1) proporcionando fibroblastos de suporte é necessário para evitar lote para lote variação de meios de cultura. Além disso, recomenda-se para isolar os embriões de ratinhos vários simultaneamente para se obter uma qualidade consistente de células. Certamente MEFs recentemente isolado pode ser mantido congelado a P0 e P1. MEFs também inactivado pode ser mantida congelada em alíquotas de quantidades apropriadas, dependendo dos requisitos para a cultura de células. Normalmente, aproximadamente 250,000 células devem ser revestida em um único poço de uma placa de 6 poços para hESCs cultura e células iPS. O método estabelecido e optimizado podendo ser utilizado rotineiramente para minimizar a variabilidade entre difealugar experimentos.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DMEM (glicose alta) </td><td> Gibco, Invitrogen </td><td> 41966-052 </td><td></td></tr><tr><td> FBS </td><td> Biochrom </td><td> S0115 </td><td></td></tr><tr><td> L-glutamina </td><td> Gibco, Invitrogen </td><td> 25030-024 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Gibco, Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Sistema de filtro a vácuo </td><td> Corning </td><td> 431097 </td><td> 500 ml, 0,22, PAS </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650 </td><td></td></tr><tr><td> Knockout DMEM </td><td> Gibco, Invitrogen </td><td> 10829-018 </td><td></td></tr><tr><td> Ser Knockouthum substituição </td><td> Gibco, Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Non-aminoácidos essenciais </td><td> Gibco, Invitrogen </td><td> 11140-035 </td><td></td></tr><tr><td> beta-mercaptoetanol </td><td> Sigma </td><td> M7522 </td><td></td></tr><tr><td> PBS </td><td> Gibco, Invitrogen </td><td> 14190-169 </td><td></td></tr><tr><td> DNase I </td><td> Sigma </td><td> D4527 </td><td></td></tr><tr><td> A mitomicina C </td><td> Roche </td><td> 10107409001 </td><td></td></tr><tr><td> Factor de crescimento de fibroblasto básico </td><td> PeproTech </td><td> 100-18B </td><td></td></tr><tr><td> Biotinilado Anti-human/mouse/rat Activina Um anticorpo </td><td> R &amp; D Systems </td><td> BAM3381 </td><td></td></tr><tr><td> Gelatina </td><td> Sigma </td><td> G9391 </td><td></td></tr><tr><td> Mouse / Humano /Rat Activina A MAb </td><td> R &amp; D Systems </td><td> MAB3381 </td><td></td></tr><tr><td> 0,05% de tripsina-EDTA </td><td> Gibco, Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Extra Fino tesoura de íris </td><td> FST </td><td> 14088-10 </td><td></td></tr><tr><td> Extra Fino Pinça Graefe </td><td> FST </td><td> 11150-10 </td><td></td></tr><tr><td> Pierse Pinças de fixação </td><td> FST </td><td> 18155-13 </td><td></td></tr></tbody></table>

preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells

Em geral, as células estaminais embrionárias (hESCs) e células estaminais pluripotentes induzidas (hiPSCs) 1 podem ser cultivadas sob condições variáveis. No entanto, não é fácil para estabelecer um sistema eficaz para a cultura dessas células. Uma vez que as condições de cultura pode influenciar a expressão do gene que confere pluripotência em hESCs e hiPSCs, a otimização e padronização do método de cultura é crucial. O estabelecimento de linhas de células estaminais embrionárias humanas foi descrita pela primeira vez utilizando MEFs como células de alimentação e de soro fetal bovino (FBS)-2 contendo meio de cultura. Em seguida, foi substituído com FBS a substituição knockout soro (KSR) e FGF2, o que aumenta a proliferação de hESCs 3. Finalmente, os sistemas de alimentação de cultura sem permitir a cultura de células em Matrigel placas revestidas em KSR contendo meio condicionado (meio condicionado por MEFs) 4. Posteriormente, as condições de cultura hESCs se mudaram para alimentador de cultura livre em definidores quimicamentecondições ed 5-7. Além disso, para evitar a contaminação potencial por patógenos e métodos de animais proteínas de cultura utilizando xeno-free componentes foram estabelecidas 8. Para se obter melhores condições células alimentadoras do rato foram substituídos com linhas de células humanas (por exemplo, músculo fetal e células da pele 9, as células adultas da pele 10, fibroblastos de prepúcio 11-12, amnióticas células mesenquimais 13). Contudo, a eficiência de manter hESCs indiferenciadas usando prepúcio humano camadas de fibroblastos derivados de alimentação não é tão elevada como a partir de células de ratinho de alimentação devido ao baixo nível de secreção de Activina A 14. Obviamente, há uma evidente diferença na produção de fator de crescimento pelo rato e células alimentadoras humanos. A análise dos transcriptomas de rato e células alimentadoras humanos revelou diferenças significativas entre as células de apoio e não apoio. Exógena FGF2 é crucial para a manutenning auto-renovação das hESCs e hiPSCs, e tem sido identificada como um factor chave regulando a expressão de TGF-p1, Uma Activina e Gremlin (um antagonista de BMP) em células de alimentação. Activina A tem sido mostrado para induzir a expressão de Oct4, SOX2, e NANOG em hESCs 15-16. Para a cultura de longo prazo, e hESCs hiPSCs podem ser cultivadas em MEFs mitoticamente inactivado ou sob condições isentas de alimentador em MEF-CM (MEF-meio condicionado) em Matrigel placas revestidas para manter o seu estado não diferenciado. Sucesso de ambas as condições de cultura depende inteiramente da qualidade das células de alimentação, uma vez que afecta directamente o crescimento de hESCs. Aqui, nós apresentamos um método optimizado para o isolamento e cultura de fibroblastos de rato embrionários (MEFs), a preparação de meio condicionado (CM) e enzyme-linked immunosorbent assay (ELISA) para avaliar os níveis de Activina A dentro dos meios de comunicação.

Watch this Scientific Journal Video about Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells at JoVE.com

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

A qualidade de fibroblastos embrionários (MEFs) é ditada pela estirpe direito de rato tal como CF-1. Pluripotência-MEFs apoio e meios condicionados (CM), obtida a partir destes deve conter concentrações ótimas de Activin A, Gremlin e TGF-p1 necessários para a Activin / Nodal e vias FGF para co-operatório manter a auto-renovação e pluripotência.

Preparação de células embrionárias de camundongos Fibroblastos adequados para a cultura de células-tronco embrionárias humanas e pluripotentes induzidas

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

preparation-mouse-embryonic-fibroblast-cells-suitable-for-culturing

Research

JoVE Journal

Biology

90.4K visualizações.  Max Planck Institute for Molecular Genetics. A qualidade de fibroblastos embrionários (MEFs) é ditada pela estirpe direito de rato tal como CF-1. Pluripotência-MEFs apoio e meios condicionados (CM), obtida a partir destes deve conter concentrações ótimas de Activin A, Gremlin e TGF-p1 necessários para a Activin / Nodal e vias FGF para co-operatório manter a auto-renovação e pluripotência.

Vídeo: Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

Cultura e Manutenção de células estaminais embrionárias humanas

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

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparação Spread cromossômica de células estaminais embrionárias humanas para Cariotipagem

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e descongelamento, células estaminais embrionárias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

Transfectando e Nucleofecting células-tronco pluripotentes induzidas

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

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...