We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

1 Preparação de Meios de Cultura, Citocinas e placas gelatinizado <ol><li> Preparar meios celulares ES utilizando Modificação de Meio de Eagle (DMEM) da Dulbecco com glucose elevada e piruvato de sódio. Adicionar 20% CES qualificado de Soro Fetal Bovino (FBS), 1% Penicilina / Estreptomicina, 1% de L-glutamina, 1% de HEPES, 1% de não-aminoácidos essenciais, 0,1% de gentamicina (50 mg / ml) e 0,1% ( 55 uM) β-mercaptoetanol. Esterilizar meios de células ES por filtração. </li><li> Prepare mídia OP9, fazendo 1 L de alfa Meio Essencial Mínimo (MEM-α) de pó de acordo com as instruções do fabricante. Adicionar 20% de soro fetal bovino (FBS) e 1% de Penicilina / Estreptomicina. Inverta para misturar. Esterilizar por filtração em duas aliquotas de 500 ml. NOTA: O uso de meios feitos de pó é recomendado e susceptível de produzir a manutenção das células OP9 melhorado e em resultados de diferenciação in vitro. Esta abordagem tornou-se o nosso procedimento padrão. No entanto, notamos também that que ter, por vezes, utilizados de líquidos pré-fabricados α-MEM com sucesso adequada. </li><li> Prepare a meio de congelação por adição de 10% de dimetilsulfóxido (DMSO) a 90% de FBS. Agitar suavemente e esterilizar por filtração. Armazenar a 4 ° C. </li><li> Prepare 2,000x Flt-3 ligando (10 pg / ml) por dissolução humana recombinante Flt-3 ligando (Flt-3L), em meio completo OP9 a 10 ug / ml. Alíquota em 1,5 ml tubos de microcentrífuga e armazenar a -80 ° C. Siga as recomendações do fornecedor sobre a estabilidade e armazenamento. NOTA: A estabilidade de Flt-3L é curto (1 mês a 4 ° C ou 3 meses a -80 ° C). </li><li> Prepare 1.000x IL-7 (1 ug / ml), utilizando meios de OP9 completa. Alíquota em 1,5 ml tubos de microcentrífuga e armazenar a -80 ° C. Seguir as recomendações do fornecedor e na estabilidade de armazenamento (IL-7 é estável durante até 12 meses a -80 ° C). </li><li> Prepare 1.000x Factor Inibidor da Leucemia (LIF) (10 ng / ml) por uma diluição de 10 7 unidades de LIF i 01:10n meios de células ES. Alíquota da loja a 4 ° C. Certifique-se de observar a data de validade fornecido pelo fabricante nos frascos alíquotas. NOTA: O produto é estável em concentrados e diluídos formar pelo menos 18 meses a partir da data de fabricação. </li><li> Prepare a placas de 6 poços gelatinizados por adicionar 1,5 ml de solução de gelatina a 0,1% em cada cavidade de uma placa de 6 poços. Deixar pratos na capa estéril à temperatura ambiente com tampas, o que permite, pelo menos, 30 minutos para o revestimento. Os pratos podem também ser deixado durante a noite numa incubadora humidificada. Remova qualquer solução de gelatina sobra pouco antes de semeadura de células. Não deixe que a solução de gelatina a secar completamente para fora do poço. </li></ol> 2 Preparação e Manutenção de células alimentadoras e mouse Células-Tronco Embrionárias (MESC) NOTA: Incubar todas as células numa atmosfera humidificada a 37 ° C incubadora com 5% de CO 2. <ol><li> Descongelamento embrionárias de camundongos fibroblastos (MEF) <ol><li> Quick-descongelar um frasco congelado (~ 5 x 10 6 </sup&gt; células / frasco) de mitomicina (ou de outra forma tratados mitoticamente-C) MEFs e transferi-las para um tubo de 15 ml. </li><li> Adicionar 8 mL de meio de células ES para células descongeladas, de uma forma gota a gota, para diluir gradualmente o DMSO a partir do meio de congelamento. </li><li> Células de centrifugação a 400 x g a 4 ° C durante 5 min. Remova cuidadosamente o sobrenadante e ressuspender o pellet celular em 3 ml de meio de células ES. </li><li> Preparar um tubo de centrífuga de 50 ml com 33 ml de meio de células pré-aquecida ES. Adicionar os 3 ml de MEFs ressuspensos para levar o volume final para 36 ml. </li><li> Distribuir 3 ml das MEFs ressuspensos em cada poço de placas de dois gelatinizado 6 poços. Colocar as placas na incubadora durante pelo menos seis horas para permitir que as células se fixem e se espalhar para fora antes de semear as mESCs sobre eles. Essas camadas alimentadoras mitoticamente pode ser usado por vários dias após a semeadura inicial, se seus meios de comunicação é trocada a cada 2-3 dias. NOTA: MEFs recentemente presas podem também ser utilizados. </li> &lt;/ Ol&gt; </li><li> MESCs Sementeira <ol><li> Quick-descongelar um frasco com um clone MESC congelado no 37 ° C banho de água e preparar células, conforme descrito no 2.1.1-2.1.3. NOTA: congelar 2 frascos de MESC de um poço de uma placa confluente de 6 poços. </li><li> Retirar mídia de um poço (MESC por clone) da placa de 6 poços com monocamadas de MEF presos (preparado no passo 2.1.5) e semear a 3 ml de mESCs em cima da monocamada de MEF. Adicione 3 mL de 1.000x LIF (10 ng / ml). Voltar pratos na incubadora. </li></ol></li><li> Manutenção de células ES e tripsina mediada passagem Nota: A alteração de mídia diariamente, ou divisão com base em confluência dos MESC. Idealmente, colheita e dividir / re-placa das células a cada dois dias. <ol><li> Remova a mídia de células ES e lavar mESCs com 2 ml de PBS. Remover PBS. Adicionar 1 ml de 0,25% de tripsina e incubação a 37 ° C durante 3-5 min. </li><li> Recolher as células tratadas com tripsina e transferi-los para um tubo de centrífuga de 15 ml. Vigorosamente pipete a suspensão de células para quebrar aglomerados de mCES. Adicionar 2 ml de meio completo de células ES para a suspensão de células para neutralizar a tripsina. </li><li> Células de rotação a 400 x g durante 5 min a 4 ° C. Remover o sobrenadante e ressuspender o sedimento em 3 ml de mídia célula ES. </li><li> Remova a mídia da placas de 6 poços contendo monocamadas MEF presos preparados. Adicionar a quantidade adequada de suspensão de células (com base na razão de separação desejado) em 3 ml de meio de células ES para cada cavidade a ser semeadas. Adicione 3 mL de 1.000x LIF. A confluente MESC bem dividida 1: 6 estará pronto para a passagem em 2 dias. </li></ol></li><li> OP9 / manutenção de células OP9-DL1 <ol><li> Descongelar um frasco de células OP9 (siga os passos 2.1.1-2.1.3 usando a mídia OP9) </li><li> Adicionar 7 ml de meio OP9 10 cm de prato de cultura de tecidos. Adicionar os 3 ml de células ressuspensas em um modo gota a gota, a distribuição de células ao longo da placa. Colocar as células durante a noite na incubadora. </li><li> Verifique a confluência das células OP9 no dia seguinte. Se o prato contém muitas células flutuantes mortos, remove os meios de comunicação e adicionar 10 ml de meio OP9 fresco. Retorne o prato para a incubadora se confluência quase cheio não é observado. Split (usando tripsina) 1: 4 a quase monocamada confluente OP9. NOTA: As placas devem chegar mesmo nível confluência novamente em cerca de 2 dias. Tome cuidado para não deixar que as células se tornam mais OP9-confluentes. </li><li> Congelar as passagens iniciais de células OP9 como ações de expansão. NOTA: Nós congelamos 2 frascos de células OP9 de um próximo confluente prato de 10 cm. Cada frasco de estoque de expansão pode ser ainda mais propagada para criar estoques de trabalho que são posteriormente utilizados na Mesc experimentos de co-cultura. Mantenha todas as ações OP9 congelados em nitrogênio líquido e não no freezer -80 ° C, uma vez que temperaturas flutuantes podem influenciar negativamente a qualidade dos congelamentos. </li></ol></li></ol> 3. OP9-DL1 Co-cultura Procedimento <ol><li> Preparações co-cultura <ol><li> Cerca de uma semana antes do início de um co-cultura, descongelar MEFs, mESCs e sto celular OP9 trabalhocks. Como as células OP9-DL1 não são necessários até que co-cultura dia 8, descongelar as células OP9-DL1 durante os três primeiros dias após o início co-cultura. Manter ou congelar todas as células, conforme necessário. </li><li> Determinar o número inicial de placas de 10 cm com monocamadas de células OP9 preparados para atingir 80% de confluência em co-cultura dia 0 com base na escala da experiência (por exemplo, número de clones a serem diferenciados Mesc e número de pontos no tempo de co-cultura para ser analisada). Para se preparar para uma co-cultura, dividida próximo confluente prato de OP9 células entre 1: 4 e 1: 6 dois dias antes de quando serão necessários os monocamadas. NOTA: Será necessário pelo menos uma placa por ESC clone. Tipicamente, um único clone CES semeadas numa única placa (como descrito abaixo), no dia 0 devem produzir células suficientes para semear placas de seis no dia 5 de passagem. Cada dia 5 chapa vai permitir que um ponto de tempo posterior análise. </li><li> Desde monocamadas OP9 será continuamente necessário para co-cultura passagem, ter o cuidado de sempre maintaem um número adequado de placas de cultura de OP9 adicionais em paralelo com a