This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

Este estudio ha sido aprobado por el comité de ética de la Regierungspräsidium Karlsruhe. Todos los animales fueron alojados en condiciones específicas libres de patógenos en el Centro Alemán de Investigación Oncológica (DKFZ). Para todos los cachorros experimentos de cultivo de ratón que van de 1 a 7 días de edad fueron utilizados. 1. Aislamiento de mTECs de timo NOTA: Se realizaron las siguientes etapas de digestión como se describió anteriormente 1 bajo condiciones estériles con algunas modificaciones de la siguiente manera. <ol><li> Decapitar a las crías de ratón y retire el timo. Coloque el timos en hielo en una placa de Petri que contiene medio RPMI 1640 (que contiene 5% de FCS). </li><li> Cortar el timos en finos, trozos pequeños y colocar en un tubo de fondo redondo con ~ 5-30 ml de medio RPMI y revuelva suavemente usando un imán durante 10 minutos a temperatura ambiente. </li><li> A partir de entonces, se decanta el sobrenadante que contiene principalmente timocitos y digerir secuencialmente el tejido restante con una ronda de collagenatipo se IV (0,2 mg / ml y 57 U / ml concentartion final) durante 15 min cada uno a 37 ° C, seguido por la colagenasa / dispasa (0,2 mg / ml y una concentración final de 1,2 U / ml) durante 25 min cada uno a 37 ° C en un baño de agua con agitación magnética hasta que el timos se digirió completamente. Usar 1 ml de enzima por dos y cincuenta y ocho timos. </li><li> Agitar el tejido una vez cada 7-10 minutos con una pipeta Pasteur. Reúnase el fracciones colagenasa / dispasa y filtrar a través de una gasa de 70 micras. </li><li> Enriquecer los mTECs por celular magnética clasificación (MACS). Realizar la purificación de mTECs por clasificación celular magnética 11 como se describió previamente, y se muestra en la Figura 1. NOTA: Para la clasificación magnética de mTECs hemos utilizado los siguientes anticuerpos anti-CD80-PE (16-10A1, uso en dilución 1: 100) y anti-EpCAM-bio (G8.8, el uso en dilución 1: 100) 12. MTECs inmaduros y maduros utilizando MACS se definieron como: CD45 - EpCAM + CD80 - y CD45 - CD80 + respectively. </li><li> Después de la purificación MACS (pureza de mTECs inmaduros = 83,1 ± 6,3% y mTECs maduros = 79,23 ± 3,42%), sembrar las mTECs en los cultivos organotípicos como se describe a continuación (Sección 2.3). </li><li> Alternativamente, ordenar mTECs por FACS (utilizando 100 boquilla micras) tras el agotamiento CD45 MACS utilizando los siguientes anticuerpos anti-CD45-PerCP (30-F11, el uso en dilución 1: 100), anti-Ly51-FITC (6C3, utilice al 1 : 100 dilución), anti-EpCAM-Alexa647 (G8.8, el uso a escala 1: 500 dilución) y el uso de anti-CD80-PE (16-10A1, en dilución 1: 100). Excluir las células muertas utilizando yoduro de propidio (1: 5000) (Día 4). MTECs inmaduros y maduros utilizando FACS se definieron como: PI - CD45 - Ly51 - EpCAM + CD80 - y PI - CD45 - Ly51 - EpCAM + CD80 +, respectivamente. </li></ol> 2. 