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 Preparación de medios de cultivo, citoquinas y Placas gelatinizada <ol><li> Preparar los medios de comunicación de células ES mediante el uso de Modificación de Medio de Eagle (DMEM) de Dulbecco con alto contenido de glucosa y piruvato de sodio. Añadir 20% ESC Calificación suero bovino fetal (FBS), 1% penicilina / estreptomicina, 1% de L-glutamina, 1% Tampón HEPES, ácidos 1% para no esencial amino, 0,1% de gentamicina (50 mg / ml) y 0,1% ( 55 M) β-mercaptoetanol. Esterilizar medio de células ES por filtración. </li><li> Preparar medios OP9 al hacer 1 L de medio esencial mínimo alfa (α-MEM) a partir de polvo de acuerdo con las instrucciones del fabricante. Añadir 20% de suero fetal bovino (FBS) y 1% penicilina / estreptomicina. Invertir para mezclar. Esterilizar por filtración en dos alícuotas de 500 ml. NOTA: Se recomienda el uso de medios elaborados a partir de polvo y es probable que para producir un mejor mantenimiento de células OP9 y en los resultados de diferenciación in vitro. Este enfoque se ha convertido en nuestro procedimiento estándar. Sin embargo, también observamos That hemos utilizado a veces líquidos pre-hechos α-MEM con éxito adecuada. </li><li> Preparar medio de congelación mediante la adición de 10% sulfóxido de dimetilo (DMSO) al 90% de FBS. Agitar suavemente y esterilizar por filtración. Almacenar a 4 ° C. </li><li> Preparar 2,000x Flt-3 ligando (10 g / ml) por disolución de Flt-3 humana recombinante ligando (Flt-3L) en medios OP9 completa a 10 g / ml. Alícuota en 1,5 ml tubos de microcentrífuga y se almacenan a -80 ° C. Siga las recomendaciones del proveedor respecto a la estabilidad y el almacenamiento. NOTA: La estabilidad de Flt-3L es corto (1 mes a 4 ° C o 3 meses a -80 ° C). </li><li> Preparar 1,000x IL-7 (1 g / ml) mediante el uso de medios de comunicación OP9 completa. Alícuota en 1,5 ml tubos de microcentrífuga y se almacenan a -80 ° C. Siga las recomendaciones del proveedor sobre la estabilidad y almacenamiento (IL-7 es estable por hasta 12 meses a -80 ° C). </li><li> Preparar 1,000x Factor Inhibidor de Leucemia (LIF) (10 g / ml) por una dilución 1:10 de 10 7 unidades de LIF in medio de células ES. Alícuota Almacenar a 4 ° C. Asegúrese de anotar la fecha de caducidad indicada por el fabricante en los viales alícuotas. NOTA: El producto es estable en forma concentrada o diluida al menos 18 meses a partir de la fecha de fabricación. </li><li> Preparar placas de 6 pocillos gelatinizados por añadir 1,5 ml de 0,1% de solución de gelatina en cada pocillo de una placa de 6 pocillos. Deja los platos en la campana estéril a temperatura ambiente con las tapas en, dejando al menos 30 minutos para el recubrimiento. Los platos también se pueden dejar en una incubadora humidificada durante la noche. Retire cualquier solución de gelatina sobrante justo antes de la siembra de células. No deje que la solución de gelatina se seque por completo en el pozo. </li></ol> 2 Preparación y mantenimiento de células alimentadoras y las células madre embrionarias de ratón (mESC) NOTA: Incubar todas las células en un 37 ° C humidificado incubadora con 5% de CO 2. <ol><li> Descongelación fibroblastos embrionarios de ratón (MEF) <ol><li> Quick-descongele un vial congelado (~ 5 x 10 6 </sup&gt; células / vial) de mitomicina C-tratado (o de otra manera detenidas mitóticamente MEFs) y los transfieren a un tubo de 15 ml. </li><li> Añadir 8 ml de medio de células ES a las células descongeladas, de una manera gota a gota para diluir gradualmente el DMSO a partir del medio de congelación. </li><li> Centrifugar las células a 400 x g a 4 ° C durante 5 min. Retire cuidadosamente el sobrenadante y resuspender el sedimento celular en 3 ml de medio de células ES. </li><li> Preparar un tubo de centrífuga de 50 ml con 33 ml de medio de células pre-calentado ES. Añadir los 3 ml de MEFs resuspendidos a llevar el volumen final a 36 ml. </li><li> Distribuir 3 ml de los MEFs resuspendidos en cada pocillo de dos placas de 6 pocillos gelatinizado. Colocar las placas en la incubadora durante al menos seis horas para permitir que las células se unan y se extienden antes de la siembra de las mESCs en ellos. Estas capas alimentadoras mitóticamente detenidas pueden ser utilizables durante varios días después de la siembra inicial, si sus medios se cambian cada 2-3 días. NOTA: MEFs recién detenidos también se pueden utilizar. </li> &lt;/ Ol&gt; </li><li> MESCs Siembra <ol><li> Quick-descongele un vial con un congelado clon