Este trabajo fue financiado de forma secuencial por un Frontiers NCCR en la adjudicación piloto de Genética, Investigación de Diabetes Juvenil Fundación Beca 41-2009-775 y Grant 12-126875 de Det Frie Forskningsråd / Sundhed og Sygdom. Los autores agradecen el laboratorio Spagnoli por acoger la grabación de vídeo.

Este protocolo tiene como objetivo crecer organoides pancreáticas derivadas de E10.5 murino disociaron células epiteliales pancreáticas. El protocolo requiere la aprobación ética para la experimentación animal. 1. Disección de dorsal de páncreas Bud de E10.5 embriones de ratón <ol><li> Medio esencial Sacrificar los ratones oportuna embarazadas en el día embrionario (E) 10,5, abrir el abdomen con un par de tijeras, retire los dos cuernos uterinos y colocarlos en un plato de Petri de 10 cm llena con tampón fosfato frío salino (PBS) o modificado por Dulbecco (DMEM) se mantuvo en hielo. El experimento total desde el sacrificio a la siembra de células se realiza en 60-90 minutos para prevenir el daño celular. </li><li> Separe el útero en segmentos de embriones individuales utilizando unas tijeras pequeñas. Transferir un embrión a una placa de 35 mm de Petri con PBS frío y visualizar bajo un microscopio de disección con iluminación desde arriba. Con fórceps de disección eliminar el músculo, la decidua y la yema que rodea saC y exponer el embrión. Coloque el embrión de nuevo en medio DMEM frío. Los embriones se pueden transferir fácilmente utilizando un 3 ml de transferencia de plástico pipeta / gotero. Aislar cada embrión individual en una manera similar antes de continuar. </li><li> Coloque un embrión en una limpia de 35 mm placa de Petri en PBS. <ol><li> Use unas pinzas finas (0,05 mm de ancho) para quitar la extremidad anterior. Inserte con cuidado la pinza en la apertura para separar el tracto digestivo, desde la región de la médula espinal (Figura 1). OPCIONAL: Para ver más cómodamente el estómago, retire la parte superior del cuerpo del embrión, hasta el corazón y la región de la cola por debajo del saco vitelino. </li><li> Localizar el estómago, el hígado y el intestino. El uso de los fórceps, aislar el tracto gastrointestinal desde el estómago hasta el intestino y colocarlo en DMEM frío en hielo (Figura 1). El brote pancreático dorsal está unido dorsalmente, posterior al estómago (Figura 1). </li><li> Aislar cada tracto gastrointestinal individuo en una simimanera lar antes de continuar. Si los embriones deben ser genotipo, recoger la cola en el momento de la disección y mantener a cada tracto gastrointestinal individual en un pozo diferente en una placa de 24 pocillos con DMEM frío en hielo. </li></ol></li><li> Coloque un tracto gastrointestinal de una manera limpia de 35 mm placa de Petri en PBS frío. Ahora utilizar la iluminación desde abajo en campo claro de visualizar y diseccionar el brote pancreático dorsal bajo el microscopio de disección. Estas condiciones optimizar la visualización de volúmenes. El uso de agujas de tungsteno afiladas electrolíticamente-o 20 G agujas de jeringa, aislar el brote pancreático con tan poco como sea posible mesénquima alrededor del epitelio (Figura 1). <ol><li> Transferir el brote aislado en una placa de Petri que contiene una solución de dispasa frío (1,25 mg / ml) durante 2-3 min. Desde este punto, la transferencia más convenientemente se puede hacer con 50 l capilares de vidrio de llama-tirado conectados a un tubo flexible boca-controlada. Alternativamente, pero con más risk de perder el brote, utilice una pipeta automática 10 l (Pipetman) con puntas de plástico adecuados. </li><li> Realizar la aspiración brote pancreático y de eyección bajo control microscópico. Ponga el brote pancreático de nuevo en PBS. Además limpiar el brote pancreático aislado a partir del mesénquima con las agujas y aspiración suave utilizando el capilar de vidrio (Figura 1). </li><li> Cuando se extirpa