3D organotípicos Co-culturas (OTC) NOTA: Las construcciones 3D dérmica para el cultivo de organotípico keratinocytES se prepararon como se ha descrito previamente 9,13. En todos los pasos células se incubaron a 37 ° C y 5% de CO 2. Las OTC utilizando mTECs se prepararon con ligeras modificaciones como sigue. <ol><li> Preparación de fibroblastos humanos NOTA: Los fibroblastos dérmicos humanos se obtuvieron a partir de cultivos de explantes de dermis de-epidermised como se describió previamente 9. <ol><li> En resumen, cortar tiras de piel humana (~ 5 cm de longitud) y tratar con termolisina (0,5 mg / ml en solución salina con 10 mM Hepes pH 7,4) O / N a 4 ° C. </li><li> A partir de entonces, separar la epidermis de la dermis utilizando fórceps. </li><li> Finamente cortar la dermis en trozos pequeños, lugar en una placa de Petri de 10 cm y dejar secar durante 1-2 horas bajo un flujo de aire estéril. Complementar los explantes regularmente con DMEM que contiene 20% de SFB. </li><li> Dividir los fibroblastos crecimiento fuera cuando confluente (normalmente alrededor de 3 semanas) utilizando 0,1% de tripsina asegurándose de que los explantes siguen adhiriendo a la placa para una mayor estafarondas consecutivas de derivación. </li><li> Expandir los fibroblastos de los mismos explantes durante un máximo de 3 veces en DMEM con 10% de SFB y crio-preservarlos 14. Utilice el mismo lote de fibroblastos para cada serie experimental. NOTA: Los fibroblastos no se irradiaron durante la puesta a punto de los equivalentes dérmicos. </li></ol></li><li> Preparación del Andamios <ol><li> Cortar los 0,4 hasta 0,6 mm de espesor de viscosa, material fibroso no tejido (los detalles del producto en el apoyo a la hoja de Excel) en círculos bien delimitadas utilizando un fuerte pegador de 11 mm de diámetro de metal para encajar exactamente en 12 inserciones bien de filtro. Luego colóquelo en el elemento filtrante de 12 pocillos (poliéster membrana de poro capilar, 3 micras de tamaño de poro) como un andamio. Coloque la configuración completa del filtro en una placa de 12 pocillos estéril. </li><li> Preparar el gel de fibrina usando un pegamento de fibrina-kit para la cirugía que consiste en una combinación de fibrinógeno y trombina. Pre-diluir el fibrinógeno así como la trombina-componente del kit a 8 mg / ml y 10unidades / ml, respectivamente. Para un solo pocillo de una placa de 12 pocillos proceder como sigue. <ol><li> Diluir 100 l de fibrinógeno (8 mg / ml) con 100 l de solución salina tamponada con fosfato (PBS) sin Ca + 2 y Mg + 2, pH 7,0. Diluir 100 l de trombina (10 unidades / ml) con 100 l FCS que contienen 270.000 fibroblastos. </li><li> Dispensar 200 l de la trombina que contiene fibroblastos de células-MIX en el andamio, a los que añaden 200 l de fibrinógeno (mezcla 1: 1), resultando en una concentración final de fibrinógeno de 2 mg / ml y de la trombina de 2,5 unidades / ml. Mezclar bien y distribuir de manera uniforme sobre toda el área del andamio por pipeteo suave (Día 1). NOTA: Después de 30 min a 37 ° C se habrá formado un coágulo que encierra los fibroblastos, llenando completamente los espacios internos del andamio y que forma una superficie superior lisa. </li></ol></li></ol></li><li> Co-cultivo con mTECs <ol><li> Para pre-cultura, sumerja los cultivos de órganos enDMEM con 10% FBS, 50 mg / ml de ácido L-ascórbico y 1 ng / ml TGF-β1 con un cambio de medio cada dos días durante 4-5 días. </li><li> En el día de la siembra MTEC, reemplazar el medio por medio rFAD (1: 1 DMEM + DMEM / F12) con 10% de FBS, 10 -10 M toxina del cólera, 0,4 mg / ml de hidrocortisona, / ml de ácido L-ascórbico 50 mg, ligando RANK (0,1 g / ml) y 500 unidades / ml de aprotinina, evitando así la fibrinólisis precoz por serina proteasas secretadas por los fibroblastos. </li><li> 7-8 horas más tarde, la configuración de los co-cultivos sembrando 250.000 mTECs (ya sea mTECs completos, o