mESC en el baño a 37 ° C y preparar las células como se describe en 2.1.1-2.1.3. NOTA: Nos congelamos 2 viales de mESC de un pozo confluente de una placa de 6 pocillos. </li><li> Retire el medio de un pozo (por clon mESC) de la placa de 6 pocillos con monocapas MEF detenidos (preparado en el paso 2.1.5) y sembrar el 3 ml de mESCs en la parte superior de la monocapa MEF. Añadir 3 l de 1,000x LIF (10 ng / ml). Volver platos en la incubadora. </li></ol></li><li> El mantenimiento de células ES y la tripsina mediada pasaje NOTA: Cambiar medios diarios o Split basado en la confluencia de mESC. En condiciones óptimas, la cosecha y de división / re-placa de las células cada dos días. <ol><li> Retire el medio de células ES y lavar mESCs con 2 ml de PBS. Retire PBS. Añadir 1 ml de 0,25% de tripsina y se incuba a 37 ° C durante 3-5 min. </li><li> Recoger las células tratadas con tripsina y transferirlos en un tubo de centrífuga de 15 ml. Pipetear vigorosamente la suspensión celular a romper los grumos de mCES. Añadir 2 ml de medio de células ES completa a la suspensión celular para neutralizar la tripsina. </li><li> Centrifugar las células a 400 x g durante 5 min a 4 ° C. Eliminar el sobrenadante y resuspender el sedimento en 3 ml de células ES medios de comunicación. </li><li> Retire el medio de placas de 6 pocillos que contienen monocapas MEF detenidos preparados. Añadir la cantidad apropiada de la suspensión celular (basado en la relación de separación deseada) en 3 ml de medio de células ES a cada pocillo para sembrar. Añadir 3 l de LIF 1,000x. Una confluencia mESC bien dividida 1: 6 estará listo para el paso en 2 días. </li></ol></li><li> OP9 / mantenimiento de células OP9-DL1 <ol><li> Descongelar un vial de células OP9 (siga los pasos 2.1.1-2.1.3 utilizando medios OP9) </li><li> Añadir 7 ml de medio OP9 a 10 cm placa de cultivo tisular. Añadir los 3 ml de células resuspendidas en una forma gota a gota, la distribución de las células de todo el plato. Colocar las células durante la noche en la incubadora. </li><li> Compruebe la confluencia de las células OP9 al día siguiente. Si el plato contiene muchas células flotando muertos, remove los medios de comunicación y añadir 10 ml de medio OP9 fresco. Devuelva el plato a la incubadora si no se observa casi llena confluencia. Split (utilizando tripsina) 1: 4 cerca de la monocapa confluente OP9. NOTA: Las placas deben llegar a mismo nivel confluencia de nuevo en unos 2 días. Tenga cuidado de no dejar que las células OP9 vuelto sobre-confluente. </li><li> Congelar los primeros pasajes de células OP9 como acciones de expansión. NOTA: Nos congelamos 2 viales de células OP9 de una cerca de la confluencia placa de 10 cm. Cada vial de expansión de valores puede ser propagado a crear reservas de trabajo que se utilizan posteriormente en mESC experimentos de co-cultivo. Mantenga todas las existencias OP9 congelados en nitrógeno líquido y no en el congelador -80 ° C, ya que las temperaturas fluctuantes pueden influir negativamente en la calidad de las heladas. </li></ol></li></ol> 3. OP9-DL1 Procedimiento Co-cultura <ol><li> Preparaciones de Co-cultura <ol><li> Aproximadamente una semana antes de la iniciación de un co-cultivo, descongelar MEFs, mESCs y sto celular OP9 trabajocks. Dado que las células OP9-DL1 no son necesarios hasta el día 8 de co-cultivo, descongelar las células OP9-DL1 durante los primeros tres días después del inicio de co-cultivo. Mantener o congelar todas las células según sea necesario. </li><li> Determinar el número inicial de placas de 10 cm con monocapas de células OP9 preparados para alcanzar el 80% de confluencia en co-cultura día 0 en base a la escala del experimento (por ejemplo, número de clones mESC para diferenciarse y el número de puntos de tiempo de co-cultivo de ser analizada). Para prepararse para una co-cultura, dividido cerca plato confluente de células OP9 entre 1: 4 y 1: 6 dos días antes de que se necesitarán las monocapas. NOTA: Uno necesitará al menos una placa por clon ESC. Típicamente, un solo clon ESC sembró en una sola placa (como se describe a continuación) en el día 0 deben producir suficientes células para sembrar placas de seis días en el paso 5. Cada placa 5 días permitirá una posterior punto de tiempo de análisis. </li><li> Desde monocapas OP9 serán continuamente necesarios para el paso de co-cultivo, tenga cuidado de siempre maintaen un número adecuado de placas de cultivo de OP9 adicionales en paralelo con el co-cultivo. </li></ol></li><li> Día 0: Inicio de la co-cultura <ol><li> Recoger mESC como en 2.3.1-2.3.4. Contar las células. </li><li> Para cada clon mESC preparar 5 x 10 4 mESC en 10 ml de medio por placa OP9. NOTA: Aunque no hemos utilizado, observamos que un medio alternativo que consiste en α-MEM, el 10% de SFB, y 5 x 10 -5 M β-mercaptoetanol También se ha informado para el uso en la diferenciación de co-cultivos 10. </li><li> Eliminar los viejos medios de OP9 platos y añadir los 10 ml de suspensión mESC a los platos teniendo cuidado para distribuir las células uniformemente a través de la placa. Colocar las cápsulas en la 37 ° C incubadora. </li></ol></li><li> Día 3: Cambio de medio <ol><li> Sacar los recipientes de la incubadora y observar bajo el microscopio. Las colonias deben comenzar a perder brillo y un aspecto apisonado. </li><li> Retire el medio de platos y añadir con cuidado 10 ml de fresco OP9medios de comunicación. </li><li> Devolver los platos de co-cultivo a la incubadora. Monitorear visualmente la formación de colonias de mesodermo, como todos los días para informar al momento de OP9 preparación monocapa alimentador para el siguiente paso (día 5), ​​que no debe llevarse a cabo hasta ~ 80-90% de las colonias mostrar visualmente mesodermo como morfología. Aplazamiento máximo de la etapa de transferencia de células 5 días es de 2 días. </li></ol></li><li> Día 5: La tripsina pasaje mediada con pre-chapado. NOTA: Preparar de antemano un número adecuado de placas de 10 cm con células OP9 óptimamente confluentes. Proceda con los siguientes pasos sólo si los co-cultivos muestran visualmente las características que se describen en el paso 3.3.3 (ver resultados también representativos). <ol><li> Retire el medio de los platos de co-cultivo. Añadir 4 ml de PBS para lavar. Agitar el PBS y quitar. Añadir 4 ml de tripsina al 0,25% y colocar los platos en la incubadora de ~ 5 min. </li><li> Interrumpir la capa de las células tratadas con tripsina por pipeteo vigoroso, con cuidado de no introducir burbujas de aire excesivas, hasta que unhomogénea se logra, sobre todo sola suspensión celular. </li><li> Añadir 4 ml de medio OP9 completos y mezclar con la pipeta. Transferencia de células en un nuevo, vacío placa de 10 cm y el lugar en la incubadora durante 30 min para permitir que las células se adhieran OP9 al plato. Este paso de &quot;pre-chapado&quot; reduce el número de OP9 células transferidas a la siguiente etapa de co-cultivo. </li><li> Preparar 50 ml tubos de centrífuga con células coladeras 40 micras. </li><li> Recoger las células no adherentes de la placa de pre-chapado y pasar a través del filtro de células en el tubo. Lave el plato suavemente con 6 ml de PBS y pasarla a través del mismo filtro, dejando las células OP9 unidos detrás. </li><li> Centrifugar las células a 400 x g 5 min a 4 ° C. Eliminar el sobrenadante y resuspender las células sedimentadas en 3 ml de medio OP9 completa. Contar las células. </li><li> Retire el papel de placas de 10 cm con células OP9 óptimamente confluentes. </li><li> Para cada punto de tiempo de análisis, sembrar 5 x 10 5 células en el mono OP9capa en volumen final de 10 ml. </li><li> Añadir 5 ng / ml humana recombinante Flt-3L y colocar los platos en la incubadora. </li></ol></li><li> Día 8: hematopoyético de células progenitoras (HPC) de recogida NOTA: Preparar de antemano un número adecuado de placas de 6 pocillos con monocapas OP9 o-DL1 OP9 celulares para que puedan ser ~ 80% de confluencia en el tiempo para este paso. <ol><li> Preparar 50 ml tubos de centrífuga con 40 micras coladores. </li><li> Quitar los platos de la incubadora y observar bajo el microscopio. Grupos brillante de HPC deben ser débilmente unidos a la monocapa OP9. Recoger la mayor cantidad de estas células como sea posible mediante el lavado (pero no demasiado interrumpir) la monocapa OP9 con una pipeta y los medios de comunicación existentes en el plato. Para evitar la formación de espuma, siempre dejar al menos 1 ml de los medios de comunicación en la punta de la pipeta durante esta colección HPC / paso de lavado. </li><li> Pasar las células a través del filtro de 40 micras en un tubo. Añadir 8 ml de PBS a la misma placa y comprobar bajo el microscopio si todos nos HPCsre recogido. Repita el paso de lavado / colección con PBS y (si es necesario) con el lavado más enérgico. Colar las células en el tubo ya que contiene las células de la misma placa. </li><li> Centrifugar las células a 400 x g durante 5 min a 4 ° C. </li><li> Preparar una mezcla maestra de 3 ml (por placa sembró en el día 5) de los