co-cultura. </li></ol></li><li> Dia 0: Iniciação de co-cultura <ol><li> Recolhe MESC como em 2.3.1-2.3.4. Contar as células. </li><li> Para cada clone MESC preparar 5 x 10 4 MESC em 10 ml de meio OP9 por placa. NOTA: Apesar de não tê-lo usado, notamos que um meio alternativo que consiste em α-MEM, 10% de FBS, e 5 x 10 -5 M β-mercaptoetanol também foi relatado para o uso em diferenciação co-culturas 10. </li><li> Remova a mídia velha de OP9 pratos e adicione os 10 ml de suspensão MESC aos pratos tendo o cuidado de distribuir as células de maneira uniforme em toda a placa. Coloque os pratos no 37 ° C incubadora. </li></ol></li><li> Dia 3: Troca de mídia <ol><li> Retirar pratos da incubadora e observar ao microscópio. Colônias deve começar a perder brilho e parecem achatadas. </li><li> Remova a mídia da pratos e adicione delicadamente 10 ml de fresco OP9mídia. </li><li> Retorne os pratos co-cultura para a incubadora. Monitorar visualmente a formação de colónias mesoderme semelhante diária para informar o tempo de preparação OP9 alimentador de monocamada para a próxima passagem (dia 5), ​​a qual não deve ser realizada até que ~ 80-90% das colónias apresentar visualmente a morfologia mesoderme semelhante. Adiamento máxima do dia 5 passo transferência de células é de 2 dias. </li></ol></li><li> Dia 5: tripsina passagem mediado com pré-galvanização. NOTA: Prepare com antecedência um número adequado de placas de 10 cm com células OP9 otimamente confluentes. Prossiga com os próximos passos somente se as co-culturas exibir visualmente os recursos descritos na etapa 3.3.3 (ver resultados também representativos). <ol><li> Remova a mídia dos pratos co-cultura. Adicionar 4 ml de PBS para lavar. Agitar o PBS e remover. Adicione 4 ml de tripsina a 0,25% e colocar os pratos na incubadora para ~ 5 min. </li><li> Romper a camada de células tripsinizados por pipetagem vigorosa, tomando cuidado para não introduzir bolhas de ar excessiva, até que um, a suspensão de célula única principalmente homogénea é atingida. </li><li> Adicione 4 ml de meio OP9 completos e misture por pipetagem. Transferência de células para um novo, vazio placa de 10 cm e lugar na incubadora durante 30 minutos para permitir que as células OP9 a aderir ao prato. Este &quot;pré-plaqueamento&quot; passo reduz o número de células OP9 transferidos para o próximo passo de co-cultura. </li><li> Prepare 50 ml tubos de centrífuga com coadores de células de 40 m. </li><li> Recolher as células não-aderentes, a partir da placa pré-preparada e passá-los através do filtro celular para dentro do tubo. Lavar o prato gentilmente com 6 ml de PBS e passá-lo através do mesmo filtro, deixando as células OP9 ligados atrás. </li><li> Células de centrifugação a 400 x g 5 min a 4 ° C. Remover o sobrenadante e ressuspender as células peletizadas em 3 ml de meio completo OP9. Contar as células. </li><li> Remova a mídia de placas de 10 cm com células OP9 otimamente confluentes. </li><li> Para cada ponto de tempo de análise, de semente de 5 x 10 5 células em mono OP9camada de 10 ml volume final. </li><li> Adicionar 5 ng / ml humana recombinante Flt-3L e colocar os pratos na incubadora. </li></ol></li><li> Dia 8: células progenitoras hematopoéticas (HPC) coleção NOTA: Prepara-se previamente uma quantidade adequada de placas de 6 poços com monocamadas OP9 ou OP9-DL1 células de modo a que eles vão ser ~ 80% confluentes em tempo para este passo. <ol><li> Prepare 50 ml tubos de centrífuga com 40 mM filtros. </li><li> Retirar as placas do incubador e observa ao microscópio. Cachos brilhantes de HPCs deve ser frouxamente ligado à monocamada OP9. Recolher o maior número dessas células quanto possível, lavando (mas não excessivamente interromper) a monocamada OP9 com uma pipeta e os meios de comunicação existente no prato. Para evitar a formação de espuma, sempre deixar pelo menos 1 ml da mídia na ponta da pipeta durante esta coleção HPC / etapa de lavagem. </li><li> Passar as células através do filtro de 40 um para um tubo. Adicione 8 ml de PBS para a mesma placa e verifique sob o microscópio, se todos nós HPCsre colhido. Repetir o passo de lavagem / recolha com PBS e (se necessário) com uma lavagem mais forte. Estirpe das células no interior do tubo que já contém as células a partir da mesma placa. </li><li> Células de centrifugação a 400 x g durante 5 min a 4 ° C. </li><li> Prepare uma mistura