CD80 y CD80 hi lo subconjuntos), en un volumen de 100 l por pocillo en la parte superior del andamio de fibroblastos. Contar las células por medio de una cámara de Neubauer (día 4). NOTA: En todo momento el timo cultivos organotípicos 3D se sumergieron en medios diferencia de OTC de la piel que son aire levantado. </li><li> Después de 24 horas de incubación, el suministro de los cultivos con medio (cambio medio total) como se mencionó anteriormente (paso 2.3.2) ahora containing redujo cantidades de aprotinina, 250 unidades / ml (día 5). </li><li> A fin de evaluar la actividad proliferativa de mTECs, añadir EdU (6,7 micras / ml, es decir, 10 M / pocillo) a OTCs durante 4 horas antes de la terminación de los cultivos. Realice la tinción de OTC crio-secciones como se describe en el Kit de EdU Imaging combinado con co-tinción de la queratina 14. Determinar los índices proliferativos de mTECs, por cualquiera de las células del K14 + EdU + contando en dos secciones de cada muestra de cultivo o por flujo citometría de (EDU citometría de flujo, la Sección 1.7, pero en lugar de Ly51-FITC utilizan CDR1-PB 15 en una dilución 1: 100 y 2.3.6.4). </li><li> Después de 4-7 días de co-cultivo, terminar las OTC y el proceso para el aislamiento de ARN, crio-seccionamiento o análisis FACS (día 8-11). <ol><li> Terminar los cultivos usando un fórceps, separando el andamio / equivalente dérmico desde el filtro del inserto así. </li><li> Para crio-seccionamiento, incrustar toda la OTC en compuesto OCT y congelar en livapor de nitrógeno quid antes de crio-seccionamiento. Preparar 5-7 micras de espesor secciones OTC utilizando un criostato y se almacena a -20 ° C hasta su uso. Por inmunohistoquímica de OTC crio-seccionó usar anti-queratina 14 (AF64, el uso a escala 1: 1000 dilución), y anti-vimentina (GP53, el uso a escala 1: 100 dilución) anticuerpos. Realice la tinción inmunohistoquímica indirecta utilizando respectivos anticuerpos secundarios. </li><li> Para el aislamiento de ARN, añadir 1 ml de solución de desnaturalización (que contiene fenol y tiocianato de guanidina) en un tapón de rosca de un tubo libre de 2 ml RNasa. Cortar toda la OTC en trozos con un bisturí y añadir en el tubo que contiene la solución desnaturalizante. Mecánicamente triturar las OTC con instrumento FastPrep dos veces durante 30 segundos a una velocidad de 6,0, colocar en el medio en hielo durante 2 min (la muestra se puede almacenar a -80 ° C después de este punto). Si se congela a -80 ° C, descongelar en hielo. Centrifugar los tubos a 11,500-13,000 rpm durante 10 min a 4 ° C. Transferir el sobrenadante a un tubo nuevo, incubar durante 5 min a RT y fprotocolos de aislamiento de ARN ollow usando ácido guanidinio tiocianato-extracción con fenol-cloroformo como se describe por el fabricante. </li><li> Para el análisis FACS, quitar el andamio y separar la membrana de la fibrina / fibroblastos / gel MTEC. Finamente cortar el gel con un escalpelo y digerir en un tubo de FACS para ~ 20 min o hasta que esté completamente digerido con 2 ml de colagenasa / dispasa a 37 ° C en un baño de agua con agitación magnética. Agitar la solución de enzima con una pipeta Pasteur una vez cada 5 min. Después de la digestión completa, se filtra la suspensión celular a través de un filtro de 70 micras; manchar la suspensión de células individuales usando los anticuerpos como se describe en 1.7 y analizar por citometría de flujo. </li></ol></li></ol></li></ol>