todo el mesénquima, enjuague el brote pancreático en PBS frío; transferir cada brote a DMEM frío en pozos individuales (60 pocillos mini-bandejas llenas de 10 l de DMEM frío). Es importante no eliminar el mesénquima en dispasa, lo que hace que el tejido muy pegajosa. </li></ol></li></ol> 2. Plating y Cultura de células dispersas <ol><li> Transferir el epitelio disecados de todos los embriones con un capilar de vidrio de la llama-tirado en pozos cónicos de 60 pocillos mini-bandejas llenas de 10 l de PBS para el enjuague. <ol><li> Transferir el brote en 10 l de tripsina al 0,05% de unnd déjelo incubado a 37 ° C durante 4 min. Inactivar la tripsina mediante la transferencia de la yema en un pozo con 10 l de DMEM + 10% de suero de ternera fetal (FCS). </li><li> Disociar la suspensión celular por aspiración a través de un capilar fino desmenuzado con un extractor de pipeta. Es importante evitar las burbujas en esta etapa mientras pipeteando arriba y abajo para disociar las células. Organoides Páncreas óptimamente empiezan desde pequeños grupos de 5 a 15 células y, por tanto, la disociación parcial se recomienda (Figura 1). </li></ol></li><li> Reunir las células de varios embriones en un tubo Eppendorf con el fin de minimizar las diferencias debido a procesamiento individual. Diluir la suspensión de células en Matrigel frío en una proporción 01:03. Alícuota de esta mezcla a una placa de 96 pocillos, 8 l / pocillo o en una placa optimizado para la imagen (ver más abajo). </li><li> Incubar la placa a 37 ° C durante 5 minutos, permitiendo que el Matrigel se espese. Llenar los pocillos con 70 l de medio de elección (organoide o esfera, véase Tables 1 y 2) y dejar en un ambiente humidificado contiene 5% de CO2 y 95% de aire a 37 ° C. </li><li> Sustituya el medio cada 4 º día. Monitorear las colonias pancreáticas crecimiento diario y documentar el proceso de obtención de imágenes. </li><li> Las moléculas pequeñas o proteínas de interés se pueden añadir al medio en esta etapa para experimentos de interferencia, como se informó anteriormente 28. </li></ol> Tabla 1: media organoide. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nombre de los Materiales </td><td> Stock Concentración </td><td> Concentración en medio final </td><td> Volumen de existencias </td></tr><tr><td> Penicilina-estreptomicina </td><td> 100% </td><td> 1% </td><td> 50 l </td></tr><tr><td> Reemplazo Serum golpe de gracia (suplemento) </td><td> 100% </td><td> 10% </td><td> 500 l </td></tr><tr><td> 2-mercaptoetanol </td><td> 14,3 M </td&gt; <td> 0,1 mM </td><td> 1 l </td></tr><tr><td> Miristato acetato de forbol (PMA) </td><td> 16 M </td><td> 16 nM </td><td> 5 l </td></tr><tr><td> Y-27632 (inhibidor de ROCK) </td><td> 50 mM </td><td> 10 M </td><td> 1 l </td></tr><tr><td> EGF </td><td> 50 g / ml </td><td> 25 ng / ml </td><td> 2.5 l </td></tr><tr><td> Recombinante Humana R-1 Spondin </td><td> 250 g / ml </td><td> 500 ng / ml </td><td> 10 l </td></tr><tr><td> - O - </td><td></td><td></td><td></td></tr><tr><td> Recombinante ratón R-1 Spondin </td><td> 250 g / ml </td><td> 500 ng / ml </td><td> 10 l </td></tr><tr><td> FGF1 humano recombinante (FCFa) </td><td> 100 g / ml </td><td> 25 g / ml </td><td> 1,25 l </td></tr><tr><td> Heparina (Liquemin) </td><td> 2500 U / ml </td><td> 2,5 U / ml </td><td> 2 l </td></tr><tr><td> RecomFGF10 humana recombinante </td><td> 100 g / ml </td><td> 100 ng / ml </td><td> 5 l </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 l </td></tr><tr><td> Total </td><td></td><td></td><td> 5000 l </td></tr></tbody></table> Tabla 2: mediano Esfera. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nombre de los Materiales </td><td> Stock Concentración </td><td> Concentración en medio final </td><td> Volumen de existencias </td></tr><tr><td> Penicilina-estreptomicina </td><td> 100% </td><td> 1% </td><td> 50 l </td></tr><tr><td> X50 B27 (suplemento) </td><td> 100% </td><td> 10% </td><td> 100 l </td></tr><tr><td> FGF2 humano recombinante (bFGF) </td><td> 100 g / ml </td><td> 64 ng / ml </td><td> 3.2 l </td></tr><tr><td> Y-27632 (inhibidor de ROCK) </td><td> 50 mM </td><td> 10 M </td><td> 1 l </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8 l </td></tr><tr><td> Total </td><td></td><td></td><td> 5000 l </td></tr></tbody></table> 3. Imagen de la progresión de Desarrollo organoide <ol><li> Organoides imagen ya sea diaria o por microscopía lapso de tiempo utilizando un microscopio de time-lapse fluorescente. Para imágenes de lapso de tiempo, utilice un gráfico XY (Z) microscopio de fluorescencia invertido automatizado. </li><li> Depósito de pequeñas gotas de 3 l en placas de 4 pocillos o placas con fondo de cristal llenos de medio de 2-5 ml. Imagen con un objetivo de larga distancia 10 veces. Nota: Los ratones transgénicos que expresan los marcadores fluorescentes se pueden utilizar. Película 1 muestra, por ejemplo, la expansión inicial de organoides de Pdx1-Ngn3-ER TM-IRES-nGFP + ratones 4. La GFP nuclear permite al usuario realizar un seguimiento de las células como objetos individuales, sino principios similares pueden ser aplicados a un seguimiento de las células con fluorescencia de membrana talescomo mT / MG ratones 29. </li><li> Comience time-lapse de imágenes de 3 horas después de la siembra de las células para evitar derivas de enfoque y ajustar el software de automatización de control para tomar 1 imagen / hr durante 3 o más días en las posiciones definidas manualmente. Para cada posición, adquirir una imagen de contraste de interferencia diferencial (DIC), así como la señal de GFP, la presentación de informes de expresión de marcador fluorescente. </li></ol> 4. Recuperación de Organoides para histología <ol><li> Coloque la placa de 96 pocillos en hielo y se elimina el medio, reemplazándolo con PBS enfriado con hielo. Esta despolimeriza parcialmente Matrigel. </li><li> Se aspira suavemente cada organoide individuo, la eliminación de la Matrigel circundante usando una punta de 1,000 l con el fin de no perturbar la arquitectura general. Transfiera cada organoide a un pozo con hielo frío PBS. Guarde la placa de hielo. La fijación directa en Matrigel también es posible. </li><li> Fijar el organoide durante 15 minutos en 4% paraformaldehido (PFA), criopreservar en sacarosa e incrustarlo en gelatina. Procesar cada unoorganoide para cryosectioning y la histología como se describe anteriormente (Johansson et al., 2007) 4. </li></ol> 5. Recuperación de Organoides para la PCR y Bioquímica <ol><li> Coloque la placa de 96 pozos en el hielo y quitar del medio. Añadir 60 l de RNAlater por pocillo con el fin de estabilizar y proteger el ARN celular. </li><li> Interrumpir el gel en cada pocillo mecánicamente por parcialmente despolimerización en hielo. Cualquiera de recuperar organoides individuales usando una punta de 1,000 l con el fin de no perturbar la arquitectura general o recuperar todo el pozo (con Matrigel) mediante la interrupción de la gel de forma mecánica con una punta de 200 l. Utilice una punta de 1.000 l para transferir el contenido bien en un libre no pegajoso tubo Eppendorf RNAsa mantener en hielo. </li><li> Lave los pocillos con 60 l RNAlater y añada el contenido restante en el mismo tubo de Eppendorf. </li><li> Haga girar los tubos durante 5 minutos a 500-1.000 xga 4 º C. </li><li> Eliminar el sobrenadante, dejando sólo 20 a 30 lde RNAlater en el tubo junto con el sedimento. Para la bioquímica, almacenar las muestras lo más seco posible. </li><li> No congelar las muestras en RNAlater inmediatamente; se almacenan a 4 ° CO / N (para permitir RNAlater penetre a fondo el tejido). El tejido puede almacenarse a -20 ° C para almacenamiento a largo plazo y más tarde se puede procesar para la interrupción del tejido y la extracción de pequeñas cantidades de ARN. </li></ol>