medios de OP9 con 5 ng / ml de Flt-3L y 1 ng / ml de IL-7. </li><li> Aspirar el sobrenadante y pellet se resuspende en medios OP9 con Flt-3L + IL-7 mezcla maestra (3 ml por cada placa de 10 cm procesado). Transferencia de las células recogidas de cada placa en un pocillo de una placa de 6 pocillos con células OP9. <ol><li> Con el fin de conducir hacia las células monocíticas, células B o linajes eritroides, sembrar las células recogidas en las células OP9. Para derivar células T, sembrar las células en monocapas de células OP9-DL1. </li></ol></li><li> Coloque las placas de 6 pocillos en incubadora. </li></ol></li><li> Día 10: Cambio de medio <ol><li> Preparar un tubo de centrífuga de 15 ml para cada distinto mESC clon-alimentador tipo de células combición en co-cultivo. </li><li> Preparar una mezcla maestra de los medios OP9 con 5 ng / ml de Flt-3L y 1 ng / ml de IL-7. Preparar 3 ml para cada pocillo. </li><li> Recoger suavemente los medios de comunicación de cada pocillo de la placa de 6 pocillos que combina los medios de comunicación de múltiples pocillos que contienen el mismo clon de alimentador de tipo de célula combinación ESC. </li><li> Añadir 2 ml de mezcla maestra medios OP9 (véase 3.6.2) en cada pocillo. </li><li> Centrifugar los medios de recogida a 400 x g durante 5 min a 4 ° C. Aspirar el sobrenadante y resuspender cada pellet (muy pequeña si es visible) en 1 ml de OP9 maestro de la mezcla de medios (ver 3.6.2) por pocillo recogido inicialmente en el paso 3.6.3. </li><li> Distribuir 1 ml de células de nuevo en cada pocillo de la que los medios de comunicación se recogió originalmente llevando el volumen en cada pocillo a 3 ml. Vuelva a poner las placas en la estufa. </li></ol></li><li> Día 12: Ningún pasaje tripsina NOTA: Preparar de antemano un número adecuado de placas de 6 pocillos con células OP9 o OP9-DL1 para que puedan ser confluentes de manera óptima en el tiempo para este step. Día 12 es ideal para el análisis de citometría de flujo de eritroide en desarrollo, monocitos y células T en fase inicial. <ol><li> Preparar una mezcla maestra (3 ml para cada pocillo que continuará en co-cultivo) de los medios de comunicación OP9 con 5 ng / ml de Flt-3L y 1 ng / ml de IL-7. </li><li> Preparar 50 ml tubos de centrífuga con 40 micras coladores (uno para cada clon-alimentador tipo de célula combinación ESC en co-cultura). </li><li> Vigorosamente pipetear los medios de comunicación existentes en cada pocillo para desagregar todas las células (incluyendo la monocapa) en el pozo. Continuar con pipeteo enérgico hasta que se alcanza un estado parecido a una suspensión de células individuales. </li><li> Pasar las células recogidas a través del filtro en el tubo. Añadir 3 ml de PBS en cada pocillo para lavar y recoger todas las células restantes. Pasar las células a través del mismo filtro. Combinar celdas de múltiples pozos que contienen el mismo clon-alimentador tipo de célula combinación ESC. </li><li> Desechar el filtro de células utilizado y eliminar una parte alícuota equivalente a un pocillo (~ 6 ml) para cualquier deseadacitometría de flujo (u otro) análisis a realizar en ese día. Guarde estas alícuotas en hielo antes de su uso. </li><li> Centrifugar las células a 400 x g durante 5 min a 4 ° C. Aspirar el sobrenadante. Resuspender el sedimento de los tubos de centrífuga de 50 ml en 3 ml de mezcla maestra por pozo a ser re-sembrado. Añadir 3 ml por pocillo de cada suspensión de células en el tipo de células de alimentación apropiada y colocar los platos en la incubadora. </li></ol></li><li> Día 14: Cambio de medio <ol><li> Siga los pasos que se indican en el apartado 3.6. </li></ol></li><li> Día 16: Ningún pasaje tripsina NOTA: Preparar con antelación placas de 6 pocillos de células OP9 o OP9-DL1 óptimamente confluentes para este paso. NOTA: El día 16 es ideal para el flujo de análisis de citometría de los linfocitos B y las células T de la etapa intermedia. <ol><li> Siga los pasos descritos en la sección 3.7. </li></ol></li><li> Día 18: Cambio de medio <ol><li> Siga los pasos que se indican en el apartado 3.6. </li></ol></li><li> Día 20: Ningún pasaje tripsina NOTA: Día 20 is ideal para citometría de flujo análisis de mediados o finales de etapa de desarrollo las células T. <ol><li> Siga los pasos descritos en la sección 3.7. Si lo desea, realizar la diferenciación de células día 20 cambio de medio alterna pasado y no tripsina pasajes cada dos días con un seguimiento diario de la tasa de expansión de los linfocitos. </li></ol></li></ol>