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Los autores declaran no tener conflicto financiera o comercial de interés.

Junto RTOCs, la OTC 3D haber sido muy superior en términos de diferenciación TEC y PGE mantenimiento / inducción (Tabla 1) en comparación con otros (i) «culturas 3D simplificados &#39;usando - fibroblastos solo sin el andamio; (Ii) sistemas 2D utilizando - células fibroblastos / alimentador co-cultivadas con TEC 10, (iii) las células 3T3-J2 en el que se desarrollan clones TEC, pero PGE se pierde, (iv) matrigel o (v) los componentes de ECM (datos no publicados). PGE se mantuvo durante u...

Timocitos en desarrollo constituyen aproximadamente el 98% del timo, mientras que el 2% restante consiste en una variedad de células que colectivamente componen el estroma tímico (es decir, células epiteliales, células dendríticas, macrófagos, células B, fibroblastos, células endoteliales). Las células epiteliales corticales exteriores (CTEC) procuran la inmigración de las células pro-T de la médula ósea, linaje de células T de inducción en multipotentes células pre-T y la selección positiva de la auto-MHC restringida timocitos inmaduros. Las células internas medulares del timo epiteliales (mTECs) están involucrados en la tolerancia a la inducción de los timocitos con un TCR de alta afinidad para los complejos de auto-péptido / MHC por cualquiera de inducir la selección negativa o su desviación en el linaje de células T reguladora. En el contexto de la inducción de la tolerancia central, mTECs son los únicos que expresan un amplio espectro de antígenos propios restringido de tejidos (TRAS) reflejando así el auto periférica. Este fenómeno se llama promiscua expresión génica (PGE)1,2. La mayoría de los estudios actuales sobre este fascinante tipo de célula se basan en células aisladas ex vivo, como diversos sistemas de cultivo 2D a corto plazo como resultado invariablemente en la pérdida de PGE y moléculas clave como regulador de MHC de clase II, Foxn1 y Aire dentro de los primeros 2 días 3-6 . Se mantuvo sin embargo no está claro, que los componentes y características de la malla 3D intacta del timo particulares faltaban en modelos 2D. El cultivo de órganos tímico re-agregación (RTOC) ha sido hasta ahora el único sistema 3D que permite el estudio de desarrollo de las células T, por un lado, y la biología de células del estroma, por otro lado, en un microambiente tímico intacta 7. Sin embargo, RTOCs tienen ciertas limitaciones, es decir, que ya contienen una mezcla compleja de células, requieren la entrada de las células del estroma fetales y soportar un período de cultivo máxima de 5 a 10 días. La falta de reduccionista en los sistemas de cultivo in vitro ha dificultado el estudio devarios aspectos del desarrollo de las células T y la organogénesis del timo no menos importante la regulación molecular de PGE y su relación con la biología del desarrollo de mTECs. Debido a la estrecha relación-de la organización estructurada de las células epiteliales de la piel y el timo, se optó por un sistema 3D cultura organotípico (OTC) que había sido desarrollado originalmente para emular a la diferenciación de los queratinocitos in vitro y por lo tanto crear un equivalente dérmico. El sistema OTC consiste en una matriz inerte andamio superpuesta con fibroblastos dérmicos que se encuentran atrapados en un gel de fibrina, en la que los queratinocitos se siembran 8,9. Aquí, hemos sustituido los queratinocitos con mTECs purificados. Manteniendo las características básicas de este modelo, hemos optimizado ciertos parámetros. En el modelo de venta libre aprobado mTECs proliferaron, se sometió a la diferenciación terminal y mantenido la identidad MTEC y PGE, imitando así de cerca en el desarrollo mTECs vivo <sup&gt; 10. Esta nota técnica proporciona un protocolo detallado que permite el paso a paso la configuración de OTC timo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 mice&#160;</td>
			<td>Charles River WIGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (G8.8 -Alexa 647 and -biotin)</td>
			<td>Ref. 12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD80-PE antibody</td>
			<td>BD Pharmingen</td>
			<td>553769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PerCP antibody</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly51-FITC antibody</td>
			<td>BD Pharmingen</td>
			<td>553160</td>
			<td></td>
		</tr>
		<tr>
			<td>CDR1-Pacific Blue</td>
			<td>Ref. 15</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 14 antibody</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Progen</td>
			<td>GP58</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated AffiniPure F(ab&#39;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>106-546-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Molecular Probes (Invitrogen GmbH)</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">Click-iT EdU Alexa Fluor 594 Imaging Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">C10339</a></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">C10425</a></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well filter inserts (thincerts)</td>
			<td>Greiner bio-one</td>
			<td>657631</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Greiner</td>
			<td>665180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Jettex 2005/45</td>
			<td>ORSA, Giorla Minore, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Serva</td>
			<td>47302.03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-049&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Gold</td>
			<td>GE Healthcare</td>
			<td>A11-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin (Trasylol)</td>
			<td>Bayer</td>
			<td>4032037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Biomol</td>
			<td>G117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Seromed (Biochrom)</td>
			<td>K3520</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#223;1</td>
			<td>Invitrogen</td>
			<td>PHG9214</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>R&#38;D systems</td>
			<td>462-TR-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma Aldrich</td>
			<td>&#160;T-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>FastPrep FP120</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV&#160;</td>
			<td>CellSystems</td>
			<td>LS004189</td>
			<td>0.2 mg/ml and 57U/ml final conc.</td>
		</tr>
		<tr>
			<td>Neutrale Protease (Dispase)</td>
			<td>CellSystems</td>
			<td>LS002104</td>
			<td>0.2 mg/ml and 1.2U/ml final conc.</td>
		</tr>
		<tr>
			<td>DNase I&#160;</td>
			<td>Roche</td>
			<td>11 284 932 001</td>
			<td>25 &#181;g/ml final conc.</td>
		</tr>
	</tbody>
</table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