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The authors have nothing to disclose.

La producción a gran escala de células beta funcionales in vitro sigue siendo ineficaz 1. En este contexto desafiante, estudios de biología del desarrollo pueden ayudar a descifrar las señales exactas que se requieren para la diferenciación de las células beta funcionales. Este protocolo permite el mantenimiento, expansión y diferenciación de células progenitoras pancreáticas embrionarias in vitro. Esto incluye la formación de células beta productoras de insulina que no co-expres...

Cultivo de órganos constituye un modelo útil que llena los huecos existentes entre la complejidad y relevancia en las investigaciones in vivo y la simulación conveniente pero aproximada de modelos de línea celular. En el caso del páncreas, no hay una línea de células perfectamente equivalente a progenitores páncreas aunque no se transforman líneas celulares que simulan células endocrinas y exocrinas. Todo el páncreas adulto no puede ser cultivado; aislados islotes endocrinos pueden mantenerse durante semanas sin la proliferación celular y rodajas de tejido se pueden mantener in vitro para algunas horas 5. Cultura páncreas embrionario ha sido ampliamente utilizado no sólo para estudiar su desarrollo, sino también para investigar las interacciones epitelio-mesenquimal 4,6,7, a la imagen procesa 8 o interferir químicamente con ellos 9. Dos métodos de cultivo de órganos se utilizan principalmente: la primera consiste en el cultivo de brotes pancreáticos en placas de fibronectina recubierto 2, que es conveniente para los propósitos de formación de imágenes; la segunda opción es la cultura de los órganos en los filtros en la interfase aire-líquido 3,4 que mejor conserva la morfogénesis. Aunque muy útil, estos métodos conducen a un cierto grado de aplanamiento; la expansión de progenitores es muy limitado en comparación con el desarrollo normal y la población de partida es complejo que comprende todos los tipos de células pancreáticas y células mesenquimales. La capacidad de la cultura y expandir las células primarias dispersas es valiosa para estudiar las relaciones de linaje y descubrir las propiedades intrínsecas de los tipos de células aisladas 10. Sugiyama et al 11. Podría mantener progenitores pancreáticos y células progenitoras endocrinas que conservaron algunos caracteres funcionales durante 3-5 días en cultivo en capas alimentadoras. Pancreatospheres, similar a neuroesferas 12 y mammospheres 13, se han ampliado de islotes adultos y células ductales aunque la naturaleza de los progenitores / células madreque generan estas esferas no está claro. Además, en contraste con el desarrollo fisiológico, los pancreatospheres contenían algunas neuronas 14,15. Esferas también se produjeron recientemente a partir de progenitores páncreas embrionario 16,17 y regenerar los páncreas de 18 años con una buena expansión de progenitores y posterior diferenciación pero no lograron recapitular la morfogénesis. Modelos 3D a partir de células dispersas y, a menudo definidos que se auto-organizan en órganos miniaturizados han florecido recientemente y simular el desarrollo o un adulto facturación de múltiples órganos, como el intestino 19,20, el estómago 21, el hígado 22, la próstata 23 y la tráquea 24. En algunos casos, la morfogénesis y la diferenciación del desarrollo se han recapitulado en 3D a partir de células ES, como es el caso de tazas 25, el intestino 26 o cerebrales 27 óptica. En este sentido, describir un método para expandir progenitores pancreáticos multipotentes disociadas en un andamio 3D Matrigel donde pueden diferenciarse y auto-organizarse.

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			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
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			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
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			<td>Phorbol Myristate Acetate (PMA)</td>
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			<td>524400-1MG</td>
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			<td>Y-27632 (ROCK inhibitor)</td>
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			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>&#160;- or -&#160;</td>
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			<td></td>
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			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
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		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
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		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
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		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
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		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
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		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
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			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
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		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
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		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
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		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
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		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
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in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

Progenitores pancreáticos dorsal E10.5 disociados y sembradas en 3D Matrigel recapitular el desarrollo del páncreas. Progenitores pueden ser más fácilmente seguidos con reporteros fluorescentes. En nuestro caso hemos utilizado un ratón transgénico que expresa una proteína GFP nuclear controlada por el promotor Pdx1 (Pdx1-Ngn3-ER TM-nGFP) (Película 1) en ausencia de tamoxifeno y por lo tanto sin necesidad de activar NEUROG3 4 (Figura 2). ...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

El método de cultivo en tres dimensiones se describe en este protocolo recapitula el desarrollo del páncreas a partir de progenitores páncreas de ratón embrionarias dispersas, incluyendo su expansión sustancial, diferenciación y morfogénesis en un órgano ramificada. Este método es susceptible de formación de imágenes, la interferencia funcional y la manipulación del nicho.

In Vitro Páncreas Organogenesis de dispersos embrionarios de ratón Progenitores

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Research

JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K vistas.  Swiss Institute for Experimental Cancer Research.  El método de cultivo en tres dimensiones se describe en este protocolo recapitula el desarrollo del páncreas a partir de progenitores páncreas de ratón embrionarias dispersas, incluyendo su expansión sustancial, diferenciación y morfogénesis en un órgano ramificada. Este método es susceptible de formación de imágenes, la interferencia funcional y la manipulación del nicho. 

Video: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

En la diferenciación in vitro de la madre embrionarias de ratón (MES), las células con el método de gota

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Investigación

Biología

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Una sola célula grabaciones de succión de los conos del ratón

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificación de los progenitores del músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A System for ex vivo Culturing of Embryonic Pancreas

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

A System for ex vivo Culturing of Embryonic Pancreas

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

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Etiquetado de la célula y de inyección en Desarrollo de embriones de ratón Corazones

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Aislamiento de ARN de ratón Páncreas: Un tejido de ribonucleasa-rica

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteína Aislamiento del desarrollo embrionario del corazón del ratón Válvula Región

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procedimiento para evaluar la eficacia de floculantes para la eliminación de partículas dispersas en extractos de plantas

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...