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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.

El sistema OP9-DL1 de co-cultivo se ha utilizado para estudiar el papel de los diversos productos de genes durante el desarrollo de tipos de células sanguíneas a partir de células madre. 8,12,13 También ha demostrado ser un modelo eficaz para estudiar la función del ADN reguladora del gen durante la diferenciación celular. 9,14 Con este enfoque como una alternativa a los modelos enteros de ratón puede producir un considerable ahorro en tiempo y coste de los experimentos que abordan muchas cu...

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.

Cuando se cultiva en MEFs en presencia de LIF, mESCs se pueden mantener en un estado indiferenciado. Bajo condiciones ideales, aparecen como colonias compactas de células rodeadas por un halo brillante en la microscopía de contraste de fases (Figura 1). Estos cultivos deben ser monitoreados diariamente. Dependiendo de la confluencia de las células, los medios de comunicación pueden ser cambiados o células se pueden dividir. Vecinas colonias mESC no deben llegar al punto de contacto entre sí. Un cu...

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.

Derivación de células T<em&gt; In Vitro</em&gt; De células madre embrionarias de ratón

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 vistas.  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.

Video: 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

Transducción retroviral de receptores de células T en ratones las 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 ...

Investigación

Inmunología e Infección

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.

Derivación de linfoma tímico líneas de células T de Atm - / -</sup Y P53 - / -</sup Ratones

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.

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

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.

Diferenciación dirigida de células madre pluripotentes inducidas hacia los linfocitos 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.

Aplicación clínica de<em&gt; La Bella Durmiente</em&gt; Artificial y células presentadoras de antígenos para modificar genéticamente las células T de la sangre del cordón umbilical y 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.

Células T Captura bacterias por transinfection de células 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.

Diferenciación in vitro de las células CD4 + humano FOXP3 + células T reguladoras (Induced iTregs) de T naive CD4 + células utilizando un protocolo de TGF-β que contienen

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 Diferenciación del Factor del Granulocyte-macrófago-estimulante de colonias (GM-CSF) de ratón-producción de linfocitos de T (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.

Purificación y expansión de células T asesinas naturales invariantes de ratón para estudios 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).

Un Método Eficiente Para La Inducción De Células Dirigidas Hepatocitos-Como De Las Células Madre Embrionarias Humanas

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