de mestre de 3 ml (por placa semeada no dia 5) de mídia OP9 com 5 ng / ml Flt-3L e 1 ng / ml de IL-7. </li><li> Remover o sobrenadante e ressuspender pellet em media OP9 com Flt-3L + IL-7 mix master (3 ml para cada 10 centímetros prato processado). Transferir as células recolhidas a partir de cada placa para um poço de uma placa de 6 poços com células OP9. <ol><li> Para dirigir as células para monocítica, B ou célula linhagens eritróide, semear as células coletadas em OP9 células. Para obter células T, semear as células em monocamadas de células OP9-DL1. </li></ol></li><li> Colocar as placas de 6 poços em incubadora. </li></ol></li><li> Dia 10: Troca de mídia <ol><li> Prepare um tubo de centrífuga de 15 ml para cada distinta MESC clone-alimentador tipo de célula Combinação em co-cultura. </li><li> Prepara-se uma mistura principal de meios OP9 com 5 ng / ml de Flt-3L e 1 ng / ml de IL-7. Preparar 3 ml para cada poço. </li><li> Suavemente recolher meio de cada poço da placa de 6 poços, combinando meios de comunicação de múltiplos poços que contêm o mesmo clone de alimentador combinação tipo de célula ESC. </li><li> Adicionar 2 ml de mistura principal mídia OP9 (ver 3.6.2) em cada poço. </li><li> Centrifugar os meios recolhidos a 400 x g durante 5 min a 4 ° C. Remover o sobrenadante e ressuspender cada pellet (muito pequena se visível) em 1 ml de OP9 master mix de mídia (ver 3.6.2) por poço originalmente coletados na etapa 3.6.3. </li><li> Distribuir 1 mL de células para cada poço de volta a partir do qual os meios foram inicialmente recolhidos elevando o volume de cada poço a 3 ml. Retorne os pratos para a incubadora. </li></ol></li><li> Dia 12: Nenhuma passagem de tripsina NOTA: Prepare com antecedência um número adequado de pratos 6 poços com células OP9 ou OP9-DL1 para que possam ser perfeitamente confluentes a tempo para este step. Dia 12 é ideal para análise de citometria de fluxo de desenvolvimento eritróide, monocítica, e as células T da fase inicial. <ol><li> Prepara-se uma mistura principal (3 ml para cada poço que continuará em co-cultura) de meios de OP9 com 5 ng / ml de Flt-3L e 1 ng / ml de IL-7. </li><li> Prepare 50 ml tubos de centrífuga com 40 mM filtros (um para cada clone-alimentador combinação tipo de célula ESC em co-cultura). </li><li> Vigorosamente pipetar os meios de comunicação existentes em cada cavidade para desagregar todas as células (incluindo a monocamada) no poço. Continue com pipetagem vigorosa até que um estado semelhante a uma suspensão de células individuais é alcançado. </li><li> Passe as células coletadas através do filtro para dentro do tubo. Adicionar 3 ml de PBS em cada poço para lavar e recolher todas as células restantes. Passa através de células do mesmo filtro. Combine as células de vários poços contendo o mesmo clone-alimentador combinação tipo de célula ESC. </li><li> Descartar o coador de células utilizadas e remover uma alíquota equivalente a uma cavidade (~ 6 ml) para qualquer pretendidacitometria de fluxo (ou outro) analisa a ser realizado no mesmo dia. Armazenar essas aliquotas sobre gelo antes da utilização. </li><li> Células de centrifugação a 400 x g durante 5 min a 4 ° C. Remover o sobrenadante. Ressuspender o sedimento dos tubos de centrífuga de 50 ml em 3 ml de mistura principal por poço para ser re-semeadas. Adicionar 3 ml por cavidade de cada suspensão de célula para o tipo de células de alimentação apropriada e colocar os pratos na incubadora. </li></ol></li><li> Dia 14: Troca de mídia <ol><li> Siga as etapas descritas na seção 3.6. </li></ol></li><li> Dia 16: No trecho de tripsina NOTA: Prepara-se por avanço pratos de 6 poços de células OP9 ou OP9-DL1 optimamente confluentes para este passo. NOTA: Dia 16 é ideal para análise de citometria de fluxo de linfócitos B e células T fase secundária. <ol><li> Siga as etapas descritas na seção 3.7. </li></ol></li><li> Dia 18: Troca de mídia <ol><li> Siga as etapas descritas na seção 3.6. </li></ol></li><li> Dia 20: Nenhuma passagem de tripsina NOTA: 20 i Dias ideal para citometria de fluxo análises de médio a fase final de desenvolvimento de células T. <ol><li> Siga as etapas descritas na seção 3.7. Se desejar, levar diferenciar células passado dia 20 de mudança media alternada e sem tripsina passagens a cada dois dias, com acompanhamento diário da taxa de expansão dos linfócitos. </li></ol></li></ol>