El desarrollo de células T Intra-tímico requiere una malla tridimensional intrincada compuesta de diversas células del estroma, es decir, las células no-T. Timocitos atraviesan este andamio en un orden temporal y espacial altamente coordinada mientras secuencialmente pasando los puntos de control obligatorios, es decir, T compromiso de linaje celular, seguido por la generación repertorio T y la selección antes de su exportación en la periferia. Los dos tipos principales de células residentes que forman este andamio son células epiteliales del timo corticales (CTEC) y medulares (mTECs). Una característica clave de mTECs es la denominada expresión promiscua de numerosos antígenos restringida de tejido. Estos antígenos restringido de tejidos se presentan a los timocitos inmaduros directa o indirectamente por mTECs o células dendríticas tímicas, respectivamente resultante en la auto-tolerancia. Modelos adecuados in vitro emulando las vías de desarrollo y las funciones de CTEC y mTECs Actualmente lacrey. Esta falta de modelos experimentales adecuados, por ejemplo, ha obstaculizado el análisis de la expresión génica promiscua, que todavía está poco conocida a nivel celular y molecular. Se adaptó un modelo de co-cultivo organotípico 3D a la cultura ex vivo mTECs aisladas. Este modelo fue originalmente ideado para cultivar queratinocitos de tal manera como para generar un equivalente de piel in vitro. El modelo 3D conserva características funcionales clave de MTEC biología: (i) la proliferación y la diferenciación terminal de CD80 he aquí-Aire negativa en CD80 hi, mTECs-Aire positivo, (ii) la capacidad de respuesta de RANKL, y (iii) la expresión sostenida de Foxn1, Aire y genes restringido de tejidos en CD80 hi mTECs.

Hemos adoptado un modelo de co-cultivo organotípico 3D (3D OTC) que había sido desarrollado originalmente para la cultura a largo plazo in vitro de queratinocitos 9. MTECs MACS enriquecidos (ver MACS enriquecimiento esquema de la figura 1) fueron sembradas en un andamio que comprende de un gel de fibrina y fibroblastos atrapadas. Los fibroblastos proporcionan la matriz extracelular esencial (ECM) que soporta mTECs in vitro. MTECs se cultivaron en OTC de 4-14 días en prese...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D organotípicos Co-cultura de apoyo medular tímica epitelial proliferación celular, la diferenciación y la Expresión Génica Promiscuo Modelo

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K vistas.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 

Video: 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Cultura y Co-cultura de ratón ovarios y los folículos ováricos

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Investigación

Biología del Desarrollo

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Predicción y validación de los elementos reguladores de genes activados durante ácido retinoico inducida por la diferenciación de células madre embrionarias

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Terapia génica adenoviral para Diabetic Queratopatía: Efectos en la curación de heridas y Stem Cell expresión del marcador en córneas de órganos cultivados humanos y células epiteliales Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Ordenamiento temporal de los datos de expresión dinámica de la expresión espacial detallada Mapas

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Diferenciación rápida y dirigida de las células epiteliales del pigmento retiniano de humanos embrionarios o inducidas pluripotenciales células

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

Protocolo de diferenciación cerebelosa 3D optimizada con modificación 2D opcional

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

Análisis clonal de precursores de células madre hematopoyéticas embrionarias usando índice célula clasificación combinada con células endoteliales nicho cultura Co

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Proliferación y diferenciación de precursores mieloides murinas 32D/G-CFS-R las células

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Reprogramación hemogénica de fibroblastos humanos por expresión forzada de factores de transcripción

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Un modelo de expresión de chemocina ectópico para probar el reclutamiento de macrófagos in Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...