<ol>
	<li>Schmitt, 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=Induction+of+T+cell+development+and+establishment+of+T+cell+competence+from+embryonic+stem+cells+differentiated+in+vitro.">Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.</a> Nat Immunol. 5, 410-417 (2004).</li><li>Pui, J. C., 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=Notch1+expression+in+early+lymphopoiesis+influences+B+versus+T+lineage+determination.">Notch1 expression in early lymphopoiesis influences B versus T lineage determination.</a> Immunity. 11, 299-308 (1999).</li><li>Nakano, T., Kodama, H., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+lymphohematopoietic+cells+from+embryonic+stem+cells+in+culture.">Generation of lymphohematopoietic cells from embryonic stem cells in culture.</a> Science. 265, 1098-1101 (1994).</li><li>Schmitt, T. M., Zuniga-Pflucker, J. C. <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+T+cell+development+from+hematopoietic+progenitor+cells+by+delta-like-1+in+vitro.">Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.</a> Immunity. 17, 749-756 (2002).</li><li>Chen, J., Lansford, R., Stewart, V., Young, F., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAG-2-deficient+blastocyst+complementation:+an+assay+of+gene+function+in+lymphocyte+development.">RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 4528-4532 (1993).</li><li>de Pooter, R. F., Cho, S. K., Carlyle, J. R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+T+lymphocytes+from+embryonic+stem+cell-derived+prehematopoietic+progenitors.">In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors.</a> Blood. 102, 1649-1653 (2003).</li><li>Holmes, R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009, (2009).</li><li>Arsov, I., 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+role+for+autophagic+protein+beclin+1+early+in+lymphocyte+development.">A role for autophagic protein beclin 1 early in lymphocyte development.</a> J Immunol. 186, 2201-2209 (2011).</li><li>Lahiji, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+TCR-alpha+gene+locus+control+region+activity+in+T+cells+derived+in+vitro+from+embryonic+stem+cells.">Complete TCR-alpha gene locus control region activity in T cells derived in vitro from embryonic stem cells.</a> J Immunol. 191, 472-479 (2013).</li><li>Hirashima, M., Kataoka, H., Nishikawa, S., Matsuyoshi, N., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+embryonic+stem+cells+into+endothelial+cells+in+an+in+vitro+model+of+vasculogenesis.">Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.</a> Blood. 93, 1253-1263 (1999).</li><li>Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., Kodama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+lineage+analysis+by+cell+sorting+and+culture+identifies+FLK1+VE-cadherin++cells+at+a+diverging+point+of+endothelial+and+hemopoietic+lineages.">Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.</a> Development. 125, 1747-1757 (1998).</li><li>de Pooter, R. 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=Notch+signaling+requires+GATA-2+to+inhibit+myelopoiesis+from+embryonic+stem+cells+and+primary+hemopoietic+progenitors.">Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.</a> J Immunol. 176, 5267-5275 (2006).</li><li>Watarai, 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=Generation+of+functional+NKT+cells+in+vitro+from+embryonic+stem+cells+bearing+rearranged+invariant+Valpha14-Jalpha18+TCRalpha.">Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha.</a> 115, 230-237 (2010).</li><li>Pipkin, M. E., 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=Chromosome+transfer+activates+and+delineates+a+locus+control+region+for+perforin.">Chromosome transfer activates and delineates a locus control region for perforin.</a> Immunity. 26, 29-41 (2007).</li></ol>

The authors have nothing to disclose.

O sistema OP9-DL1 co-cultura foi utilizada para estudar o papel de vários produtos de genes durante o desenvolvimento de tipos de células do sangue a partir de células estaminais. 8,12,13 Provou também um modelo eficaz para estudar a função de genes de DNA regulador durante a diferenciação celular 9,14. Usando essa abordagem como uma alternativa aos modelos inteiros rato pode render uma considerável economia de tempo e custo de experimentos que abordam muitas questões básicas na hematopo...

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture &#8220;microenvironment&#8221; that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals. 
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this &#8220;day 5&#8221; passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:355px;">MATERIALS</td>
			<td style="width:127px;">COMPANY</td>
			<td style="width:141px;">CATALOG NUMBER</td>
			<td style="width:432px;">COMMENTS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Stem cell qualified FBS</td>
			<td>Gemini</td>
			<td>100-125</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-alanyl L-glutamine</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HEPES buffer&#160;</td>
			<td>Millipore</td>
			<td>TMS-003-C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Non-Essential Amino Acids</td>
			<td>ThermoScientific</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gentamicin Regent Solution (50 mg/mL)</td>
			<td>Life Technologies&#160;</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#946;-mercaptoethanol (55 mM) in DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22&#956;m PES membrane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>25-055-CM</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#945;-MEM&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>12000-022</td>
			<td>Powder, reconstitute per manufacture recommendation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>ThermoScientific</td>
			<td>SH 30396.03</td>
			<td>Testing of individual lots required&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dimethyl Sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Human Flt-3 Ligand</td>
			<td>R&#38;D Systems</td>
			<td>308-FK</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Murine IL-7</td>
			<td>PeproTech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LIF&#160;</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Utrapure water with 0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEFs mitomycin C treated</td>
			<td>Millipore</td>
			<td>PMEF-CF</td>
			<td>Any mitotically arresteded MEFs can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypsin EDTA, 1X</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell strainer (40 &#956;m)</td>
			<td>Fisher</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue culture dish 100 X 20 mm</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiwell 6-well</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 ml microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>Tubes for FACS&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ES R1 cells</td>
			<td>ATCC</td>
			<td>SCRC-1011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9 cells</td>
			<td></td>
			<td></td>
			<td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9-DL1 cells</td>
			<td>&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td>Cells can be requested from the Z&#250;&#241;iga-Pfl&#252;cker laboratory.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>FACS data analyses</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Flow Cytometer</td>
			<td>BD</td>
			<td></td>
			<td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td>
		</tr>
	</tbody>
</table>

derivation of t cells in vitro from mouse embryonic stem cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Quando cultivado em MEFs na presença de LIF, mESCs pode ser mantido num estado indiferenciado. Em condições ideais, eles aparecem como colônias compactas de células rodeadas por um halo brilhante em microscopia de contraste de fase (Figura 1). Estas culturas devem ser monitorizados diariamente. Dependendo da confluência das células, a mídia pode ser alterado ou células podem ser divididos. Vizinhas colônias Mesc não deve vir para o ponto de contato com o outro. Uma cultura normal, saudável d...

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivação de células T<em&gt; In Vitro</em&gt; De Mouse Células-Tronco Embrionárias

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Research

JoVE Journal

Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

10.9K visualizações.  City University of New York. Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Vídeo: Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Imunologia e Infecção

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Derivação de Tímico linfoma de células T Linhas de Atm - / -</sup E<emP53> - / -</sup Ratos

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Diferenciação de células Dirigido tronco pluripotentes induzidas no sentido de Linfócitos T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

As células T Capturar Bactérias por Transinfection das dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Na diferenciação in vitro de células T CD4 + humano FOXP3 + Induzida T reguladoras (iTregs) a partir de células naive T CD4 + usando um protocolo de TGF-β contendo

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Diferenciação de Mouse Granulócito-macrófago-colônia-estimulando Factor (GM-CSF)-produzindo pilhas de T Helper (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Purificação e Expansão de Células T Assassinos Naturais Invariantes para Estudos In vitro e In vivo

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

Um método eficiente para indução celular direcionada à hepatocito de células-tronco embrionárias humanas

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...